Leishmania, is treated with drugs associated with hightoxicity and limited efficacy, in addition to constant reports of theemergence of resistant parasites. In this context, snake serums emerge asgood candidates since they are natural sources with the potential to yieldnovel drugs. Leishmaniasis, a neglected disease caused by the parasite2 inhibitor from Crotalus durissuscollilineatus snake serum, against Leishmania(Leishmania) amazonensis. We aimed to show the antileishmanial effects of γCdcPLI, a phospholipaseA Promastigotes forms were exposed to γCdcPLI, and we assessed the parasiteviability and cell cycle, as well as invasion and proliferation assays.2 inhibitor reduced the parasite infectivity whenpromastigotes were pre-treated. Also, we demonstrated that the γCdcPLItreatment modulated the host cell environment impairing early and late stepsof the parasitism. Despite the low cytotoxicity effect on macrophages, our data indicate thatγCdcPLI has a direct effect on parasites promoting an arrest in the G1 phaseand reduction in the G2/M phase at the highest dose tested. Moreover, thisPLA γCdcPLI is an interesting tool for the discovery of new essential targets onthe parasite, as well as an alternative compound to improve theeffectiveness of the leishmaniasis treatment. Leishmania.Leishmaniasis is a set of complex and multifaceted syndromes, with different clinicalmanifestations, caused by protozoa of the species of the genusLeishmaniaspecies, belonging to the subgenera Viannia,Leishmania and Mundinia, allocated into thesubfamily Leishmaniinae.,,,,,Leishmania can cause damage to the skin, mucosa, and visceral organs.The main forms are Cutaneous Leishmaniasis (CL), Diffuse Cutaneous Leishmaniasis (DCL),Mucocutaneous Leishmaniasis (MCL), Anergic Diffuse Cutaneous Leishmaniasis (ADCL) andVisceral Leishmaniasis (VL).,,In the Americas, leishmaniasis is associated with at least 15 Leishmania that are transmitted by sandflies.Leishmania (Leishmania)amazonensis, which affects skin, causing ulcers characteristic of cutaneousleishmaniasis.,L. (L.) amazonensis has been also associated with aremarkably diverse clinical manifestations, such as DCL, ADCL and, less frequently, withMCL and VL.,CL is a widespread tropical infection caused by numerous different species ofL. (L.) amazonensis. Evidencessuggest that this species has a particular capacity to negatively interfere with severalimmunological mechanisms necessary for the generation of an effective immuneresponse.L.(L.) amazonensis.In Brazil, the anergic-multiparasitic end of the leishmaniasis spectrum is exclusivelyassociated with infection caused by Drug therapy for all forms of leishmaniasis aims at ensuring adherence to treatment,alleviating symptoms caused by the disease, safely administering indicated medications,controlling and/or minimising the occurrence of adverse effects.Trypanosoma cruzi andL. (L.) amazonensis increases the ability of the parasites toinfect host cells.,,The complexity of leishmaniasis symptoms is largely a result of the parasite’s virulence,which among several actions can induce responses in the host cell, such as changes inlipid metabolism that lead to plasma membrane remodelling by membrane phospholipidturnover, then modulating the process of adhesion and invasion of the parasite.2 (PLA2s) from snakevenom, demonstrated that these antibodies possibly recognise PLA2s present inthe L. (L.) amazonensis, compromising its ability to establishinfection in the host. Similarly, Bordon and collaborators2(PLA2) inhibitors against L. (L.) amazonensis, which wasillustrated by a reduction in the lesion size and skin parasitism in infected BALB/cmice.Literature findings have demonstrated the role of some molecules with anti-phospholipaseaction on leishmaniasis. Borges et al.,2activity, especially compounds from plants extracts, marine organisms and snakes.2 inhibitory proteins,named PLIs, which are serum globular proteins and possess the unique ability toneutralise the enzymatic and toxic components of snake venom PLA2s.,,2recognition.Natural products have been extensively studied in regards its anti-PLA2 inhibitor isolated from Crotalus durissuscollilineatus snake serum, has been explored for its therapeuticproperties.,,2-induced biologicalactivities, such as oedema and myotoxicity.2s.γCdcPLI, a γ-type PLA2s can be involved in parasite virulence and maintenancein vertebrate hosts, they can be considered a possible target for studies that aim tobetter understand the parasite infection process that leads to leishmaniasis.,,,,L. (L.)amazonensis promastigotes. Our findings may pave the way forfurther investigations on the pathogenesis of the parasitic disease, as well as for thedevelopment of new therapeutic approaches against leishmaniasis.Since endogenous PLACrotalus durissus collilineatus serum and Bothrops pauloensis crudevenom - The serum of C. d. collilineatus (Cdc) wasobtained from specimens maintained in the Reptiles Sector of the Federal Universityof Uberlândia, Minas Gerais, Brazil. This serpentarium was registered in theBrazilian Institute of Environment and Renewable Natural Resources - IBAMA (nº301286). The snake blood was collected periodically and the serum was obtained bycentrifugation at 5,000 g for 10 min at 4ºC. The serum was storedat -20ºC. B. pauloensis crude venom was collected from snakes keptat the Ceta serpentarium, Animal Toxin Extraction Center, Ltda. - CNPJ:08.972.260/0001-30, Morungaba, SP, Brazil. This serpentarium has undergone IBAMAregistration and obtained authorisation for the use of renewable natural resources(nº 2087163).Isolation of PLA2inhibitor (γCdcPLI) from Cdc serum - γCdcPLI was purified from thesame specimens of Cdc serum in two sequential chromatographic steps, as previouslypublished by Gimenes et al.2 (datanot shown) was further submitted to NHS Hitrap (N-hydroxysuccinimide) affinitycolumn immobilised with PLA2 BnSP-7.Q4 fraction (8.5 mg) with inhibitory activity on phospholipases A2 datanot shownThe protein concentration was determined using Bradford reagent according to Bradford (1976).Inhibition of PLA2activity - The PLA2 inhibition assay was determinedaccording to De Haas and Postema.2. In order toevaluate the inhibitory effect of γCdcPLI on PLA2 activity, the γCdcPLIwas incubated with B. pauloensis venom at different venom:γCdcPLI(m/m) ratios for 30 min at 37ºC. Results were in triplicate and expressed inmEqNaOH/mg/min.Cell culture and parasite maintenance - Immortalised macrophages(macrophages C57) were derived from the bone marrow of C57BL/6 mice and maintainedaccording to Araujo et al.L. (L.) amazonensis promastigotes (IFLA/BR/67/PH8) were maintainedin Brain Heart Infusion - HiMedia medium supplemented with 10% foetal bovine serum(FBS) , 100 mg of gentamicin/mL and 2 mM L-glutamine at 23ºC. Promastigotes in the stationaryphase (metacyclic) were used