Aleiodes Wesmael in the gastritor Thunberg species-group is described and illustrated. The new species, Aleiodes ceres Shimbori sp.n., occurs in Southern Brazil and Argentina. It is a potential biological control agent of caterpillars in the genus Spodoptera Guenée, namely S. cosmioides , S. eridania (Stoll), and S. frugiperda (J.E. Smith), which are serious pests on major crops including maize and soybean. The position of the new species within Aleiodes is discussed in relation to existing proposals of subdivisions of the genus into species-groups. In agreement with recent publications, the gastritor species-group is treated as separate from the circumscriptus Nees/bicolor Spinola group, as evidence suggests the latter is absent in the New World. We also propose the provisional shakirae Shimbori & Shaw species-group, to accommodate a clade of Neotropical species with geometrid hosts and morphological features similar to the gastritor and circumscriptus groups. A key to Neotropical species in the gastritor group and similar species is provided, except for species recently named without a morphological description.A new species of The online version contains supplementary material available at 10.1007/s13744-023-01076-8. Aleiodes Wesmael is the most diverse lineage within Rogadinae , and has a worldwide distribution. All rogadines are koinobiont endoparasitoids of lepidopteran larvae and mummify their host caterpillar while pupating inside it  comprises 31 species, of which 15 are considered pests in the Western and Eastern hemispheres , S. androgea , S. cosmioides , S. dolichos , S. eridania , S. evanida Schaus, 1914, S. frugiperda , and S. ornithogalli  (Pogue Bacillus thuringiensis Berliner (Bt)  Pogue , and som2) Pogue . Since t) CTNBio , there hSpodoptera aiming at its control has been high for at least a century, especially for the fall armyworm  is the most commonly reported, mainly in North America (USA and Mexico)  in S. frugiperda and S. eridania, in Central and South America  Merrill) crop season in São José dos Pinhais (25°36′49.0″S 49°08′01″W), Paraná, Brazil. In the 2017/2018 and 2018/2019 soybean crop seasons, between January and March, a survey of pest caterpillars was conducted weekly by the beat cloth method  which confers resistance to target lepidopteran species.Specimens of Spodoptera complex in Brazil, namely Spodoptera cosmioides , Spodoptera eridania (Stoll), and Spodoptera frugiperda (J.E. Smith). The caterpillars were observed daily for parasitism. After emergence of the adult parasitoids, lab-reared Spodoptera eridania caterpillars were provided aiming to establish a laboratory rearing of the parasitoids. Specimens of the parasitoid were killed at low temperature (− 20 °C) and preserved in 99% ethanol for identification.Collected caterpillars were separated individually in plastic containers, identified at species level, and fed with soybean leaves from the same variety in which they were collected. Within the species collected, we identified the most common species of the Zea mays L.) crop season, in Pinhais (25°24′01″ S 49°07′01″ W), Paraná, Brazil, in an experimental area with the variety 30F53 Pioneer. The methodology used was similar to the soybean sampling.Additional samplings were conducted in the 2019 maize (S. eridania (n = 15) were provided for each female, and after 24 h, caterpillars were removed and new specimens were offered. This procedure was repeated for 3 days.Fifteen 48-h-old parasitoid females previously mated were kept individually in plastic containers (Æ 98.5 × 210 mm) with a drop of honey in an incubator room . Second instar larvae of Larvae were individualized in plastic containers (500 mL) and daily, soybean leaves from the variety BRS 1003IPRO which express tolerance to the herbicide glyphosate and the Cry1Ac protein of Bt were provided. We evaluated the total number of larvae parasitized (by observing the formation of mummies), development time (in days), and emergence rate.Aleiodes adopted here follows that of van Achterberg , see van Achterberg  or Sharkhterberg  and Shawhterberg . For idew et al. , b and Fw et al. . The ideOOL: shortest distance between eye and lateral ocellusOD: maximum diameter of lateral ocellusPOL: shortest distance between lateral ocelliT1: metasomal tergite 1T2: metasomal tergite 2T3: metasomal tergite 3.Morphological terminology for descriptions follows that of Sharkey and Wharton , S.R. ShColeção Entomológica do Departamento de Ecologia e Biologia Evolutiva da Universidade Federal de São Carlos, São Carlos, Brazil (DCBU)Coleção Entomológica Padre Jesus S. Moure, Departamento de Zoologia da Universidade Federal do Paraná, Curitiba, Brazil (DZUP)University of Wyoming Insect Museum, Department of Ecosystem Science and Management, University of Wyoming, Laramie, WY, USA (UWIM)Examined specimens are deposited in the following collections:2, 0.2 mM dNTP Mix, 0.2 µM each primer, 1 × HOT FIREPol® Buffer B2, and 1 U HOT FIREPol® DNA Polymerase, Solis Biodyne) and followed a cycling process of initial denaturation at 95°C for 15 min; 35 cycles of denaturation at 95°C for 45 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min; and finally a final extension at 72°C for 5 min. PCR products were purified by polyethylene glycol precipitation  following the manufacturer’s instructions. Subsequent DNA purification was performed by Ethanol Precipitation  is recovered as a monophyletic clade rather than a grade leading to the subgenus Aleiodes , and metasomal terga usually yellow , as opposed to the smaller ocelli (OD < OOL) and metasomal terga always yellow medially and black laterally in circumscriptus. In addition, the circumscriptus group is broadly associated with Noctuoidea hosts, while gastritor group species are mostly associated and Geometridae , and many unassigned species  is likely a monophyletic group absent in the New World . It should be treated as its own group, as proposed by van Achterberg et al.  group, and if the biogeographical distribution of circumscriptus is indeed restricted to the Old World, the species from the New World are better placed in another group. The homoplasious nature of the characters defining pallidator seems corroborated by all available evidence, and by the fact that the only described neotropical species in the pallidator group, A. luteosicarius, is recovered in the gastritor group in our phylogeny  was recovered and should be considered for a possible subdivision of the subgenus. Here, we provisionally name this clade the shakirae group, and include species with similar morphology or DNA barcode. This group is not yet distinguishable from neotropical members of the gastritor group; however, all species have larger eyes and ocelli, the ocelli-ocular distance shorter than the diameter of lateral ocelli, and are parasitoids on Geometridae. The metasoma is at least partly dark brown or black in described species. Species without morphological descriptions  shakirae, or (3) unassigned). These are neotropical species that would key in the gastritor and/or circumscriptus groups following the key provided by Shaw et al. , herein included in the gastritor group. These species do not comprise a monophyletic group but are recognizable by the following set of characters:Diagnosis. Apex of hind tibia without comb of specialized adpressed setae; hind wing vein RS slightly sinuate and enclosing a marginal cell which is narrowest around its middle length; metasomal tergites 1–3 with finely rugose or rugose costate sculpturing, never smooth or strongly costate; tarsal claws without distinct blackish pecten, but sometimes a yellowish pecten with thinner spines present; metasomal tergite 2 without a distinct smooth triangular area medio-basally; ocelli size variable.The key presented below includes only neotropical species in the subgenus w et al. , b ; cacuangoi Shimbori & Shaw 2014; falloni Shimbori & Shaw 2014; gossypii (Muesebeck laphygmae (Viereck 1912); leptocarina Fortier luteosicarius Shimbori & Shaw 2014; mirandae Shimbori & Shaw 2014; napo Shimbori & Shaw 2014; nubicola Shimbori & Shaw 2014; onyx Shimbori & Shaw 2014; yanayacu Shimbori & Shaw 2014; and ceres Shimbori sp. n.1.  Species included in the Neotropical shakirae species-group: based on morphology—shakirae Shimbori & Shaw 2014; speciosus Townsend, 2009; townsendi Shimbori & Shaw 2014; japi Shimbori & Penteado-Dias 2014; based on DNA barcode: alejandromasisi Sharkey 2021; michelledsouzae Sharkey 2021; davefurthi Sharkey 2021.2. Neotropical species in the subgenus Aleiodes not assigned to a group: kingmani Shimbori & Shaw 2014; tzantza Shimbori & Shaw 2014. Both species are morphologically compatible with the shakirae species-group; however, kingmani is not assigned to shakirae based on mummy morphology, resembling the buzurae group, and tzantza because it is a parasitoid of Noctuidae, whereas all species in the shakirae species-group are parasitoids of Geometridae.3.The species included in the key are, therefore, members of at least two separate clades, for which a morphological distinction is currently unavailable, also including species that are not assigned to any of the two species-groups:1.  Ocelli small, ocelli–ocular distance longer than width of lateral ocellus Fig A, B … 2Ocelli moderate-sized, ocelli–ocular distance equal to or shorter than width of lateral ocellus Fig C, D … 142(1). First and/or second metasomal terga with median carina present Fig A; oviposalbiterminus Townsend– First and second metasomal terga with median carina absent Fig B; oviposarbitrium Townsend3(2). Malar space about as long as width of mandible base Fig. A; head m– Malar space at least 1.25× width of mandible base Fig. B; head a4(3). Occipital carina weak and interrupted mid-dorsally Fig A, C … 5– Occipital carina complete and well-defined at mid-dorsally Fig. B, D … 105(4). Mesopleuron with central disc lacking setae, smooth and shining . Tergite 2 mostly black with white markings, hind coxa black Fig. C… 7yanayacu Shimbori & Shaw– Tergite 2 entirely whitish yellow, hind coxa yellowish Fig. B; hind wmirandae Shimbori & Shaw7(6). Head, pronotum, propleuron, and scutellum orangish yellow . Entirely honey yellow except ocellar triangle dark brown . Head orange, except for black ocellar triangle, contrasting with mostly black body Fig. D; Zygaenatripileatus Townsend– Head mostly yellowish brown with large black semicircular spot on occiput, vertex and ocellar triangle also black Fig. A; mesopl10(4). Pronotal collar yellowish brown or honey yellow , 17(G)) … 11– P ronotal collar black Fig. B, D … 13bimaculatus Shimbori & Shaw11(10). 