in the experiments.Cellular viability in promastigote forms of L. (L.) amazonensis -Cytotoxicity assays in the presence or absence of γCdcPLI were performed onpromastigote forms by the 3--2,5-diphenyl tetrazoliumbromide (MTT) assay, as previously described.,,6 parasites/well) were placed in 96-wellculture plates and incubated in two-fold serial dilution of γCdcPLI (from 0.781 to50 µg/mL) for 24 h at 23ºC. Control parasites were incubated with medium only. After24 h, promastigotes were incubated with 5 mg/mL MTT for 3 h at 23ºC. Formazancrystals were dissolved by adding 100 μL of PBS  containing10% SDS and 0.01 M HCl. After 18 h, the absorbance was measured in a multi-wellscanning spectrophotometer  at 570 nm. Results wereexpressed as the percentages of viable cells in relation to untreated control (100%viability). Dose response inhibition curves [Log (inhibitor) vs. normalised response- Variable slope] were calculated.Cellular viability in macrophages C57 - Cell viability in thepresence or absence of γCdcPLI was evaluated in macrophages C57 by MTT assay.Briefly, cells were cultured in 96-well plates (3.0 x 104 cells/200µL/well) in a medium supplemented with FBS for 24 h at 37ºC and 5% CO2.Then, cells were treated in two-fold serial dilution of γCdcPLI (from 0.781 to 50µg/mL) in RPMI 1640 medium. After 24 h, supernatants were discarded and 10 µL of MTT(5 mg/mL) and 90 µL of 10% FBS medium were added to each well for 3 h, under thesame culture conditions. Subsequently, formazan crystals were dissolved by adding100 μL of PBS containing 10% SDS and 0.01 M HCl and, after 18 h, the optical densitywas determined at 570 nm on a plate reader. Cell viability was reported inpercentages (viability %), with the absorbance of cells incubated only with culturemedium considered as 100% viable. Dose response inhibition curves [Log (inhibitor)vs. normalised response - Variable slope] were calculated. The selectivity index(SI) was determined as the ratio between the half-maximal cytotoxic concentration(CC50) for macrophages C57 and the half-maximal inhibitoryconcentration (IC50) against L. (L.) amazonensis.Inference of the cell cycle phases based on DNA content analysis -The cell cycle phases were inferred based on the DNA content analysis, as previouslydescribed.,,,L. (L.) amazonensis(1.0 × 106 cells/well) were plated in 96-well micro-plates and treatedwith γCdcPLI (10 and 50 μg/mL) or culture medium (control group) for 24 h at 23ºC.Then, parasites were harvested and fixed in 70% ethanol for 18 h at 4ºC. To ensurethat only the DNA was stained, parasites were incubated with RNase A (100 μg/mL) andpropidium iodide (PI) (10 μg/mL) for 45 min in the dark at 37ºC. Cell cycle wasanalysed by a FACS CantoII (BD), and the data were obtained using Flow Jo software(version 7.6.3).Invasion assays - Invasion assays were carried out following apublished study, with minor modifications.5 cells/well) were cultured in a 24-wellplate containing 13-mm coverslips in each well. After adhesion, the cells weresubmitted to two distinct experimental models: (i) macrophages C57 were pre-treatedor not with 10 and 50 µg/mL of γCdcPLI for 24 h, and then infected with L.(L.) amazonensis promastigotes, with a multiplicity of infection (MOI)of 10 parasites per 1 cell, for 4 h at 23ºC; (ii) Promastigote forms of L.(L.) amazonensis (with a MOI of 10:1) were pre-treated or not with 10and 50 µg/mL of γCdcPLI for 1 h, and allowed to invade the cells for 4 h at 23ºC.For both protocols, cells were fixed with Bouin’s solution (HT10132 Sigma Aldrich)and stained with Giemsa (1:20 - P3288 Sigma Aldrich).Intracellular killing assay - Macrophages C57 (2.0 x 105cells/well) were cultured in a 24-well plate containing 13-mm coverslips in eachwell, and infected with a MOI of 10:1 of promastigote forms of L. (L.)amazonensis for 4 h at 23ºC. Cells were carefully rinsed several timeswith PBS to remove the excess of extracellular parasites. Next, cells were incubatedwith twofold serial dilutions of γCdcPLI (ranging from 50 to 1.56 μg/mL), or culturemedium for 24 h 37ºC and 5% CO2. Also, the present study used theamphotericin B (1 μg/mL) (Sigma #1397-89-3) as a gold standard drug againstleishmaniasis.Statistical analysis - Data are expressed as mean ± standarddeviation (SD) of experiments performed at least three times in triplicate. All datawere first checked for normal distribution. Differences between two groups weredetermined by Student’s t test (two-tailed) and Mann-Whitney test for parametric ornon-parametric data, respectively. Differences among multiple groups were assessedby one-way analysis of variance (ANOVA) test with Dunnett’s multiple comparisonstest, for parametric data, or by Kruskal-Wallis test with Dunn’s multiple comparisonpost-test, in the case of non-parametric data (GraphPad Prism Software version8.00). Data were considered statistically significant at p < 0.05.Isolation of γCdcPLI from Cdc serum - Here, we described theantileishmanial effects induced by γCdcPLI isolated from Cdc snake serum. Initially,we isolated γCdcPLI according to Gimenes et al.,2-BnSP-7 [Supplementarydata ]. Affinity chromatography has beenemployed by other authors to isolate different classes of inhibitors.2 activity was tested (data notshown). The Q4 fraction, which showed PLA2 activity inhibition, wasapplied to an NHS Hitrap (N- hydroxysuccinimide) affinity column immobilised withPLA2-BnSP-7, resulting in two fractions, NHS-1 and NHS-2  . Theauthors demonstrated that γCdcPLI can inhibit 100% of the activity of differenttypes these PLA2. Furthermore, Gimenes et al.,2 from honey beeand the recombinant form of γCdcPLI. The authors demonstrated a higher capacity ofinhibitor to interact with the PLA2, which Kd value around 1.48 μM. Here,we observed a lower inhibition percentage of inhibitor against the whole snakevenom. This finding suggests that due to snake venom is a rich mixture of adifferent types of toxins, among them different classes of PLA2,2 activities has beenalready described in the current literature. Oliveira et al.γBjussuMIP isolated from B.jararacussu plasma was able to inhibit oedema, myotoxic, cytotoxic andbactericidal effects induced by bothropic PLA2s, which show theremarkable scenario for the use of these molecules as tools for the treatment ofdiseases.The fractionation of Cdc serum on a Q-Sepharose Fast Flow column produced five majorprotein peaks, called Q1 to Q5 A. Theinnd NHS-2 B. Similaly 23kDaC. This pLI, m/m) D. GimeneγCdcPLI decreases the viability of promastigote forms of L. (L). amazonensisand macrophages C57 - The viability assays were performed on bothpromastigote forms and macrophages cultivated in absence (control) or presence ofincreasing concentrations of γCdcPLI (0.78 -50 μg/mL) for 24 h. Regarding thecontrol group (untreated group), γCdcPLI was able to reduce 20% and 50% of theparasite’s viability at 25 and 50 µg/mL, respectively  against L. (L.)amazonensis is 48.9 µg/mL, while the half-maximal cytotoxicconcentration (CC50) for macrophages C57 is higher than 50 µg/mL (highestconcentration tested), revealing a SI > 1.0. These results suggest that γCdcPLIhad a possible selective action against parasites, as well as an ideal concentrationto cause a better effect on promastigote forms of L. (L.)amazonensis.2 inhibitors has been described inthe literature. Alvarez et al.,L. (L)infantum promastigotes. The most active derivativeImidazolidin-2-one showed an important activity against the clinically relevantstage of parasites in comparison with Glucantime® and did not inducetoxicity on human fibroblast cells. This same study suggested thatImidazolidin-2-one compounds have a direct antiparasitic effect through aperturbation of phospholipid membrane homeostasis and through the inhibition of theparasite protein kinase C (PKC), an important enzyme that regulates early events ofthe parasite-macrophage interaction process.2) and methyl arachidonyl fluorophosphonate  induced cytotoxicity to 50% of promastigoteforms of L. (L.) amazonensis at 15.1 ± 3.7 μM and 50.5 ± 7.8 μM,respectively. Moreover, these PLA2 inhibitors did not induce cytotoxicityto peritoneal macrophages.Antileishmanial activity induced by PLAγCdcPLI treatment promoted a cell cycle arrest at G0/G1 phase inpromastigote forms of L. (L.) amazonensis - Our previous resultsdemonstrated that γCdcPLI was able to interfere in parasite’s viability. Thus, weaimed to assess the impact of γCdcPLI treatment on the cell cycle progression ofpromastigote forms of L. (L.) amazonensis. This measurement wascarried out indirectly, through the analysis of DNA content by flow cytometry usinga DNA intercalant (PI). Of note, the inference of cell cycle phases  through DNA content is a standardised approach, commonly used in severalcellular models,,,,,Briefly, promastigotes were treated  with 10 and 50 µg/mLof γCdcPLI for 24 h, and the DNA content was analysed by flow cytometry. Our resultsdemonstrated that γCdcPLI at 50 µg/mL arrested the cells at G1 phase of the cellcycle and, as a consequence, there was a decrease in the percentage of cells in theG2/M phases 24 h post-treatment .,,,,,One possible explanation for this phenomenon, based in other study,γCdcPLI interferes directly in the infective capacity of promastigote formsand modulates the host cell environment - To gain insights into theleishmanicidal activity of γCdcPLI, we verified the capacity of this protein tointerfere with the parasite invasion process. For this purpose, we assessed whetherγCdcPLI would be able to target the parasites and/or the cells, by treating infectedmacrophages, or pre-treating both the promastigotes and macrophages prior toinfection. Our data showed that the pre-treatment of promastigote forms ofL. (L.) amazonensis with γCdcPLI (10 and 50 μg/mL) for 1 hreduced the number of intracellular parasites, as well as the percentage of infectedcells in comparison with the untreated control group amazonensis. Representative images are shown , in the absence of Ca2+, culminated in a reduction ofparasite invasion into macrophages. Moreover, the activities of the cyclooxygenaseand lipoxygenase pathways were down-modulated when macrophages were pre-treated withthese PLA2 inhibitors, which led the authors to suggest that theparasites activated the host cell PLA2.Several prokaryotic and eukaryotic pathogens produce PLALeishmania spp. are intracellular protozoans capable of scavengingglycerophospholipids from host cells and degrading them via thePLA2 activity, which suggests that these parasites can remodelexogenous lipids into their own via the Lands cycle.,,2s were also reported inLeishmania, and they could be involved in the biosynthesis oflipophosphoglycan, the main macromolecule on the surface of the procyclicpromastigotes.2 on themacrophage.L. (L.)amazonensis treated with PLA2 had more intracellularamastigotes relative to the control group. Furthermore, this study showed anassociation between intracellular parasitism and PGE2 production byinfected macrophages.Passero et al.,2 can be considered as an additional mechanism bywhich L. (L.) amazonensis parasites infect, modulate inflammationand persist in the host, suggesting that the inhibition of this molecule can shedlight on important targets for the host-parasite interaction.,,2s inparasitism.The involvement of PLAL.(L.) amazonensis could involve the modulation of the host cellenvironment, since the treatment of macrophages prior or after infection alsoimpaired the parasite growth.In summary, our data indicate that γCdcPLI has a direct effect on parasites, sincethis protein was highly toxic against promastigote forms, culminating in a reductionof the parasite invasion rate. However, even though the mechanism of action ofγCdcPLI appears to be specific against the parasites, an additional andnon-exclusive hypothesis to explain the impact of this inhibitor against 2 inhibitor isolated from Cdc snake serum, against L. (L.)amazonensis. γCdcPLI presented high toxicity against the parasites,inducing cell cycle arrest that is probably a consequence of DNA damage.Furthermore, γCdcPLI treatment interfered with parasite invasion and intracellularproliferation in host cells. Thus, our results provide evidence of the potential useof PLA2 inhibitors as an interesting approach to studying the pathogenesis ofinfectious diseases, as well as a model for the discovery of relevant targets inparasites and/or host cells, for the design of new compounds against parasiticillnesses.In conclusion, the present study demonstrated the antileishmanial effects of γCdcPLI,a PLA


Plasma glucose, glucagon-like peptide-1 (GLP-1), glucose independent insulinotropic polypeptide (GIP) and insulin were measured, and participants classified according to the pattern of their glucose response. Gastric emptying was measured on breath samples (stable isotope breath test). Results: At baseline, 22 participants had a “monophasic” and 14 a “biphasic” glucose response. The 1 h plasma glucose response curve was greater and the GLP-1 AUC0–120 min and insulin secretion lower in the monophasic group. There were no differences in gastric emptying, GIP or insulin sensitivity. At the follow-up, the 1 h glucose response curve was greater again, while GLP-1 AUC0–120 min was lower in the monophasic group. Conclusions: A biphasic curve is associated with a higher 60 min glucose response curve and increases in GLP-1, but no difference in either GIP or gastric emptying.Background: The pattern of the plasma glucose response curve during an oral glucose tolerance test (OGTT) is of prognostic significance with “biphasic” when compared with “monophasic” patterns being associated with greater insulin sensitivity/secretion and a reduced risk of progression