39–46 antennomeres; hind coxa rugose dorsally … – 31–36-antennomeres; hind coxa granulate dorsally … 12ceres sp. n.12(11). Ocelli small, ocell-ocular distance about 2× longer than diameter of lateral ocellus – Ocelli larger, ocell-ocular distance about as long as diameter of lateral ocellus . Mesoscutum with square orangish brown mark postero-medially . Mesosoma and metasoma mostly honey yellow Fig. A–C, someA. shakirae species-group)– Propodeum and most of metasomal terga black or dark brown Fig. A, B, C, 15(14). Fore wing vein at least 1CUa 2.0× vein 1cu-a; antenna with 43–51 flagellomeres …16gossypii Muesebeck2– Fore wing vein 1CUa 1.5× vein 1cu-a; antenna with 36–39 flagellomeres; Erebidae hosts … 16(15). Hind wing vein m-cu present as pigmented not tubular vein; fore wing vein 1CUa 1.8× 1CUb, vein r 0.5–0.7× vein 2RS; T1 and T2 with complete median carina … 17leptocarina Fortier– Hind wing vein m-cu absent; fore wing vein 1CUa 1.3× vein 1CUb, vein r 0.9× vein 2RS; T1 and T2 with median carina weak or absent apically; a gregarious parasitoid of Erebidae… falloni Shimbori & Shaw17(16). Hind wing vein 2-1A absent; diameter of lateral ocellus roughly as long as ocell–ocular distance Fig. B … falloluteosicarius Shimbori & Shaw– Hind wing vein 2-1A present Fig. B; diametaclydis Townsend18(14). Malar space short, length 0.7× width of mandibular base Fig. A; median– Malar space moderately wide, at least slightly longer than width of mandibular base Fig. B; median19(18). Mesoscutum and scutellum honey yellow, hind coxa bicolored black and white Fig. A, C, E …– Mesoscutum and scutellum partially to mostly black, hind coxa one color, either black Fig. D or whit20(19). Head honey yellow, ocellar triangle dark brown Fig. C, E; foralbigena Shimbori & Shaw– Head dark brown, gena white Fig. A; fore wshakirae Shimbori & Shaw21(20). First metasomal tergite about 2× longer than its apical width, dark brown to black Fig. A; hind ctownsendi Shimbori & Shaw– First metasomal tergite about as long as apical width, white with small black spot mid-apically; hind coxa black basally and apically white Fig. E; Geomet22(19). First metasomal tergite dark brown, not contrasting with reminder terga (14C) … 23– First metasomal tergite white, contrasting with remainder mostly dark brown metasoma Figs. D, 14B … tzantza Shimbori & Shaw23(22). Fore wing vein 1CUa about 0.45× as long as vein 1CUb . Mesopleuron, metapleuron, hind coxa, and propodeum medially and anteriorly smooth; mesopleuron except for anterior corner and hind coxa orangish; Geometridae hosts … kingmani Shimbori & Shaw– Mesopleuron, metapleuron, hind coxa and propodeum granulate; mesopleuron and hind coxa black Fig. D; GeometComments on biology of Neotropical species. Information regarding association with hosts is quite abundant. Of the 36 species, 24 have host records .Holotype, female. “Brazil, Paraná, São José dos Pinhais. 25°36′49″S 49°08′01″W. 01.II.2018. Emerged from Paratypes: 2 females and 1 male, same data as holotype (DCBU 518.067–518.069); 4 females and 4 males, same data as holotype except “1.I.2018…” (DCBU 518.070–518.077); 2 females and 1 male, same data as holotype except “25°36′44.68″ S 49°08′17.31″ W. 1–15.II.2017. Soja trans (bt-rr2)…” (DCBU 518.078–518.080).Female. Body length 3.9–4.5 mm. Fore wing length 3.2–3.7 mm.Head . Head dark brown, except palpi light pale yellow and mandibles pale brown. Antenna with flagellum brown; scape and pedicel mostly honey yellow, light brown dorsally. Mesosoma mostly honey yellow; mesoscutum and propodeum dark brown; metanotum and scutellum light brown, darker medially. Metasoma light pale yellow ventrally; T1–2 dark brown with large pale yellow mark medially, starting at posterior 1/4 of T1 and extending posteriorly to cover most of T2 medially, and reaching anterior 1/2 of T3 as a small triangular spot; T3 mostly brown to dark brown with honey yellow borders, remainder terga mostly honey yellow with brown spots on T5–7 medially. Wings faintly tinged brown; veins mostly brown; stigma pale yellow with infuscate borders. Legs honey yellow basally , apical half of femur, tibia and 5th tarsomere brown, tarsomeres 1–4 light brown. Ovipositor sheaths dark brown.Male. Body length 3.7–4.5; fore wing length 2.9–3.7. Similar to females in most aspects, except for the antenna which is longer than body (about 1.2 ×), compared with the antenna about as long as body in females. Eyes and ocelli relatively larger in males, OOL/OD 1.92–2.12 and eye length/temple 1.46–1.67.Biology. Specimens of A. ceres collected in 2019/2020 soybean crop season have been kept in the laboratory for over 64 generations without losing their biological traits. In the laboratory, A. ceres can develop in the hosts: S. cosmioides, S. eridania, and S. frugiperda with their hosts being reared on a natural or artificial diet. Females of A. ceres can parasitize caterpillars from the first to third instar; however, better parasitism and emergence rates were observed in caterpillars of the first and second larval instar. When offered the second instar of S. eridania, A. ceres parasitized on average 9.07 ± 0.78 caterpillars in 24 h. In 3 days, a total of 26.0 ± 1.47 caterpillars were parasitized. The development time (egg-adult) was 16.58 ± 0.09 days and the emergence rate was 87.44 ± 2.59 at 25 ± 2 °C, 70 ± 10% RH.Spodoptera complex because no parasitism was recorded in other species like Anticarsia gemmatalis Hübner, Chrysodeixis includens , Ephestia kuehniella (Zeller), Helicoverpa armigera (Hübner), and Mythimna sequax (Franclemont).This species seems to be closely associated with caterpillars of the Etymology. The choice for the name of the new species was made in a public contest, with the objective of raising awareness and interest of the general public, especially middle and high school students, for taxonomic research and its relevance to biological control. The contest was held during a community outreach fair “ESALQSHOW,” on the 6th and 7th of October, 2022, on the campus of the “Luiz de Queiroz College of Agriculture (ESALQ),” of the University of São Paulo. For all participants, a brief explanation of the rules of zoological nomenclature was given, in addition to the economic and ecologic importance of the new species and the consequences of the gap in taxonomic knowledge. The name chosen by one of the public contest participants was given in reference to the goddess of agriculture, Ceres, of Roman mythology.Diagnosis. Very small ocelli, ocell-ocular distance more than 2 × longer than diameter of lateral ocellus in females ; body color variable, mostly honey yellow with dark brown marking at mesoscutum, propodeum and metasoma, pronotum and mesopleuron always honey yellow, head nearly always black; stigma entirely pale brown; longitudinal carina at metasomal tergum 3 absent or shortly indicated anteriorly, complete at terga 1 and 2.Comments. The new species differs from A. laphygmae in having much smaller ocelli. The ocell-ocular distance is more than two times longer than the diameter of the lateral ocellus . The metasomal tergum 3 has a nearly complete longitudinal carina in A. laphygmae, but in the new species, the longitudinal carina is absent or only weakly indicated anteriorly. The new species also has a much more extensive darker coloration, including head , mesonotum, propodeum, and metasomal terga 1–4, compared with the entire honey-yellow body of A. laphygmae. The new species also resembles Aleiodes nubicola Shimbori and Shaw A. nubicola, as compared to entirely yellow in A. ceres sp.n. A species of Geometridae is the only known host for A. nubicola, while A. ceressp.n. is known to attack Spodoptera eridania (Noctuidae), which also results in very different caterpillar mummies produced by each parasitoid species .Records from Argentina are based on DNA Barcode sequences deposited in public databases . The similarity between sequences from Argentinian and Brazilian specimens ranges from 98.3 to 99.1%. Our ML phylogeny also supports the conspecificity of Argentinian and Brazilian specimens. All Argentinian specimens are deposited at Museo Argentino de Ciencias Naturales Bernardino Rivadavia, in Buenos Aires, Argentina, and are assigned a single BIN (Barcode Index Number), BOLD:ACN2401 Figure Rogas laphygmae Viereck 1912, 43: 581.Ocelli moderate-sized, ocell-ocular distance about as long as the diameter of lateral ocellus; body color entirely honey yellow; stigma brown with yellow spots at base and apex; longitudinal carina at metasomal tergum 3 present and extending nearly over the entire length of tergite; body length 4.0–5.0 mm; antenna with 33–35 antennomeres; the angle between fore wing vein r and basal ventral line of stigma about 160 degrees Fig. , both apBiology. The species is a parasitoid of the early stages of a few caterpillar species in the family Noctuidae, namely Mythmna unipuncta (Haw.), Spodoptera exigua (Hbn.), S. frugiperda (Smith), and S. orthogonali (Guen.).Distribution. Southern USA, Cuba, and Nicaragua. Recent records from Argentina are most likely of Aleiodes ceressp.n.TAXONOMIC NOTESAleiodes leptocarina Fortier 2000Aleiodes normwoodleyi Sharkey, 2021 NEW SYNONYMYAleiodes normwoodleyi Sharkey, 2021 is a junior synonym of the previously described species Aleiodes leptocarina Fortier Aleiodes leptocarina from Costa Rica, which was notable (at that time) in being the only known Aleiodes from Costa Rica to develop gregariously inside large caterpillars. The identity of the host caterpillar was not known at that time, but Fortier (Dysschema vidua (Erebidae)). Sharkey’s “minimalist” species description was based on a DNA sequence, one photograph, the host association with Dysschema vidua, and only one stated morphological difference: the antennal flagellum was stated to be “melanic” (black) in A. normwoodleyi, as opposed to being “honey yellow” in A. leptocarina. During this study, we re-examined the holotype and paratype series for A. leptocarina Fortier, and observed that the flagellum is not yellow but instead is entirely black in all the type specimens. We believe that confusion was created by an error in Fortier’s  has previously identified in 2005 a long series of gregarious specimens reared from a mummified caterpillar of Dysschema vidua as being A. leptocarina (by direct comparison with the type series). This host association was reported to Dan Janzen and was posted on the Caterpillars of the ACG website for many years (until the species association was later changed to A. normwoodleyi), presumably on the basis of the wasps having a black flagellum (and the erroneous assumption that A. leptocarina specimens have a yellow flagellum). Therefore, we regard Dysschema vidua to be a previously known host record for A. leptocarina . We regard the species concept of A. normwoodleyi to be based on an (understandable) misidentification; therefore, the molecular sequence presented by Sharkey Below is the link to the electronic supplementary material.