to diabetes. The relationships of the glucose response curves with gastric emptying and incretin hormone secretion are not known. Methods: Thirty-six adults (age > 65 years) without known diabetes consumed a 300 mL drink containing 75 g glucose and 150 mg C It is not widely appreciated that, in addition to the diagnostic implications of baseline, 60 min and 120 min plasma glucose levels ,4,5,6. TGE, for which there is a substantial inter-  but lesser intra-individual variation, is now recognised to be a major determinant of postprandial glucose, accounting for ~35% of the post-prandial blood glucose response . In someGlucagon-like peptide-1 (GLP-1) and glucose-independent insulinotropic polypeptide (GIP), released from the small intestine, increase insulin sensitivity and secretion and, in the case of GLP-1, suppress glucagon . An incrWe performed, in older individuals (>65 years) without known diabetes, a cross-sectional, longitudinal evaluation of the association of the shape of the glucose response curve with GLP-1 and GIP secretion, and GE as well as insulin secretion and sensitivity. We hypothesised that a relatively more rapid rate of GE would be associated with a monophasic rather than a biphasic curve, reflecting the relatively faster influx of glucose into the small intestine. We also hypothesised that plasma GLP-1 and GIP concentrations would be greater in the monophasic group in this cohort as a compensatory response to the glycaemic excursion.Information relating to the relationship of blood pressure to the rate of gastric emptying of a glucose drink in this cohort of older individuals has been previously reported . At the Following the initial study, participants were invited to attend a follow-up study after a mean interval of 5.8 ± 0.1 (SEM) years.All participants gave informed consent for their participation. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Human Research Ethics Committee of the Royal Adelaide Hospital.The protocol at the initial and follow-up studies was identical. Participants presented at 8.30 am after an overnight fast (14 h for solids and 12 h for liquids) when an intravenous cannula was inserted into the antecubital vein to facilitate blood sampling. A drink containing 75 g of glucose and 150 mg of 13C-acetate , made up to 300 mL with water at room temperature was then consumed within 5 min—t = 0 was defined as the time of completion of the drink. Plasma glucose (hexokinase method), insulin , GLP-1  and GIP  were measured at baseline and t = 15, 30, 45, 60, 90 and 120 min.Insulin secretion was estimated using the “insulinogenic index” of Δinsulin0–30/Δglucose0–30, insulin sensitivity using 1/fasting insulin and the oral disposition index (oDI) calculated using the product of insulin secretion and insulin sensitivity, i.e., 1/fasting insulin × Δinsulin0–30/Δglucose0–30 .Exhaled breath samples were collected before ingestion of the drink (t = −3 min) and then every 5 min for the first hour (commencing at t = 5 min) followed by every 15 min for the next 3 h. The 13CO2 concentration in the breath samples was measured using an isotope ratio mass spectrometer , and the gastric 50% emptying time (T50) was calculated . Wagner–t-test and shown as means ± SEM. A p-value of less than 0.05 was considered a significant difference.Glucose tolerance was classified according to The Expert Committee on Diagnosis and Classification of Diabetes Mellitus definitions . ImpaireParticipant characteristics are summarised in the table below . The monp < 0.001). While fasting, glucose was not different in both groups (p = 0.67); the 1 h post-OGTT plasma glucose response was greater in the monophasic vs. biphasic group 9.5 ± 0.5 mmol/L vs. 8.0 ± 0.5 mmol/L (p = 0.04). Forty-one participants  were recruited: 19 (46%) had normal fasting glucose and normal glucose tolerance, 2 (5%) had impaired fasting glucose, 12 (29%) had impaired glucose tolerance, 3 (7%) had both impaired fasting glucose and impaired glucose tolerance and 5 (12%) had undiagnosed type 2 diabetes—in these 5 participants, their general practitioners were notified of the results and they were excluded from the analysis. Of the remaining 36 individuals , 22 participants (61%) had a “monophasic” and 14 (39%) a “biphasic” glucose response. None had either an incessantly rising pattern or a more complex pattern. The monophasic group was modestly older (p = 0.03) group. There were no differences in either insulin sensitivity or the oral disposition index between the two groups [In the monophasic, there was an approximate two-fold reduction in insulin secretion compared to the biphasic  and the GLP-1 AUC0–120 min OGTT (p = 0.02) were lower. In contrast, there were no differences in plasma GIP between the two groups.In the monophasic group, both peak GLP-1 (p = 0.45) or as the gastric 50% emptying time (T50) .Gastric emptying was comparable in the two groups, whether expressed as a caloric rate  individuals with an initial monophasic response had a biphasic response and 8 (22%) individuals with a biphasic response had a monophasic response; the remaining 22 (61%) had the same response curve. p = 0.036) at the follow-up visit, but no difference in 1 h/2 h plasma glucose responses, glucose peaks or AUCs. As with the initial visit, a monophasic glucose pattern was associated with a higher 1 h plasma glucose response (p = 0.01) but not 2 h (p = 0.78) or fasting glucose (p = 0.09). There was, again, no difference in GIP, while GLP-1 was lower in the monophasic compared with the biphasic group. There were no differences in insulin secretion (p = 0.20), sensitivity (p = 0.44) and the oral disposition index (p = 0.15) [There was a modest reduction in fasting glucose  .p = 0.16) and the caloric rate was 1.78 ± 0.16 vs. 1.36 ± 0.16 kcal/min (p = 0.11). Gastric emptying, again, was not significantly different in the monophasic vs. biphasic group. The gastric 50% emptying time was 104 ± 10 min vs. 131 ± 17 min  is now recognised as a strong predictor of future type 2 diabetes, outperforming both the 2 h glucose response and HbA1c . In the Our study is the first to evaluate the shape of the glucose curve longitudinally, concurrently with measurements of incretin hormones and GE. Furthermore, Wagner–Nelson analysis was used to evaluate GE as this method enables isotope breath tests to be more comparable to the “gold-standard” of scintigraphy .We had a modest sample size and used surrogate markers of insulin secretion and sensitivity rather than the hyperinsulinaemic, euglycaemic clamp study . We alsoIn summary, a biphasic plasma glucose curve following a 75 g oral glucose tolerance test is associated with a reduction in the 60 min glucose response and increases in GLP-1, but no difference in GIP or gastric emptying when compared to a monophasic response in older individuals without diabetes.


Citrus × paradisi) grapefruit trees grafted on ‘Kuharske’ citrange (Citrus × sinensis × Citrus trifoliata). The study consisted of 4 foliar fertilizer treatments, which included 0×, 1.5×, 3× and 6× the University of Florida Institute of Food and Agriculture (UF/IFAS) recommended guidelines for B, Mn and Zn. Additionally, 2 ground-applied fertilizer treatments were used, specifically controlled-release fertilizer (CRF1): 12−3−14 + B, Fe, Mn and Zn micronutrients at 1× UF/IFAS recommendation, and (CRF2): 12−3−14 + 2× Mg + 3× B, Fe, Mn and Zn micronutrients, with micronutrients applied as sulfur-coated products. The planting densities implemented were low (300 trees ha−1), medium (440 trees ha−1) and high (975 trees ha−1). The CRF fertilizer resulted in greater soil nutrient concentrations through all of the time sampling points, with significant differences in soil Zn and Mn. Grapefruit treated with ground-applied CRF2 and 3× foliar fertilizers resulted in the greatest bacterial alpha and beta diversity in the rhizosphere. Significantly greater abundances of Rhizobiales and Vicinamibacterales were found in the grapefruit rhizosphere of trees treated with 0× UF/IFAS foliar fertilizer compared to higher doses of foliar fertilizers.Huanglongbing (HLB) disease has caused a severe decline in citrus production globally over the past decade. There is a need for improved nutrient regimens to better manage the productivity of HLB-affected trees, as current guidelines are based on healthy trees. The aim of this study was to evaluate the effects of different fertilizer application methods and rates with different planting densities on HLB-affected citrus root and soil health. Plant material consisted of ‘Ray Ruby’ ( Candidatus Liberibacter asiaticus (CLas), bacteria transmitted to tree hosts by a vector called the Asian Citrus Psyllid  [Much citrus production worldwide has significantly declined due to a disease known as huanglongbing  . In Florri, ACP) ,6,7. As ri, ACP) . Symptomri, ACP) . Grapefrri, ACP) . Most ofri, ACP) .There are several approaches toward dealing with HLB and alleviating symptoms, including the development of disease-tolerant rootstocks ,11, the Citrus × sinensis) when evaluating varying rates of manganese (Mn) via foliar application at 3× the recommended rate [The development of improved nutrient guidelines has proven to be beneficial since most of the established guidelines are based on healthy noninfected citrus trees rather than HLB-affected trees . Severalded rate . Additioded rate . The effded rate . Furtherded rate  found thBacillus subtilis, can assist plant hosts directly in the defense against pathogens [The application of various types and rates of fertilizers may also have subsequent effects on the microbial communities in the soil, notably those that reside within the rhizosphere (a portion of the soil that encompasses the roots of plants). When studying the effects of various N fertilizer treatments on wheat health and rhizosphere composition, Ref.  found thathogens . Furtherathogens . Further−1) had significantly greater soil Mg . However, trees planted in a medium density had significantly greater soil P  had significantly greater soil Zn . Rhizosphere bacterial alpha diversity did vary according to treatments according to the Shannon index. Grapefruit trees treated with CRF 2 had a greater bacterial alpha diversity than those treated with CRF 1 A. GrapefBeta diversity analyses included principal coordinate analysis (PCoA) on Bray–Curtis distances. An ANOSIM test was performed to determine significant differences in beta diversity between treatments. Rhizosphere bacterial beta diversity did vary according to treatments according to the Shannon index. Grapefruit trees treated with CRF 2 had greater bacterial beta diversity than those treated with CRF 1 B. Grapefp < 0.05), Rhizobiales (p < 0.05) and Rickettsiales (p < 0.05) compared to those from the 1.5× foliar treatment. Additionally, grapefruit trees at 0× foliar fertilizer had a rhizosphere bacterial community with a significantly greater abundance of Nitrosotaleales (p  <  0.05), Vicinamibacterales (p < 0.05), Tistrellales (p < 0.05) and Solirubrobacterales (p  <  0.05) compared to those from the 3× foliar fertilizer treatment. Furthermore, grapefruit trees with 0× foliar fertilizer had a rhizosphere bacterial community with a significantly greater abundance of Nitrososphaerales (p < 0.05), Clostridiales (p  <  0.05), Caulobacterales (p  <  0.05) and Rickettsiales (p  <  0.05) compared to 6× foliar treatment.There was variation in the relative abundance of bacterial taxonomic orders among treatments. Grapefruit trees treated with 0× foliar fertilizer had a rhizosphere bacterial community with a significantly greater abundance of Reyranellales , when compared to foliar fertilizers, affecting the availability of plant essential nutrients ,37. FurtWhen comparing the impact of ground-applied CRF on root health, the CRF2 treatment had an overall greater beneficial effect compared to the CRF1 treatment, notably in May 2021, with significant increases in the total root area and total root volume of grapefruit. Similarly,  found thBoth foliar and ground-applied fertilizer treatments had