Additionally, a 3D-spheroid study was performed where a single dose of Cela MP at 1.0 µM significantly inhibited spheroid growth. Cela MP was also able to retain the antioxidant activity of Cela only while mechanistic studies revealed triggered autophagy and an induction of apoptosis. Therefore, these studies highlight the anti-mesothelioma activity of Cela and demonstrate that Cela MPs are a promising inhalable medicine for MPM treatment.Malignant pleural mesothelioma (MPM) is a rare and aggressive cancer affecting the pleural lining of the lungs. Celastrol (Cela), a pentacyclic triterpenoid, has demonstrated promising therapeutic potential as an antioxidant, anti-inflammatory, neuroprotective agent, and anti-cancer agent. In this study, we developed inhaled surface-modified Cela-loaded poly(lactic-co-glycolic) acid (PLGA) microparticles (Cela MPs) for the treatment of MPM using a double emulsion solvent evaporation method. The optimized Cela MPs exhibited high entrapment efficiency (72.8 ± 6.1%) and possessed a wrinkled surface with a mean geometric diameter of ~2 µm and an aerodynamic diameter of 4.5 ± 0.1 µm, suggesting them to be suitable for pulmonary delivery. A subsequent release study showed an initial burst release up to 59.9 ± 2.9%, followed by sustained release. The therapeutic efficacy of Cela MPs was evaluated against four mesothelioma cell lines, where Cela MP exhibited significant reduction in IC Malignant mesothelioma (MM) is a rare, incurable, and malignant type of cancer arising from the mesothelial or sub-mesothelial cells of the pleura (lung lining), peritoneum , pericardium (heart sac), or testes . MM is cTripterygium wilfordii Hook F (Thunder God Vine), which has demonstrated interesting and promising bioactivity [Celastrol (Cela) is an extensively studied phytochemical derived from the root extract of activity . Numerouactivity ,9, neuroactivity ,11, and activity ,13. Addiactivity ,15. Celaactivity ,17. Despactivity . TherefoPulmonary drug delivery presents various advantages over oral and parenteral routes for localized treatment of chronic lung disorders. This route of administration provides targeted lung delivery, local accumulation, rapid onset, reduced systemic exposure, extensive surface area for drug absorption, and reduced side effects, and requires lower doses for similar lung deposition of therapies ,19. The This is the first report demonstrating Cela’s efficacy for MPM treatment. In addition, the present study explores surface-modified microparticles as effective inhaled carriers possessing deep lung deposition properties. Cela-loaded PLGA microparticles (Cela MPs) were prepared using pore-forming agent sodium chloride (NaCl) to create corrugated surface morphology that provides favorable aerodynamic properties while efficiently avoiding macrophage uptake. Optimized Cela MPs were analyzed for their aerodynamic performance as well as their 2D anti-proliferative properties and 3D anti-tumor efficacy against mesothelioma cell lines. Finally, multiple anti-cancer mechanisms of action of Cela MPs were elucidated. 3 and NaCl), were examined. Incorporating NaHCO3 (F5) in the inner aqueous phase resulted in a reduced %EE of 34.8%, particle size of 2341 nm, and zeta potential of −21.6 mV, along with a very high PDI of 0.791. However, the addition of NaCl (F6) as a porogen resulted in a relatively higher %EE of 72.8 ± 6.1%, lower PDI of 0.3 ± 0.2, and a stable zeta potential of −36.6 ± 3.8 mV, along with a particle size of 2076 ± 390 nm geometric size and a PDI of 0.3 ± 0.2. F6 formulation was selected for further studies.Cela-loaded microparticles were successfully prepared using a double w/o/w emulsion solvent evaporation method . The F1 The morphology of Cela MPs was examined by SEM. A representative image is presented in The release profile of Cela MPs in 1% SLS in SLF at 37 °C is shown in The DSC thermogram of Cela C shows aDiffractometry studies revealed multiple peaks representing the crystalline nature of Cela, shown in The stability of optimized Cela MP was determined in terms of particle size, zeta potential, and percent drug entrapment as a function of storage time and conditions. As seen in In vitro lung deposition studies were performed using a Next Generation Impactor (NGI). Additionally, %EE, PS, PDI, and zeta potential were evaluated for Cela MPs after nebulization. As presented in 50 values of Cela and Cela MPs were calculated using GraphPad Prism®, and it was observed that microparticle encapsulation reduced the IC50 of Cela ~1.3-fold against MTSO-211H cells   . Similarectively .To determine whether blank MPs imparted any notable toxicities, blank MPs were evaluated against MSTO-211H cells and normal human lung fibroblast (NHLF) cells. As shown in p < 0.0001). These results suggest that the antioxidant activity of Cela was either retained or enhanced upon encapsulation into the PLGA microparticles.Cela is a known antioxidant that may boost its anti-inflammatory and anti-cancer properties. To test the ability of Cela and Cela MPs in scavenging free radicals, a DPPH assay was performed. As shown in p < 0.01) and ~3.5-fold increase compared to 1.0 µM Cela (143 ± 19.7%) (p < 0.01) (Caspase-3 activity was then monitored to examine the effects of Cela and Cela MP treatment on apoptotic pathways.  < 0.01) C. Theref® Autophagy Detection Kit was used to determine any effect on LC3 levels, where increased levels indicated induced autophagy. The effects of Cela and Cela MP were investigated in the MSTO-211H cell line. As shown in p < 0.01) and ~1.5-fold relative to Cela (p < 0.001). No significant difference was found between the control and Cela only treatments. This signifies that Cela MP induced autophagosome formation in mesothelioma cells at a concentration of just 1.0 µM.Cela has also been reported to trigger autophagic pathways in which promote cell death in cancer ,34,35. Ip < 0.01) as compared to the control. Additionally, a higher concentration of Cela MP (1.0 µM) suppressed growth by ~13-fold as compared to the control (p < 0.0001) and ~10-fold as compared to 1.0 µM Cela only (p < 0.0001).An in vitro tumor simulation model was utilized to mimic in vivo tumor conditions and better predict the clinical effectiveness of Cela MP. The single-dose study involved drug treatment once throughout the 15-day experimental period. Representative tumor spheroid images from single dose treatment are shown in p < 0.0001). Therefore, the 3D spheroid volume data concludes that enhanced anti-tumorigenic efficacy is achieved for our formulation against MPM.A multiple-dose study was then performed to mimic the physiological conditions a treatment may undergo  ,37. Simip < 0.0001). The multiple-dose treatment regimen proved that all four treatment groups significantly reduced cell viability as compared to the control (p < 0.0001) and no statistical difference was found between the treatment groups , is known to be efficacious against a variety of cancers, including liver cancer, breast cancer, prostate cancer, multiple myeloma, glioma, etc. [Celastrol (Cela), a natural product from ma, etc. . This prma, etc. ,39,40. Hma, etc. ,42. To aThe optimized Cela MP (F6 formulation) resulted in high drug entrapment (72.8 ± 6.1%), which is equivalent to 1.5 ± 0.12 mg (or 3.2 ± 0.3 mM), which represents a significant improvement in Celastrol solubilization and encapsulation of about ≈0.8 mM, as shown by Shukla et al. using cyclodextrin complexation . The geoRelease of the drug from the microparticles is important to predict the in vivo performance of the formulation. The developed formulation displayed a biphasic release pattern characterized by an initial burst release due to the drug being present near the particle surface , followeDSC studies showed that Cela had a characteristic endothermic peak at 155 °C, corresponding to its melting point, and an exothermic peak at 211 °C, likely due to degradation of Cela . Both DSmax with lower systemic AUC, and was well-tolerated and effective at reducing bleomycin-induced pulmonary fibrosis [Conventional routes of administration, such as oral and intravenous, are not ideal for Cela delivery. For instance, a pharmacokinetic study done on rats demonstrated the mean oral absolute bioavailability after oral administration (3 mg/kg) was very low at 3.14% . Even thfibrosis . Howeverfibrosis . NGI is fibrosis ,58,59. Afibrosis . Multiplfibrosis ,62,63. TThe in vitro cytotoxicity studies demonstrated that microparticles can be used as prospective carriers for MPM treatment by improving therapeutic efficacy at reduced doses. This may be explained by better cell attachment that may cause enhanced cellular uptake and subsequent cytotoxicity. Particles with wrinkled surfaces are widely found in nature, such as plant pollens and microorganisms . These wrinkles with their significantly enlarged surface areas provide enhanced survival tools, including pollen adhesion and hydration and cell signaling ,65. InspThe antioxidant activity of Cela has been reported in various diseases ,67,68. PCaspase-3 is a cysteine protease that plays a vital role in the terminal course of programmed cell death (apoptosis). As a proteolytic enzyme, caspase-3 is responsible for the cleavage of DEVD peptide, poly(ADP-ribose) polymerase (PARP), DNA-dependent protein kinase, etc. ,73. FailWhile the in vitro assays performed provided great insight into the efficacy of Cela MPs, they were likely inadequate in fully predicting the preclinical behavior of MPs. Tumors in the human body exist as a solid mass of cells that proliferate uncontrollably and grow exponentially into a three-dimensional structure. The in vitro monolayer studies lack the behavioral properties of a solid tumor, including cell-cell interactions, cellular heterogeneity, spatial architecture, and establishment of unique gene expression patterns ,80. Ther® RG 502H   was purchased from Evonik Corporation . Branched polyethyleneimine , Poly (PVA) , and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich . Sodium chloride (NaCl), sodium bicarbonate (NaHCO₃), 3--2,5-diphenyltetrazolium bromide (MTT), dichloromethane (DCM), dimethyl sulfoxide (DMSO), acetonitrile (ACN), other UPLC grade chemicals, and LC-MS grade water were purchased from Fisher Scientific . Molecular biology kits and supplies were acquired from other commercial vendors as listed at suitable places throughout the manuscript.Celastrol (Cela) was purchased from Adooq Bioscience ; ResomerTM basal medium