a greater impact on soil micronutrient concentrations compared to planting density at all timepoints, as confirmed through Pearson correlation coefficients. This was expected, as micronutrients were the main component that changed through the foliar and ground-applied fertilizer treatments.CLas. Greater concentrations of micronutrients, such as Mn and Zn, have been shown to exhibit antimicrobial functions [Excess amounts of micronutrients provided by the CRF2 treatment may have improved HLB-affected root health by reducing the activity of pathogenic organisms, such as unctions . For insunctions ,43, thusunctions .Significantly greater abundances of Rhizobiales and Vicinamibacterales may have been recruited from excess root exudates released from stressed HLB-affected grapefruit treated with 0× UF/IFAS foliar fertilizer compared to the other trees treated with higher doses of foliar fertilizers. Rhizobiales are a bacterial order of interest as they can interact with host plants to produce auxins, vitamins and N fixation, and protect the plants against stress . AdditioCitrus × paradisi) grafted on ‘Kuharske’ citrange (Citrus × sinensis × Citrus trifoliata). Trees were planted in September 2013 in flatwood soils  which are poorly drained and consist of 96% sand, 2.5% silt and 1.5% clay and have an argillic soil layer at 90 cm below the soil surface. The average soil pH was 5.8 and the cation exchange capacity (CEC) was 3.5 cmol kg−1. Trees were grown on raised beds roughly 1 m tall to facilitate drainage, with swales 15 m between beds. Irrigation was delivered using 39.7 L h−1 microjet sprinklers . The original experiment, which only focused on aboveground parameters, has been described in [−1), medium (440 trees ha−1) and high (975 trees ha−1). The study consisted of two ground-applied fertilizer treatments, specifically, controlled-release fertilizer blends 1 (CRF1): 12.00N-1.31P-11.62K and micronutrients at 1× the UF/IFAS recommendation with micronutrients as sulfates (12-3-14 1× Micro) and CRF2: enhanced 12.00N-1.31P-11.62K with 2× Mg and 2.5× the UF/IFAS recommendation with micronutrients as sulfur-coated products  Indian River Research and Education Center in Fort Pierce, FL . Plant material consisted of ‘Ray Ruby’ grapefruit trees  7 cm in diameter and 10 cm in depth was used to collect soil samples for nutrient concentrations. A single core was taken within the irrigated zone from one tree per experimental plot  across eight experimental blocks. Soil samples were analyzed for extractable N, P, K, Mg, Ca, S, B, Zn, Mn, Fe and Cu. 3COOH + 0.015 M NH4F + 0.013 M HNO3 + 0.001 M EDTA + 0.25 M H4NO3) was pipetted into extraction tubes containing 2.5 ± 0.05 g of soil. Soil nutrient concentrations were measured using inductively coupled plasma optical emission spectroscopy  [Soil samples were dried overnight at 80 ℃, and nutrient concentrations were determined using Mehlich III extraction . A totalMA, USA) .Root parameters were measured using a minirhizotron system . Each tube consisted of three scannable windows at different depths , and all three windows in every tube were scanned for each sampling point. After images were acquired, average root length and density were calculated using commercial software .2PO4 and 0.24 g KH2PO4) was added to the sample and shaken by hand for 15 s. Roots were removed with forceps and discarded, the remaining soil was centrifuged at 3000 g for 15 min and the supernatant was discarded. Soil DNA was extracted from 0.25 g of the soil pellet using the DNeasy PowerSoil Kit  according to the manufacturer’s instructions. A fluorometer  was used to quantify the extracted DNA and determine whether the DNA was concentrated enough for sequencing (>1 ng/µL). Rhizosphere DNA was amplified with 515Fa/926R universal bacterial [Rhizosphere samples were taken shortly after bulk soil sampling, which consisted of rhizosphere soil (located around the roots) being lightly shaken from the roots and placed in 50 mL sterile tubes. Approximately, 50 g of the soil was collected and stored at −20 °C before DNA extraction. Approximately, 15 mL of 1× sterile phosphate-buffered saline  was performed using statistical software . The main effect means were separated using Tukey’s honestly significant difference post hoc test. Differences were considered to be significant when In this study, we examined the impact of planting densities, ground-applied controlled-release fertilizers and foliar fertilizer dosages on both soil and plant parameters, including rhizosphere community composition. The use of the CRF2 ground-applied fertilizer resulted in higher soil nutrient concentrations through all time points of the study, notably with significant differences in Zn and Mn. Additionally, increased micronutrient concentrations provided by the CRF2 ground-applied fertilizer may have provided additional nutrients required to assist the root health of HLB-affected grapefruit trees. Furthermore, HLB-affected grapefruit at 0× the UF/IFAS recommended foliar fertilizer resulted in a significantly greater abundance of Vicinamibacterales and Rhizobiales, whereby both of which are PGPR that may have been recruited to assist grapefruit health under nutrient-deprived conditions.


BCR::ABL1 mRNA levels, while α-SMA expression was only partially reduced. Interestingly, the number of MKs was increased in the spleen of CML mice and elevated in both BM and spleen upon nilotinib treatment. Analysis of human CML-vs healthy donor (HD)-derived MSCs showed an altered expression of gene signatures reflecting fibrosis as well as hematopoietic support, thus suggesting MSCs as a potential player in these two processes. Finally, in our cohort, 12 patients qualified for TKI discontinuation, and here we observed that all patients who failed TFR had BM fibrosis at diagnosis, whereas this was only the case in 25% of patients with achieved TFR, further supporting the link between fibrosis and LSC persistence.The management of patients with chronic myeloid leukemia (CML) has been revolutionized by the introduction of tyrosine kinase inhibitors (TKIs), which induce deep molecular responses so that treatment can eventually be discontinued, leading to treatment-free remission (TFR) in a subset of patients. Unfortunately, leukemic stem cells (LSCs) often persist and a fraction of these can again expand in about half of patients that attempt TKI discontinuation. In this study, we show that presence of myelofibrosis (MF) at the time of diagnosis is a factor associating with TFR failure. Fibrotic transformation is governed by the action of several cytokines, and interestingly, some of them have also been described to support LSC persistence. At the cellular level, these could be produced by both malignant cells and by components of the bone marrow (BM) niche, including megakaryocytes (MKs) and mesenchymal stromal cells (MSCs). In our cohort of 57 patients, around 40% presented with MF at diagnosis and the number of blasts in the peripheral blood and BM was significantly elevated in patients with higher grade of MF. Employing a CML transgenic mouse model, we could observe higher levels of alpha-smooth muscle actin (α-SMA) in the BM when compared to control mice. Short-term treatment with the TKI nilotinib, efficiently reduced spleen weight and  BCR::ABL1-expression in CML patients in major or deep molecular response , which often persist at a very low level despite long-term treatment. LSC persistence is a major obstacle for one of the main goals of CML therapy, i.e., treatment-free remission (TFR), where patients remain in MMR or DMR after discontinuation of TKI therapy. The first large clinical trial on TFR has shown that about half of CML patients relapse, usually within the first 6 months (Chronic myeloid leukemia (CML) is a prime example of the success of molecular-targeted therapy. The implementation of tyrosine kinase inhibitors (TKIs) that bind to and inhibit the oncogenic driver BCR::ABL1 has revolutionized treatment outcomes in this cancer entity. However, primary or secondary resistance, as well as leukemic stem cell (LSC) persistence, remain a problem that hinders the cure of this disease. The persistence of LSCs is clinically reflected by residual 6 months . Further6 months ]. Some p6 months ]. Based A variety of mechanisms that could be responsible for the persistence of LSCs have been described. Looking at the microenvironment, we now know that the inflammatory signature of LSCs is driven not only by autocrine but also by paracrine mechanisms . Interes−) myeloproliferative neoplasms (MPNs), Gli+ and LepR1+ MSCs are driven into differentiation by a proinflammatory environment into a pro-fibrotic, myofibroblast-like cell type that then deposits excessive reticulin and collagen fibers (BM fibrosis or myelofibrosis (MF) is a complex process that leads to the deposition of excessive extracellular matrix (ECM) within the BM, which, upon progression slowly replaces hematopoietic cells. The development of MF is potentially possible in all myeloid neoplasms  and is tn fibers . The sevn fibers . In CML,n fibers . Interesn fibers . An openn fibers . ImportaGiven the supposed impact of MF on TKI response and the overlapping spectrum of cytokines that influence this process as well as LSC persistence, we analyzed here the relationship between BM fibrosis and TFR outcome in a small cohort of patients.BCR::ABL1 <0.01%). Success of discontinuation was defined as stable MMR for at least 1 year without need for re-treatment. TFR failure was defined as loss of MMR. Optimal response (OR) at 12 months was achieved if the patient’s BCR::ABL1 levels were below 0.1% . MF grades were assessed by the local pathology department of the RWTH Aachen University Hospital for all patients except 3. Median age ± standard deviation of patients was 54.7 ± 15.4 years, 42 (74%) were male. Correlations between MF grade at diagnosis and TFR were assessed for patients undergoing discontinuation of the TKI after being at least 1.5 years in deep molecular remission  12 montSCLtTA/BCR::ABL1  mice and FVB/N SCLtTA  siblings were bred in-house and tetracycline was added to the drinking water  in order to avoid the expression of BCR::ABL1 . BM cells of control or CML mice were isolated from femur and tibia, separated by cell straining and 2 × 106 cells were transplanted into the wildtype 10 Gy irradiated FVB/N CD45.1+ recipient mice by tail vain injection (Janvier Labs®). Mice were treated with cotrimoxazole  for 2 weeks after transplantation. One week after transplantation, BCR::ABL1 expression was induced by withdrawing tetracycline. After an additional week, treatment of CML mice with nilotinib  by daily gavage over 3 weeks was started. NIL  was resolved in 10% N-Methyl-2-pyrrolidon (Carl Roth) and 90% polyethylene glycol 300 (Sigma-Aldrich). Treatment controls were performed on both control and CML mice by administering the vehicle following the same procedure. All animal experiments were approved by the local authorities of North Rhine-Westphalia, Germany (LANUV Az. 84–02.04.2013.A072).FVB/N ®). To perform the staining, sections were de-paraffinized and hydrated, followed by antigen retrieval with citrate buffer. Sections were then blocked by peroxidase followed by serum blocking. Sections were incubated overnight at 4°C with an anti-alpha-smooth muscle actin (α-SMA) primary antibody (add antibody number) at a 1:800 dilution. Next, slides were washed and incubated for 30 min with the secondary antibody . Finally, sections were developed with diaminobenzidine, counterstained with hematoxylin and mounted with coverslips. Quantification of IHC staining was carried out using Fiji software (ImageJ®), after performing a color deconvolution (H DAB)  .After Formalin preservation, decalcification (EDTA) and paraffin embedding, 2.5–4 µm slides were produced by a microtome. For staining, a silver impregnation technique was used. The sections were de-paraffinized and hydrated and workflow was performed according to the following protocol: potassium permanganate (1%) for 5 min, oxalid acid (2%) for 5 min, iron alum for 5 min, silver solution ammoniacal for 10 s, formalin (5%) for 3 min, sodium thiosulphate (2%) for 2 