supplemented with fibroblast growth medium (FGMTM)-2 SingleQuotsTM supplements from Lonza. All cell lines were incubated at 37 °C/5% CO2 and 90–100% relative humidity. Three immortalized malignant pleural mesothelioma cell lines, MSTO-211H, H28, and H2452 were procured from American Type Culture Collection . One primary patient-derived mesothelioma cell line, ROB, was obtained from Dr. Raffit Hassan at the National Cancer Institute  under an executed material transfer agreement and approved IRB protocol # 0818-034 from St. John’s University. This primary cell line was isolated from neoplastic effusions of patients undergoing therapeutic paracenteses, originally provided by The Stehlin Foundation  to the NIH, and were established as ROB in a study by Li et al. [® BEH Shield RP18 2.5µm (3.0 × 100 mm) . The mobile phase was 0.1% orthophosphoric acid (OPA) in HPLC grade water: acetonitrile (ACN) at a 10:90 ratio with a flow rate of 0.8 mL/min. The wavelength used for detection was 425 nm and data were collected and processed using EMPOWER 3.0 software . A representative UPLC chromatogram of Cela is shown in A reverse-phase liquid chromatography technique was established for measuring Cela content using a Waters Acquity series UPLC . The column used was an XBridgew/v PVA solution and homogenized  for 1 min at 8000 rpm to obtain the double emulsion (w/o/w), followed by overnight stirring to remove DCM. The next day, smaller particles remaining in the supernatant were removed by centrifugation at 3000 rpm for 3 min. The remaining pellet containing the MPs were reconstituted in milli-Q water and washed three times by centrifugation at 21,000× g for 15 min. The final formulation was reconstituted in 1 mL of water. F1-F6 formulations were synthesized with minor alterations, including various polymers included in the inner aqueous phase (cationic polymer PEI and porogen NaHCO₃) and stabilizer PVA concentration. A summary of the formulation details is provided in Cela MPs were fabricated using a double emulsion solvent evaporation method previously published with some modifications . SpecifiA total of 20 µL of the formulation samples were separately diluted by 150× in HPLC water. Particle size (PS), polydispersity index (PDI), and zeta potential were measured using a Malvern Zetasizer .g (4 °C). The supernatant containing free Cela was injected into the UPLC for analysis using the method defined in The percent encapsulation efficiency (%EE) and percent drug loading (%DL) of Cela in MPs were determined using a MP lysis method. Briefly, 20 µL of the formulation was lysed using a mixture of 10 µL DMSO, 20 µL DCM, and 1950 µL ACN, followed by centrifugation for 45 min at 21,191× The morphology of Cela MPs was examined using Helios Nano Lab 660 . The microparticle suspension was dried on the SEM pin stub  and then sputter coated with gold for 90 s and imaged at 5 kV.g for 15 min. The supernatant was then analyzed by UPLC, as described in Samples for an in vitro release study were prepared by diluting 50 µL Cela MPs with 950 µL of 1% sodium lauryl sulfate (SLS) in Simulated Lung Fluid (SLF), which was prepared according to the components in Gamble’s Solution . The sam® freeze dryer system. DSC analyses were performed on a DSC 6000 , using samples of 2–5 mg inside sealed pans. The samples were heated in sealed aluminum pans at a rate of 10 °C/min from 25–250 °C under a nitrogen flow rate of 50 mL/min. A blank sealed aluminum pan was used as the reference.Various samples were first lyophilized into dry powders using a Labconco FreeZoneXRD analysis was performed using XRD-6000 . The diffraction patterns were measured with a voltage of 40 kV and a current of 30 mA. The freeze-dried samples were uniformly spread on a glass sample holder and analyzed over a range of 2θ or 10–60° at a scan speed of 2 °/min.Triplicate samples of Cela MP suspensions were stored at 4 °C for a 4-week period. A total of 20 µL aliquots of each sample were withdrawn each week to determine the PS, PDI, and zeta potential by a Malvern Zetasizer as previously described. The %EE was analyzed by UPLC.® 170 Next Generation ImpactorTM , following a previous report [® LC plus nebulizer . The vacuum pump  was set at a flow rate of 15 L/min for 4 min as the sample travelled into the NGI. After the run, ACN was used to wash and collect the samples from each stage plate, followed by centrifugation at 21,191× g for 45 min to lyse the MPs. The resulting supernatant was analyzed using the UPLC method as mentioned in The in vitro aerodynamic assessment of Cela MPs was performed using the Copleys report . Brieflys report . A totalae < 5.39 μm or amount of drug deposited from stage 3–8) to the total emitted dose (ED) (amount of drug emitted from mouthpiece to stage 8) deposited in the NGI. The mass median aerodynamic diameter  was determined by acquiring the diameter corresponding to 50% of the cumulative deposition. The geometric standard deviation (GSD) was calculated using Equation (3) as shown below:The Fine Particle Fraction  was calculated as the ratio of fine particle dose . To further evaluate the toxicity of blank MPs, toxicity studies were performed in MSTO-211H and NHLF cell lines at corresponding celastrol concentrations (0.10–6.25 µM) after a 48-h incubation period.Cell viability studies for Cela and Cela MP were evaluated against MSTO-211H, H28, and H2452 cells, in addition to using a primary patient-derived cell line ROB using an MTT -2,5-diphenyl tetrazolium bromide) assay, following the methods published in our earlier studies ,85. DetaThe potent antioxidant and anti-inflammatory activity of Cela has been shown to be useful to treat a multitude of diseases such as cancers  ,87,88,895 cells/TC dish (100 mm diameter) . Treatments of Cela and Cela MP (0.5 and 1.0 μM) were incubated for 6 h, after which cells were harvested and washed with sterile PBS, followed by resuspending in 1X cell-lysis buffer and centrifugation. A total of 50 μL of the supernatant and 50 μL of a 2X substrate working solution (10 mM Z-DEVD-AMC substrate + 2X reaction buffer) were added to a 96-well plate while using 50 μL of lysis buffer, and a substrate working solution was added as a no enzyme control. The fluorescence was measured after incubating for 20 min at excitation/emission of 360/460 nm.To evaluate the apoptotic properties of Cela and Cela MPs, the apoptosis mediator (caspase-3) activity was determined using an EnzChek Caspase-3 assay kit . MSTO-211H cells were seeded at a density of 5 × 10® Autophagy Detection Kit  was used to determine the effect of Cela and Cela MP on autophagy in MPM cells by following a previously published protocol [The CYTO-IDprotocol . MSTO-21® ultra-low attachment spheroid 96-well plate  at a density of 2.0 × 103 cells/well and incubated at 37 °C/5% CO2 for 3 days to allow spheroids to grow into a solid tumor mass, following which treatments were started (Day 0 for treatment). Totals of 0.5 and 1.0 µM celastrol concentrations were selected for treatment. For a single dose study, half the media (100 µL) was replaced with fresh media on each imaging day (except for Day 0). For the multiple dose study, half of the media was replaced with respective concentrations of Cela or Cela MP or fresh media (control). Images of the spheroids were taken using an inverted microscope  on days 0, 3, 6, 9, 12, and 15. NIH ImageJ software (Version 1.44) was used to measure the diameter of the spheroids, which was then used to calculate the spheroid volumes.The anti-cancer efficacy of Cela MPs was further evaluated by determining the penetrability and efficacy of nanoparticles into solid tumors. As previous reports have indicated, 3D spheroid cell culture studies have been used in an attempt to bridge the gap between in vitro and in vivo systems by partially representing the tumor structure and microenvironment ,91,92. O® kit . Briefly, 100 µL of treatment was removed and replaced with 100 µL of CellTiter-Glo® reagent in each well. The contents were mixed for a few minutes, followed by incubation at room temperature for 30 min. Luminescence was measured using a Spark 10M plate reader .At the end of Day 15, cell viability within the spheroid core was assessed using Cell Titer-GloA Live/Dead cell assay was performed using a fluorescent Viability/Cytotoxicity assay kit  to visualize the live and dead cells on the surface of the spheroids. At day 15, the treatments were completely removed from each well and replaced with 100 µL of 2 µM calcein AM/4 µM EthD-III staining solution. The plate was then incubated in the dark at room temperature for 30 min and subsequently imaged on an EVOS-FL Cell Imaging fluorescence microscopy at 4X magnification . Green fluorescent protein  is representative of viable cells, whereas red fluorescent protein  indicates necrotic/dead cells. Fluorescent intensity was analyzed by NIH ImageJ software (Version 1.53c).t-test or one-way ANOVA followed by Tukey’s multiple comparisons test using GraphPad Prism software . A p value of <0.05 was considered statistically significant and was presented in data figures as a single asterisk (*). However, some studies demonstrated a smaller p-value of 0.01 or less, which is indicated at respective places.All data were addressed as mean ± SD or SEM, with n = 3 unless otherwise mentioned. At least three trials of cytotoxicity studies were performed for each control or treatment with n = 6 for each trial. All data were evaluated by an unpaired student’s In this study, surface-wrinkled Cela-loaded microparticles were developed with a suitable aerodynamic diameter for efficient deep lung delivery. Cela MP was able to retain the antioxidant activity of the drug, trigger autophagy, and induce apoptosis. These mechanisms, in combination with an efficient delivery system, resulted in significantly enhanced efficacy against MPM in both 2D cell culture and a 3D tumor spheroid model. This study lays the groundwork to pursue the development of Celastrol and Celastrol-encapsulated delivery systems in preclinical and potentially clinical settings. These findings also suggest that the successful encapsulation of Cela in polymeric microparticles addresses certain limitations of Cela, such as low aqueous solubility and potential toxicity at higher doses. Going forward, we plan to study the benefits of Cela MPs in vivo and evaluate their long-term safety and stability profiles. In conclusion, inhaled Cela MPs are a promising anti-cancer standalone therapy for MPM treatment.


Early detection of rare mutations through liquid biopsy can provide real-time information related to cancer diagnosis, prognosis, and treatment outcomes. Cell-free DNA samples used in liquid biopsies contain single-nucleotide variants (SNVs) with a variant allele frequency (VAF) of approximately ≤1%. Droplet digital polymerase chain reaction (ddPCR) is considered the gold standard of sequencing using liquid samples, generating amplicons from samples containing mutations with 0.001–0.005% VAF; however, it requires expensive equipment and time-consuming protocols. Therefore, various PCR methods for discriminating SNVs have been developed; nonetheless, non-specific amplification cannot be avoided even in the absence of mutations, which hampers the accurate diagnosis of SNVs. In this study, we introduce single-nucleotide variant on–off discrimination–PCR (Soo-PCR), a highly accurate and practical method that uses a 3′-end tailing primer for the on–off discrimination of low-abundance mutant-type targets, including SNVs. Soo-PCR minimizes the chance of incorrect judgments owing to its high discriminating power. Cancer markers, such as KRAS G12D, EGFR L858R, and EGFR T790M mutations, containing 0.1% VAF, were clearly detected in under 2 h with a high reliability comparable with that of ddPCR. This new method serves as a practical approach to accurately detect and evaluate low-abundance mutations in a user-friendly manner. Early detection of rare mutations in the blood plasma provides real-time information related to tumor progression, treatment effectiveness, and cancer metastasis risk, and it has been a long-term goal in medical research ,2,3,4. ALiquid biopsies enable the detection and assessment of slight variations in DNA sequences and are convenient, minimally invasive, and reproducible ,9,10. NeIn this study, to address the background signal issue of the engineered PCR methods, we focused on and improved a conventional 3′ mismatch primer strategy. Existing methods were not able to maximize the discriminating power of Taq polymerase, resulting in signal amplification even when only the WT was present. However, we significantly improved Taq polymerase’s SNV discrimination ability by adding a tail to the 3′-end, preventing amplification when only the WT is present. We named this method a single-nucleotide variant on–off discrimination–PCR (Soo-PCR). This method reduces associated costs, time, and labor while maintaining strong discrimination ability; it is capable of distinguishing low-abundance MT targets containing SNVs from the abundant WT background. Low-abundance mutations with 0.1% VAF were easily detected through on–off discrimination using Soo-PCR by optimizing the length of the primer’s 3′-end non-complementary nucleotides (tailing structure), annealing the temperature, and screening various Taq DNA polymerases. We used Horizon’s cfDNA  derived from human cell lines, which are fragmented to an average size of 160 bp to closely resemble cfDNA extracted from human plasma ,35. Low-Oligonucleotides were designed and commercially synthesized using the PrimerQuest™ Tool (Integrated DNA Technologies (IDT), Coralville, IA, USA) for each target (KRAS G12D (c.35G > A), EGFR L858R (c.2573T > G), and EGFR T790M) c.2369C > T)). For designing primers (excluding the 3′-end tailing structure), the parameters were as follows: amplicon sizes between 60 and 80 nt, primer melting temperatures between 60 and 65 °C, and primer sizes between 18 and 25 nt. For designing the TaqMan probe, the parameters were as follows: Affinity Plus Probes (IDT) comprising 4–6 LNAs, probe melting temperatures between 66 and 72 °C, and probe sizes between 10 and 15 nt. All oligonucleotides used in this study are listed in ® Green Master Mix —were screened for each of the two Soo-PCR primers (one that was a perfect match with the template at the penultimate (–2) base and another that was a mismatch at the same base) with a 3′-end tailing structure . The experiments were performed in triplicate. A total of 1500 copies (5 ng) of 100% KRAS wild-type gDNA  containing only WT allelic frequency (AF) of the KRAS G12D target was used as a template.Five different Taq DNA polymerases—AmpliTaq Gold™ 360 Master Mix , Hot Start Taq 2× Master Mix , EzWay Hot Taq PCR Master Mix , DreamTaq™ Hot Start PCR Master Mix , and GoTaq−1), 10 μL of nuclease-free water , 12.5 μL of Taq master mix , 0.5 μL of TaqMan probe , 0.5 μL of forward primer , and 0.5 μL of reverse primer . The following constituents were used for all PCRs, regardless of the Taq DNA polymerase used: 1 μL of DNA template (5 ng μL−1), 10 μL of nuclease-free water (Invitrogen), 12.5 μL of AmpliTaq Gold™ 360 master mix , 0.5 μL of TaqMan probe , 0.5 μL of forward primer , and 0.5 μL of reverse primer .Based on the Taq DNA polymerase used, the PCR cycling conditions were as follows. AmpliTaq: 95 °C for 10 min, followed by 70 cycles at 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 15 s, and then at 72 °C for 7 min. Hot Start Taq: 95 °C for 30 s, followed by 70 cycles at 95 °C for 30 s, 56 °C for 30 s, and 68 °C for 15 s, and then at 68 °C for 5 min. EzWay: 95 °C for 15 min, followed by 70 cycles at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 15 s, and then at 72 °C for 10 min. DreamTaq: 95 °C for 3 min, followed by 70 cycles at 95 °C for 30 s, 61 °C for 30 s, and 72 °C for 1 min, and then at 72 °C for 7 min. GoTaq: 95 °C for 2 min, followed by 70 cycles at 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 15 s, and then at 72 °C for 5 min . We optiThe following PCR cycling conditions were used: 95 °C for 30 s, annealing temperature X for 30 s , and 72 °C for 15 s, followed by 72 °C for 7 min.−1) for each SNV allele. WT and mutant gDNA standards were mixed to a total of approximately 15,000 copies (50 ng) to obtain samples with 0%, 0.1%, 1%, 10%, and 50% VAFs for each SNV allele.The performance of Soo-PCR in amplifying low-abundance SNVs was assessed by using Soo-PCR to detect KRAS G12D, EGFR L858R, or EGFR T790M from gDNA and cfDNA standards (Horizon Discovery). Samples consisted of 0%, 0.1%, 1%, and 5% VAFs , nuclease-free water , 12.5 μL of AmpliTaq Gold 360 master mix , 0.5 μL of TaqMan probe , 0.5 μL of forward primer , and 0.5 μL of reverse primer .The following PCR cycling conditions were used: 95 °C for 10 min, followed by 70 cycles at 95 °C for 30 s, annealing temperature X for 30 s , and 72 °C for 15 s, followed by 72 °C for 7 min.The process of liquid biopsy used for early cancer diagnosis involves the purification of cfDNA from patient blood samples and the detection of specific mutations in the samples using NGS analysis or PCR techniques, such as ddPCR. The Soo-PCR method that we developed in this study by preparing specially designed PCR primers detected low-abundance cancer markers KRAS G12D, EGFR L858R, and EGFR T790M through on–off discrimination in under 2 h A.It has been reported that Taq polymerase has a characteristic of ignoring short 3′ mismatches and proceeding with extension ,37,38. TTo address this issue, we focused on the tailing of the primer to create conditions where the 3′-end region can exist in an entirely ssDNA form. The Soo-PCR primer design is as follows: First, position the SNV at the penultimate position to create a 2 nt overhang structure due to the mismatch caused by the SNV. Second, add a non-complementary tail sequence to the 3′ end to make the 3′ end completely ssDNA. In this way, the primer forms a longer ssDNA 3′-end tailing structure when there is a mismatch, which cannot be ignored by Taq DNA polymerase, leading to significant suppression of amplification.Therefore, it is important to choose which nucleotide to use as a tail to make the 3′ end as stable as possible in ssDNA form. The G and C nucleotides form a triple hydrogen bond, exhibiting a stronger hydrogen bond force than that between A and T ,39,40, aThe Soo-PCR primers are designed to maximize the differential recognition of mismatched dsDNA by Taq polymerase. We anticipated that the structural stability of the primer/template hybrid would be greatly influenced by buffer composition and engineered active domains of the polymerase. Therefore, we conducted a screening process to optimize specificity conditions for on–off discrimination between the perfect-match target at the penultimate base (PMP template) and the mismatch target at the penultimate base (MP template) with various tail lengths of Soo-PCR primers using Taq DNA polymerases from multiple companies including AmpliTaq, Hot Start Taq, EzWay, DreamTaq, and GoTaq. This screening process also demonstrated experiments that clearly show specificity.High specificity, with regard to the Soo-PCR assay, refers to a stable on–off SNV discrimination performance. Here, on–off discrimination describes the ability to stably induce the amplification reaction in the PMP primer/template hybrid through the 3′-end tailing structure but not in the MP primer/template hybrid . To compThrough Taq polymerase screening experiments, we were able to identify polymerase types and tailing lengths that can distinguish SNVs as on–off. Existing PCR methods that distinguish SNVs have low discriminating power, making it challenging to diagnose mutations accurately in extremely low amounts. In this regard, Soo-PCR, which demonstrates extremely high specificity through the tailing effect, is significant in the field of precision diagnosis that requires high accuracy.To evaluate the potential for cancer diagnosis with Soo-PCR, we selected KRAS G12D (c.35G > A), EGFR L858R (c.2573T > G), and EGFR T790M (c.2369C > T) among the cancer markers of major cancers such as stomach, colon, lung, thyroid, and breast cancer. For on–off discrimination of each target, we designed the target and Soo-PCR primers as follows. In both PMP and MP primer/template hybrid structures, the template for each target was prepared using wild-type genomic DNA (Horizon) containing only the WT allelic frequency (AF), with 1500 copies used. For each target, Soo-PCR primers were designed with ‘PMP’ and ‘MP’ primers that include tailing of various lengths (0–4 nucleotides). The ‘PMP’ primer was designed to have a perfect match with the template sequence at the penultimate (−2) base  was observed with 1 nt and 2 nt tailing of KRAS G12D: 0 nt , 1 nt , and 2 nt ; EGFR L858R: 0 nt , 1 nt , and 2 nt ; and EGFR T790M: 0 nt , 1 nt , and 2 nt  . InteresThis observation is because the mismatch between the primer and the template affects both the stability and priming efficiency of the primer–template duplex. Structurally, a mismatch in the 3′-end structure is considered to interfere with the polymerase active site ,37,38. IIn liquid biopsy, cfDNA containing SNVs has a VAF of approximately 1% or lower ,12,13. HOne Soo-PCR primer with a complementary structure for MT sequence at the penultimate base was used, whereas a 3′-end 2 nt tailing structure was selected from the optimization experiment to determine the length of the 3′-end tailing structure of Soo-PCR primers for KRAS G12D (c.35G > A), EGFR L858R (c.2573T > G), and EGFR T790M (c.2369C > T). Validation was first performed with gDNA and then with the cfDNA. MT gDNA forming a PMP structure with the primer and WT gDNA forming a MP structure were mixed at various ratios for a total of 15,000 copies to produce different VAF concentrations. The VAF percentage was determined using the MT gDNA copy number. For instance, a 50% VAF indicates that 7500 copies of both MT and WT gDNA are present, and 10% VAF indicates that 1500 copies of MT gDNA and 13,500 copies of WT gDNA are present. A total copy number of 15,000 was selected to correspond with the maximum amount of cfDNA existing in blood plasma (liquid biopsy) according to previous reports ,42,43.The 0% VAF sample consisting of WT gDNA alone was not amplified for KRAS G12D, EGFR L858R, or EGFR T790M. Furthermore, the 0% VAF sample consisting of 135,000 copies of KRAS G12D WT gDNA was not amplified . This suBased on the results obtained from gDNA (Horizon), we designed an experiment to validate the sensitivity and specificity of optimized Soo-PCR primers for cfDNA (Horizon) that closely resemble the form of clinical samples. Similar to the gDNA experiment, the total copy number of each sample was set to 15,000 and the VAF percentage was determined by the copy number of MT cfDNA; VAFs of 5%, 1%, 0.1%, and 0% were used. The results were quantified and validated using ddPCR, which is considered the gold standard of sequencing using liquid samples . The on–2, which indicate quantitative measures, were not close to 1, with values of 0.8171, 0.9458, and 0.9085, respectively  present in low abundance  in cfDNA samples closely resembling clinical samples in under 2 h. These results were comparable with those from ddPCR, which is recognized as the gold standard of sequencing using liquid samples. Furthermore, a 3′-end-tailing-structure-dependent amplification reaction can reduce cost by simplifying the primer design and only requires regular real-time PCR equipment. As it does not produce any background signals resulting in on–off discrimination, this new technique can serve as a practical tool to accurately detect and analyze low-abundance mutations in the cfDNA from plasma samples and has strong clinical application potential. Moreover, Soo-PCR is user-friendly and can be used as an endpoint diagnosis without requiring a qPCR device, thanks to its on–off system. Therefore, it can be combined with various nano-based diagnostic platforms, leading to potential synergistic effects ,45.


Humans can be exposed to endocrine disruptors (EDs) in numerous ways. EDs can interfere with endogenous hormones at different levels, resulting in numerous adverse human health outcomes, including immunotoxicity. In this regard, this study aimed to investigate in vitro the possible effects of EDs on immune cells and possible gender differences. Peripheral blood mononuclear cells from healthy humans, both males and females, were exposed to 6 different EDs, namely atrazine (herbicide), cypermethrin (insecticide), diethyl phthalate (plasticizer), 17α-ethynylestradiol (contraceptive drug), perfluorooctanesulfonic acid (persistent organic pollutant), and vinclozolin (fungicide). We evaluated the effect of EDs on RACK1 (receptor for activated C kinase 1) expression, considering it as a bridge between the endocrine and the immune system, and putatively used as screening tool of immunotoxic effects of EDs. The exposure to EDs resulted at different extent in alteration in RACK1 expression, pro-inflammatory activity, natural killer lytic ability, and lymphocyte differentiation, with sex-related differences. In particular, diethyl phthalate and perfluorooctanesulfonic acid resulted the most active EDs tested, with gender differences in terms of effects and magnitude. The results from our study evidenced the ability of EDs to directly affect immune cells.The online version contains supplementary material available at 10.1007/s00204-023-03592-3. According to the definition by World Health Organization (WHO), ‘an endocrine disruptor (ED) is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations WHO ’. CompouAdverse effects on the immune system were observed with several EDs, including bisphenols, phthalates, and several pesticides Patisaul . These sThe EDs have been selected based on their different endocrine targets . Indeed, EE is able to interfere with the estrogen pathway, DEP can act on estrogen and glucocorticoid pathway, while PFOS can impair the estrogen, glucocorticoid and thyroid signaling . Their ability to modulate RACK1 expression, to interfere with natural killer (NK) cell activity, and lymphocyte differentiation, focusing on CD4The selected EDs are listed in Table All the substances were purchased from Sigma-Aldrich  at the highest purity available. They were dissolved in dimethyl sulfoxide  at 10 mM stocks that were stored at − 20 ℃. Working concentrations were then obtained diluting stock solutions for each treatment. The final DMSO concentration in culture medium was ≤ 0.2%, and it was used as solvent control. Concentrations were selected based on previous studies conducted on THP-1 cell line  and cultured at 37 ℃ in a 5% CO2 incubator.PBMCs were obtained by Ficoll gradient centrifugation from buffy coats from anonymous healthy blood donors of both sexes, purchased from the Niguarda Hospital in Milan . Following centrifugation, PBMC layers were removed, and after washing with Dulbecco’s Phosphate-Buffered Saline (PBS), isolated cells were diluted to 106 cells/mL) were exposed to the different EDs or DMSO (vehicle control) for 24 h at 37 °C in a 5% CO2 incubator. RACK1 protein expression was evaluated by Western blot analysis and normalized to β-tubulin expression.For the evaluation of RACK1 expression, PBMCs (106 cells/mL), following 24 h of exposure to EDs or DMSO, were stimulated with lipopolysaccharide (LPS) from Escherichia coli serotype 0127:B8 (Sigma-Aldrich) at the final concentration of 100 ng/mL for further 24 h at 37 ℃ in a 5% CO2 incubator.To evaluate the expression of CD86 and CD54, and the release of IL-8 and TNF-α, PBMCs (106 cells/mL) were exposed to the different EDs or DMSO for 24 h. As target cells, K562 cells  stained with CellTrace™ CFSE  were used. Briefly, 500 µL of K562 at the concentration of 106 cells/mL were centrifuged, the CellTrace™ CFSE was added to the cell pellet (1:1000 dilution) and incubated for 15 min at 37 ℃ protected from light. After the incubation time, the reaction was stopped by adding culture medium containing 5% heat-inactivated dialyzed fetal bovine serum. CellTrace™ CFSE-stained K562 cells were then kept to the concentration of 105 cells/mL and co-cultured together with EDs/DMSO-exposed PBMCs. Three different ratios of effector (PBMC) and target (K562) cells were used: 50:1, 25:1, 12.5;1, maintaining fixed concentration of K562 cells. The cellular concentrations are reported in Table To evaluate NK-cell lytic activity, PBMCs  was added to 10After 24 h of treatment, cells were harvested, washed, and lysed in homogenization buffer . Protein content was assessed using the Bradford method. Cell lysates were mixed with sample buffer  and denatured at 95 ℃ for 10 min. 10 µg of extracted proteins were electrophoresed into 10% SDS-PAGE under reducing conditions and then transferred to PVDF membranes. The membranes were blocked with 1X TBS, 0.1% Tween-20, and BSA (5% w/v), and the expression of RACK1 and β-tubulin assessed following over-night incubation of the relative antibodies (dilution 1:1000) and following 1-h incubation of secondary IgG peroxidase-conjugated antibodies . Anti-human-RACK1 mouse antibody was purchased from Santa Cruz Biotechnology , anti-human-β-tubulin rabbit antibody was purchased from Novus Bio , goat anti-mouse IgG was purchased from Sigma-Aldrich, and goat anti-rabbit IgG was purchased from Bio-Rad. All the antibodies were diluted in 1X TBS, 0.1% Tween-20, and BSA 5% w/v. The band visualization was performed using Clarity western ECL blotting substrates . Blot images were acquired with Image Lab Software version 4.0 (Bio-Rad) using the Molecular Imager Gel Doc XR (Bio-Rad) and quantified normalizing on β-tubulin expression levels. The stimulation index (SI) was calculated on DMSO-treated PBMCs (vehicle control) set at 100.After 48 h of treatment, PBMCs were centrifuged, and the supernatants were stored at -20° C for the assessment of cytokine release. Cell pellets were washed with PBS, suspended in 200 µl of PBS, and stained at 4° C in the dark for 30 min with specific PE-conjugated antibody against human CD54 or FITC-conjugated antibody against human CD86 or with isotype control antibodies, following supplier’s instructions. All the antibodies were purchased from BD Biosciences . After incubation, cells were centrifuged and suspended in 500 µL of PBS. The % of positive cells was analyzed using Novocyte 3000 flow cytometer  and data were quantified using Novocyte software (Acea Bioscience Inc.). 10′000 viable cells were analyzed for % of positivity to the respective marker. The % of isotype control was subtracted from the % of CD86/CD54 stained cells. Changes in CD86/CD54 expression are reported as SI calculated on DMSO-treated PBMC (vehicle control) set at 1. The gating strategy is reported in Supplementary Fig. 1 and representative dot plots are reported in Supplementary Fig. 2.From the same treatments in which surface markers expression was assessed, the cell-free supernatants were kept at – 20 ℃ for cytokine evaluation through commercially available ELISA kits. The ELISA kits to assess the release of IL-8 and TNF-α were purchased from ImmunoTools  and R&D Systems, respectively. The limits of detection were 8 pg/mL for IL-8 and 7.8 pg/mL for TNF-α, respectively. Changes in IL-8/TNF-α release are reported as SI calculated on DMSO-treated PBMC (vehicle control) which was set at 1.After the treatment with EDs and the co-culture with CellTrace™ CFSE-stained K562 cells, the plate content was transferred to flow cytometrical tubes and PI (5 nM) was added to each tube. The % of cells positive to PI within CFSE-stained K562 cells was acquired using Novocyte 3000 flow cytometer. 1′000 CFSE-positive cells were analyzed for % of positivity to PI, indicative of dead K562 cells. The gating strategy is reported in Supplementary Fig. 3 and representative dot plots are reported in Supplementary Fig. 4.2 incubator. Following incubation, cells were harvested and washed, magnetic beads were removed using DynaMag 15 (Invitrogen) and washed again. The staining for surface markers was then performed, according to Table After 4 days of treatment, the Protein Transport Inhibitor Cocktail (Invitrogen) was added to each well, to stop cytokine secretion (1 µL every 500 µL of cell culture), for 5 h at 37 ℃ in a 5% COAfter incubation, cells were washed and fixed using the fixation reagent for 45 min on ice avoiding the light. After washing, cells were permeabilized using permeabilization reagent for 5 min on ice avoiding the light and washed again, following supplier’s instruction (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set—Invitrogen). Then, cells were stained for intracellular proteins, according to Table + cells, as performed for the conventional analysis , the t-SNE was run. Briefly, a Barnes-Hut t-SNE method, with a perplexity of 50 and 3000 iterations was chosen. The results were visualized in 2D t-SNE maps. The single treatment conditions were successively gated through the sample ID. Clusters of cells based on the expression level of the different analyzed markers were manually created on merged data, and the % of gated cells in each cluster was further analyzed for the single treatment conditions. Within CD4+ cells, it was possible to recognize different subpopulations for IFN-γ, IL-4, IL-9, FoxP3, and GITR. For IL-10, IL-17A, IL-22, and CD25 it was not possible to define distinct subpopulations. Only the subpopulations common to all the 6 donors were further evaluated, and the % of cells present in the clusters was expressed as a Log2 value calculated on DMSO-treated PBMC, which is set at 0. The t-SNE density plots are reported in Supplementary Figs. 6 and 7.Following conventional analysis, through FlowJo, to deeply investigate T-cell subpopulations, the t-distributed stochastic neighbor embedding (t-SNE) algorithm was applied. All the.FCS files were merged in two concatenated files as follows: a) DMSO, DEP, EE, and PFOS from 6 donors  due to their high presence in the environment and high exposure levels to humans to and b) DMSO, ATR, CYP, and VIN from 6 donors due to their belonging to the pesticide class. Samples were down-sampled to 10′000 cells, and after the gating of the single populations within CD4t test was applied, after the assessment of the normal distribution of the data through the Shapiro–Wilk test. Differences were considered statistically significant at p ≤ 0.05.Statistical analysis was performed using Prism version 9.4.0 . Data were reported as mean ± standard error (SEM) or as median of 3 (only for T cells differentiation) or 5 male and female donors, as reported in figure legends. To calculate differences between the treatment, First, to confirm previous results obtained in THP-1 cells  for 4 h Fig. .Fig. 3EfDifferent effector and target cell ratios were used: 50:1 Fig. A, 25:1 B, and 1235), meaning the ratio of effector cells necessary to kill the 35% of target cells. The LU35 are shown in Table 35, reducing, therefore, the lytic ability of female effector cells. Regarding sex differences, it can be noted that DEP, EE, PFOS, and VIN show different trend based on donors’ sex, generally reducing LU35 in males and increasing them in females, meaning their ability to increase lytic activity in males, while reducing it in females.To better express the lytic activity, integrating the three different ratios used, the results are also expressed as lytic unit 35  cells and cytotoxic T cells, based on their expression of CD4 and CD8, respectively. Both populations can be further divided into subsets based on the expression of cytokines, transcription factors, and surface markers. In particular, within circulating T helper cells, Th1, Th2, Th9, Th17, Th22, and regulatory T (Treg) cells can be found. Following activation, the first 5 populations may be recognized by the expression of IFN-γ, IL-4, IL-9, IL-17, and IL-22, respectively. Whereas Treg can be recognized by the expression of CD25, GITR, FoxP3, and IL-10. However, conventional activated CD4+ lymphocytes polarization, EE and VIN induced a slight increase in the percentage of cells CD4+IL-17+ in PBMC from male donors only  and pesticides .+ cells: 8 IFN-γ+ subsets, 3 IL-4+ subsets, 14 IL-9+ subsets, 14 FoxP3+ subsets, and 11 GITR+ subsets. Some of these subsets express more than one marker, as can be observed in Supplementary Fig. 6. The mean percentage of cells in each subset after treatment is divided by that in the control solvent for males and females and the values are reported in Fig. + cells was decreased following EE exposure. Whereas, DEP exposure in general increased the percentage of IL-9+ cells (mainly #9 and 10) and of FoxP3+ cells (#5), while decreased subset #9 of GITR+ cells. Also PFOS increased subset #9 of IL-9+ cells. In females, instead, more modulations were observed. In detail, EE reduced the percentage of cells present in subset #7 of FoxP3+ cells. DEP exposure reduced the percentage of cells present in subset #7 of IFN-γ+ cells, reduced the percentage of IL-9+ cells , of FoxP3+ cells , and of GITR+ cells . PFOS also reduced the percentage of IL-9+ cells (#3 and 4), #11 of FoxP3+ cells, and #3 and 5 of GITR+ cells. Even more interestingly, neither DEP, nor PFOS, nor EE had the same effects in females and males in each subset. In particular, looking at the subsets of cells positive to IL-9 and GITR  was increased following CYP exposure and also subset #10 of FoxP3+ cells. VIN exposure to male donors induced a slight increase of subset #10 of GITR+ cells. In females, ATR was able to increase the percentage of cells positive to IFN-γ present in subset #6, and to decrease the percentage of cells present in subset #14 of FoxP3+ cells. VIN exposure to female donors, instead, statistically significantly increased the percentage of IL-9+ cells (#2) and of GITR+ cells (#2). Differently, from the three highly persistent EDs, the three pesticides here analyzed do not show a strong difference between male and female donors. The only statistical differences were observed on subset #5 of IFN-γ+ cells following CYP exposure, which increased the percentage of cells in males and decreased it in females  and irrelevant or absent in males Below is the link to the electronic supplementary material.


A previous small-sample study verified that a blunt-tip antenna reduced hemorrhage during microwave ablation. We conducted this large-sample, multicenter, case–control study to further verify the efficacy and safety of microwave ablation with a blunt-tip antenna for ground-glass nodules.Patients with pulmonary ground-glass nodules were treated with either a sharp-tip (Group A) or blunt-tip antenna (Group B). A total of 147 and 150 patients were retrospectively allocated to Groups A and Group B, respectively. Group A patients underwent 151 procedures, and Group B patients underwent 153 procedures. We assessed the technical success, technique efficacy, and complications.p < 0.001). Minor complications, such as intrapulmonary hemorrhage  and hemothorax , occurred less frequently in Group B compared to Group A.Technical success and overall technique efficacy were achieved in all patients (100%). Major complications of pneumothorax were more commonly observed in Group A than in Group B (19.7% vs. 2.0%, In the treatment of ground-glass nodules, microwave ablation with a blunt-tip antenna had equal efficacy compared to microwave ablation with a sharp-tip antenna but had a decreased number of hemorrhage and hemothorax complications. Lung cancer remains the leading cause of malignant tumor-related mortality and morbidity in China . Non-smaCurrently, lobectomy plus mediastinal lymph node dissection or systematic lymph node sampling is the standard treatment for early-stage  NSCLC. However, lobectomy may not be the preferred choice for patients with GGN-like lung cancer , 10. PatNearly 25% of patients are high-risk for surgery, for whom, stereotactic radiotherapy and image-guided thermal ablation may be alternative treatments –15. MicrAs GGNs are usually adjacent to pulmonary vessels or have intertubercular vessels, routine MWA with sharp-tip antennae undoubtedly has a relatively high risk of hemorrhage. Hemoptysis is estimated to occur in 28.8–32.4% of patients , 21. OncFrom January 2018 to December 2021, patients who met the inclusion criteria were allocated sequentially into groups: (1) pulmonary GGNs with a maximum diameter of 30 mm; (2) pathologically verified AIS or MIA; (3) pathologically confirmed invasive adenocarcinoma (IA) unsuitable for radical surgery; (4) pure GGNs or mixed GGNs without histological diagnoses but with no decrease found during follow-up for at least 6 months; (5) GGNs located in the peripheral lung; and (6) adequate cardiopulmonary, hepatic, and renal functions.The exclusion criteria were as follows: (1) pulmonary lesions larger than 30 mm; (2) pulmonary solid nodules; (3) GGNs identified for less than 6 months; (4) pathologically verified atypical adenomatous hyperplasia or other benign disease; (5) GGNs located in the center of the lung; (6) antiplatelet treatment discontinued for less than 5 days; (7) acute myocardial or acute cerebral infarction during the past 30 days; (8) moderate or serious interstitial pulmonary disease; (9) platelet < 50*10^9/L and abnormal prothrombin/activated partial thromboplastin time. Patients who underwent MWA with the routine sharp-tip antenna or a blunt-tip antenna were assigned to Group A or Group B, respectively.MWA was performed using CT guidance . An ECO-100A1 MWA system with either an ECO-100AL6 blunt antenna or ECO-100AI16 sharp antenna  or a MTC-3C MWA system with a MTC-3CA-II7 blunt antenna or MTC-3CA-II25 sharp antenna  was used for MWA. We used a frequency of 2450 ± 50 MHz, and the output power ranged from 0 to 100 W. The commonly applied output powers were 30 W, 40 W, 50 W, and 60 W. For the microwave antenna, the effective length included both 150 mm and 180 mm (two styles) and the dominant outside diameter was 18 G. A water circulation cooling system was employed to cool the surface temperature of the antennae. First, we planned the puncture point and “target skin distance” of the target lesion. Local anesthesia and sedation were achieved with 100 mg of lidocaine and 10 mg of diazepam, respectively. Once satisfactory anesthesia was achieved, MWA was performed by operators from four centers with at least 10 years of experience  who inserted the antenna into the target lesion according to the plan. The cold circulating system worked simultaneously during the ablation period. The ablation was terminated when the post-ablation ground-glass opacity surpassed the target lesion by at least 5 mm.For patients who underwent MWA with a blunt-tip antenna Fig. , a coordCT was conducted 24 h post-ablation to identify any related complications. Major complications, such as pneumothorax, hemorrhage, and pleural effusion, were treated if necessary. Contrast-enhanced CT (CECT) was conducted 1-month post-ablation and then with an interval of 3–6 months for two years and with 6–12-month intervals for the next 3 years. Thereafter, CECT was performed annually. The last follow up date was April 9, 2022. The follow-up CT images were assessed by both operators and radiologists with at least ten years of experience.Technical success and technique efficacy were used to evaluate the response to MWA. Technical success primarily indicated that the procedure was conducted in accordance with the protocol. In contrast to technical success, technique efficacy rate was the proportion of patients who achieved complete ablation . CompletWe assessed MWA complications using the Society of Interventional Radiology (SIR) criteria, which divides all complications into major and minor complications, with grades ranging from SIR A to SIR E. Minor complications were classified as SIR A or B. SIR C and D included major complications. SIR E referred to death, the most serious complication .2 test or Fisher exact test. All statistical analyses were two-sided, and p < 0.05 indicated statistical significance.SPSS software  was used for statistical analyses. Percentage was used to describe categorical variables. Mean and standard deviation/median and interquartile range were used for the description of numerical variables according to the normality test. Two independent samples t-test and nonparametric Mann–Whitney U test were used for the comparison of age and the remaining numerical variables between the groups, respectively. The distribution of baseline characteristics and GGN lesions and incidence of complications between the groups were analyzed using Pearson’s χA total of 147 and 150 patients were retrospectively allocated to Group A and Group B, respectively. In Group A, the mean age was 58.2 years (range: 29–85 years). Most patients  were younger than 65 years of age. Ninety-three patients (63.3%) were women and 122 (83.0%) were nonsmokers. Moreover, 52 patients (35.4%) had comorbidities. Sixty-nine patients (46.9%) were pathologically diagnosed, and IA  was the most common histological type.In Group B, the mean age was 61.2 years (range: 30–87 years). Most patients  were younger than 65 years of age. Seventy-six patients (50.7%) were women, and 126 (85.3%) were nonsmokers; 36 (24.0%) had comorbidities. Pathology was performed in 96 (64.0%) patients. AIS, MIA, and IA were confirmed in 36 (24.0%), 33 (22.0%), and 25 (16.7%) patients, respectively. Group A and B differed in baseline characteristics of mean age, sex, comorbidities, and pathology (Table p = 0.032), which mainly included pulmonary disease  and type 2 diabetes .As for the comorbidities, patients of Group A were more common than Group B . Both the lesion depth (76 mm vs. 77 mm) and the involvement of surrounding pulmonary vessels (91.8% vs. 91.3%) were similar between the two groups. The median power and ablation time in both groups were 40 W and 5 min, respectively . Moreover, hemothorax was observed in four (2.7%) and zero (0.0%) patients in Groups A and B, respectively (p = 0.049). These differences indicate that the blunt-tip antenna decreases the incidence of hemorrhage during ablation. When treated with a blunt-tip antenna, pneumothorax was more commonly identified  as a minor complication, but fewer patients in Group B than in Group A with pneumothorax as a major complication required chest tube insertion   in Group B, and no interventions were needed; however, 14 patients (9.5%) in Group A experienced intrapulmonary hemorrhage  and hemothorax in patients compared to those who underwent a sharp-tip antenna application . Nearly all lesions had surrounding pulmonary vessels in both groups. However, the sharp-tip antenna application commonly induced vessel injury and led to bleeding compared with the blunt antenna application.In previous studies, where patients were treated with the conventional sharp-tip antenna, although no major complications of hemorrhage were reported, minor complications of hemorrhage ranged from 19.7 to 28.8% , 25. In p = 0.025), but the incidence of major complications of pneumothorax was lower in this group compared to patients treated with the sharp-tip antenna . Increased incidence at baseline of pulmonary disease in Group A led to more severe cases of pneumothorax than the more common pneumothorax cases. Previous studies have shown that patients with pulmonary disease more commonly experience major complication of pneumothorax, for which chest tube insertion is needed.Similar to our findings, previous studies have reported the occurrence of other complications, mainly pneumothorax, in 9.8–39.0% of patients –21, 25 aOther treatment modalities also serve as alternatives, including the cone-beam CT (CBCT). Compared with CT, CBCT could reduce the time of the thermal ablation procedure in the treatment of lung cancer dramatically ; howeverThis study had several limitations. First, this was a retrospective rather than a prospective, randomized study. Second, 53.1% and 36% of patients in both groups did not have histological diagnoses. Thirdly, the median follow-up interval was less than 3 years, which prevented the evaluation of long-term survival. Fourth, different baseline characteristics between the groups may have influenced efficacy and safety. Finally, the patients underwent MWA in four different hospitals and operators, therefore, operator bias may have existed.In conclusion, in the treatment of GGNs, MWA performed with a blunt-tip antenna had equal efficacy as the use of a sharp-tip antenna, but was associated with fewer complications of hemorrhage and hemothorax.


Liver cancer is the third most deadly malignant tumor in the world with poor prognosis and lacks early diagnostic markers. It is urgent need to explore new biomarkers and prognostic factors. The oxysterol-binding protein-like family proteins (OSBPLs) are essential mediators of lipid transportation and cholesterol balancing which has been reported to participate in cancer progression. So far, the expression, immune infiltration, and prognosis of OSBPLs have not been elucidated in liver cancer.The differential expressions of OSBPLs between liver tumor and normal tissues were assessed by analyzing RNA-seq data from TCGA and protein data from CPTAC, respectively. Subsequently, genetic variations, potential functional enrichment analysis, and immune cell infiltration were analyzed. Further, the prognostic effects of OSBPLs were identified via constructing lasso models and performing receiver operating characteristic curve (ROC) analysis. Moreover, 10 local liver cancer specimens were involved to validate the expression of OSBPL3 via immunohistochemistry (IHC) assay. Finally, CCK-8, cell cycle, apoptosis, transwell assays, real time qPCR (RT-qPCR), and western blot assays were conducted to explore the function of OSBPL3 in liver cancer cells.The mRNA of OSBPL2, OSBPL3, and OSBPL8 were highly expressed while OSBPL6 was lowly expressed in liver cancer samples compared with normal samples. As to the protein expression, OSBPL2 and OSBPL3 were significantly elevated and OSBPL5, OSBPL6, OSBPL9, OSBPL10, OSBPL11 were downregulated in tumor samples. A positive correlation was found between copy number variations (CNV) and the expression of OSBPL2, OSBPL8, OSBPL9, OSBPL11, while DNA methylation was negatively associated with the expressions of OSBPLs. Of these, CNV amplification mainly contributed to the overexpression of OSBPL2 and DNA methylation may be responsible for the high expression of OSBPL3. Interestingly, OSBPL3, OSBPL5, SOBPL7, and OSBPL10 were significantly positively correlated with immune infiltration. Notably, OSBPL3 was identified correlated to overall survival (OS) and disease specific survival (DSS) in liver cancer. Functionally, knocking down OSBPL3 reduced liver cancer cell viability, induced a G2/M cell cycle arrest, promoted apoptosis, and restrained cell migration.In aggregate, we reported a heretofore undescribed role of OSBPLs in liver cancer by analyzing multi-omics data. Importantly, we identified OSBPL3 was overexpressed in liver tumor compared with normal and its high expression was correlated with poor OS and DSS. Inhibition of OSBPL3 resulted in a pronounced decrease in cell proliferation and migration.The online version contains supplementary material available at 10.1186/s12885-023-10713-9. Liver cancer is the third cause of cancer related death with an estimate of 830,180 deaths in 2020. Alpha-fThe oxysterol-binding protein-like (OSBPL) protein family includes nine members namely OSBPL2, OSBPL3, OSBPL5, OSBPL6, OSBPL7, OSBPL8, OSBPL9, OSBPL10, and OSBPL11. MutatioIn the present study, the transcriptome, genomic, epigenetic, immune infiltration, and prognosis of OSBPLs were explored in liver cancer. Immunohistochemistry (IHC) was applied to validate the expression of OSBPL3 in liver cancer. CCK-8, cell cycle, apoptosis, transwell assays, real time qPCR (RT-qPCR), and western blot assays were conducted to investigate the biologic functions of OSBPL3 in liver cancer cells Fig. . Our finLiver cancer tissues and matched normal adjacent tissues (≥ 2 cm away from cancer) were obtained from the First Affiliated Hospital of Guangxi University of Chinese Medicine with written informed consent of patients. Tumor cells account for approximately 80% of liver cancer tissue. This study was approved by Ethics Committee of the First Affiliated Hospital of Guangxi University of Chinese Medicine (2020-009-02). All experiments in this study were conducted in accordance with the guidelines in the Declaration of Helsinki.The liver cancer cells  were purchased from NANJING COBIOER BIOSCIENCES CO., LTD and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. PLC/PRF/5 Cells were transfected with negative control (NC) or small interfering RNA of OSBPL3 (siOSBPL3)  by using lipofectamine 3000 (Invitrogen) in 12 well plate (0.66 µg per well). Twenty-four hours later, the cells were harvested for cell viability, cell cycle, transwell assays, and western blot assays.https://xenabrowser.net/datapages/?host=https%3 A%2 F%2Ftoil.xenahubs.net) and analyzed via applying R package “stats” and “car”. The microarray data  was downloaded from GEO database (https://www.ncbi.nlm.nih.gov/geo/) and processed with R package “RMA”. Subsequently, data were processed by running R package “limma” to obtain DEGs. Genes with a criterion of p < 0.05 and |Fold change| >2 were considered as DEGs between normal and tumor samples.RNA seq data of liver cancer  preprocessed by python package “toil” were retrieved from UCSC XENA database [The protein expressions of OSBPLs between liver tumor and normal tissues were determined via using CPTAC database (rot.html). Brieflyhttp://bioinfo.life.hust.edu.cn/GSCA/#/) which assembles various tumor genome data including CNV, mutation, methylation and immune [Online database of Gene Set Cancer Analysis (GSCA) (d immune  was applhttps://cistrome.shinyapps.io/timer/)[The correlation between expression of OSBPLs and immune infiltrates were determined using TIMER database (o/timer/). The GenRNA-seq data (level 3) of liver cancer filtered with normal control samples was downloaded from TCGA. First, the expression data  of OSBPLs was converted to Transcripts Per kilobase per Million (TPM) following a log2 transformation. Then, the expression matrix of OSBPLs with clinical information was processed via running R packages “glmnet” and “survival” to construct a risk score system.The median risk score of the combining of OSBPL3 and OSBPL10 was used to divide group into high and low risk groups for OS. Also, the median risk score of the combining of OSBPL3 and OSBPL6 was used to divide group into high and low risk groups for DSS. Then, the Kaplan-Meier (K-M) survival analysis was conducted via running R package “survminer”. The time-related receiver operating characteristic curve (ROC) was performed to assess the OS predictive ability of OSBPL3 in liver cancer by running R package “pROC”.RNA-seq data (level 3) of liver cancer was downloaded from TCGA via running R package “TCGAbiolinks”. Then DEGs between OSBPL3 highly and lowly expressed groups were identified via running R package “DESeq2” with a criterion of p < 0.05 and |Fold change| >2. Meanwhile, expression correlation analysis of OSBPL3 was conducted via running R package “stat”. Subsequently, up-regulated DEGs with a correlation value r > 0.5 were involved in the GSEA analysis by running R package “clusterProfiler”.A total of 10 liver cancer samples with matched normal adjacent tissues were involved to validate the expression of OSBPL3 by IHC assays as previously . BrieflyPLC/PRF/5 Cells transfected with NC or siOSBPL3 were plated in 96-well plates with 5000 cells per well. CCK-8 was added after indicated time and incubated for additional 2 h. Then, the plates were assayed by testing the absorbance at 450 and 600 nm.For cell cycle analysis, PLC/PRF/5 cells were transfected with NC or siOSBPL3 in 12-well plate. After 24 h, cells were harvested and cell cycle was determined by using a cell cycle detection kit  according to the kit instruction. For apoptosis assay, PLC/PRF/5 cells were transfected with NC or siOSBPL3 for 48 h. Then, cells were collected and cell apoptosis was detected using an Annexin V/7-AAD Apoptosis Detection kit (BioLegend (USA), Cat: 640,922) according to the kit instruction.RNA extraction and real-time qPCR were conducted as reported . BrieflyPLC/PRF/5 Cells transfected with NC or siOSBPL3 for 48 h were harvested and lysed in RIPA buffer. Then, cell lysates were centrifuged at 12,000 x g for 10 min at 4 °C to obtain proteins. Next, proteins were subjected to 8–15% SDS-PAGE, electrophoresed, and transferred on to a PVDF membrane following blocking with 5% non-fat milk dissolved in trisbuffered saline. After washing with trisbuffered saline, the membrane was incubated with indicated primary and secondary antibodies. Finally, proteins were detected using the Luminescent Image Analyser LSA 4000 . The primary antibodies involved in this study include Bax , PARP , Caspase-3 , and β-actin .Transwell inserts  were pretreated with serum-free medium for 1 h. Cells transfected with NC or siOSBPL3 for 24 h were plated into each insert (the upper chamber). Medium with 10% of FBS was loaded into the bottom chamber. Twenty-four hours later, cells in the upper surface of the insert membrane were removed, while cells in the lower surface were fixed with methanol, stained with crystal violet, and counted by microscopy.Differences between data groups were evaluated for significance using Student t-test. P values < 0.05 were considered statistically significant. All experiments were repeated at least three times and data are presented as the mean ± SD unless noted otherwise. *, p < 0.05; **, p < 0.01; ***, p < 0.001.The mRNA expressions of OSBPLs in liver tumor and matched normal tissues (50 pairs) were examined first by analyzing data from TCGA which demonstrated that OSBPL2, OSBPL3, OSBPL5, OSBPL7, OSBPL8, and OSBPL9 were upregulated  analysis. A total of 2986 genes with |r|>0.5 were identified including 2978 positively and 8 negatively OSBPL3 correlated genes (Fig. Deregulated cell cycle and extracellular matrix remodeling are important hallmarks of . Given tAbnormal lipid metabolism occurs frequently in tumor patients which could regulate gene expressions and modify signaling transduction . AccumulImmune cells infiltration is gradually recognized as one of the main obstacles of cancer . The rolIn order to evaluate the prognostic of OSBPLs in liver cancer, OS and DSS related lasso models were constructed which exerted that OSBPL3 was a key prognostic factor to both OS and DSS Fig. . The ROCIn conclusion, we assessed the expression, genomic, and epigenomic variation in liver cancer. Especially, we found OSBPL3 was overexpressed and a prognostic factor in liver cancer. Downregulating OSBPL3 induced a G2/M cell cycle arrest and apoptosis and suppressed cell migration.Below is the link to the electronic supplementary material.Supplementary Material 1