min. Between each step, washing with distilled water was performed. Afterwards, nuclear fast red solution was used for 10 min for counterstaining. After immersing in alcohol  and xylol, the slides were mounted with coverslips.Grading occurred according to the WHO grading system (semiquantitative bone marrow fibrosis (MF) grading system proposed by WHO Classification of Tumours of Hematopoetic and Lymphoid Tissues ). The fo®) + 2% fetal bovine serum  and then incubated for 20 min at 4°C with a 1:100 dilution with anti-CD41 conjugated with APC Cy7 fluorochrome (BioLegend®) in the dark. Next, cells were washed and resuspended in PBS + 2% FBS. In addition, FACS staining of B-cells (B220+), granulocytes (GR-1+/CD11b+), T-cells (CD3+), blasts (KIT+), and erythrocytes (Ter119+) was performed. Flow cytometer measurements were carried out using Gallios flow cytometer (Beckman Coulter®) and analyzed with Kaluza analysis software (Beckman Coulter®).BM and spleen cells were harvested and lysis of red blood cells was performed with a ammonium-chloride-potassium solution (AKC). Subsequently, cells were washed with PBS (Pan Biotech2 in Dulbecco’s Modified Eagle Medium (DMEM) low glucose (Gibco®) supplemented with 10% of FBS, 1% each of L-glutamine, penicillin/streptomycin (both Gibco®) and cultured at 37°C with 5% CO2. After 24 h, the medium was replaced and non-adherent cells removed by washing with phosphate-buffered saline . Afterwards, adherent cells were maintained with medium replacement twice a week, detached with 0.25% trypsin-EDTA (Gibco®) after reaching confluence. MSCs were used for our experiments at passage 2 (p2).Human MSCs were obtained from BM aspirates of five CML patients at time of diagnosis and from hip bone donated after hip replacement surgeries of five healthy donors (HD). Cells were isolated by tissue culture plastic adherence after the mononuclear cell fraction was separated by Ficoll. Cells were seeded at 160,000 cells per cmin vitro into adipocytes, osteocytes and chondrocytes (®), 10% FBS, 1 µM dexamethasone, 200 µM indomethacin, 500 µM 3-isobutyl-1-methylxantine and 10 μg/mL insulin . On day 14–21, adipogenic cultures were washed with PBS, fixed in PFA 4% and stained with Oil Red O (Sigma®). For the osteogenic differentiation, cells were cultured in medium consisting of DMEM high glucose (Gibco®) supplemented with 10% of FBS (PAN Biotech®), 1% L-glutamine, 1% penicillin/streptomycin (both Gibco®), 10 nM dexamethasone, 10 mM β-glycerolphosphate and 50 µM ascorbic acid (Sigma®). After 21 days, cells were washed with PBS, fixed in PFA 4% and stained with Alizarin Red (Sigma®). For the chondrogenic differentiation, cells were cultured in medium consisting of DMEM high glucose (Gibco®), 1% L-glutamine, 1% penicillin/streptomycin (both Gibco®), 100 nM dexamethasone, 0.17 mM L-Ascorbic acid-2-PO4, 100 μg/mL sodium pyruvate, Insulin Transferrin Selenium supplement  and 10 ng/mL TGF-β. On day 14, cultures were washed with PBS, fixed in PFA 4% and stained with Alcian blue (Sigma®). After visual inspection and image acquisition, quantification of the three stainings was performed using the following solvents: isopropanol for adipogenic staining, 10%-cetylpyridinium chloride solution for osteogenic staining and 6M Guanidine HCl solution for chondrogenic staining, the relative absorbance was measured with a plate reader .MSCs were tested for their ability to differentiate drocytes . Briefly®); anti-CD34-FITC ; anti-CD45-PE (BD®); anti-CD90-PerCP-Cy5.5 (eBioscience®); anti-CD13-PE (eBioscience®); anti-CD11b -FITC (BD®) anti-CD44-APC and anti-HLA-DR-FITC (BD®). Staining of at least 50,000 cells was performed at 4°C for 15 min in the dark in presence of 1% human serum to avoid unspecific binding. Samples were acquired with a Gallios flow cytometer (Beckman Coulter®) and analyses were performed using FlowJo Software (BD®).MSC phenotype was assessed at passage 2 (p2) using the following monoclonal antibodies: anti-CD73-FITC (eBioscience®), where the wells were stained with Giemsa (Merck) according to manufacturer’s instruction. Colonies were then counted manually. The frequency of progenitor cells in the undiluted starting population was calculated manually.Log serial dilutions of the MSC at p2 were placed into 10 to 20 replicates in 96-well plates in the presence of MesenPure (STEMCELL Technologies) and 0.5% penicillin-streptomycin. After 14 days, colony-forming unit-fibroblast (CFU-F) were performed using MesenCult Expansion Kit (STEMCELL Technologies®) as previously described. RNA was hybridized on Human Clariom D Assay gene chip (Affymetrix®). Further steps, such as sample labeling, hybridization, washing and scanning were performed according to manufacturer’s protocols. Downstream analysis was performed in R (v4.2.2), using packages oligo (v1.62.2), Biobase (v2.58), affycoretools , limma (v3.54.2), PCAtools (v2.10), progeny (v1.20), msigdbr (v7.5.1) and clusterProfiler (v4.6) for analysis; data.table (v1.14.8), tibble (v3.2.1) and dplyr (v1.1.1) for data wrangling; and ggplot2 (v3.4.1), ggrepel (v0.9.2), ComplexHeatmap (v2.14), patchwork (v1.1.2) and cowplot (v1.1.1) for visualization.RNA of four HD- and four CML-derived MSCs was isolated using TRIzol™ Reagent  and Gene Set Expression Analysis (GSEA) were performed on the results from the differential expression analysis. For ORA, the background probes considered were the same as the ones present in the differential expression analysis. Only enrichment for under-expressed probes was performed as no probe passed the over-expression significance threshold during differential expression analysis. For ORA and GSEA, t-test was used to compare the means of two groups. Fisher’s exact test was used for Contingency Table. Statistical calculations were performed with GraphPad Prism . Data are presented as mean ± standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were considered statistically significant.1-way or 2-way analysis of variance (ANOVA) were applied for multiple comparison analysis, with Tukey’s or Bonferroni’s post-hoc-test. Unpaired Based on the conflicting reports on the frequency of MF, we first assessed the proportion of patients with MF at diagnosis in our CML cohort (n = 57). Here, 38.6% (n = 22) had MF at the time of diagnosis, with 31.6% having MF stage 1 (n = 18) and 7.0% (n = 4) having stage 2 . No grad6 BM cells, isolated from either SCLtTA/BCR::ABL1 CML or SCLtTA control mice into 10 Gy irradiated recipients and allowed these cells to engraft for 1 week before inducing BCR::ABL1 expression. 2 weeks after induction of the oncogene, mice were treated with either nilotinib (NIL) or vehicle (V) control for 3 weeks  were either isolated from BM of CML patients or the hip bone of HDs via plastic adherence  and theiTo determine the gene expression signature of CML-vs HD-MSCs, we performed a microarray analysis. Overall changes where rather mild in these MSCs that were expanded until p2, as shown by the volcano plot . HoweverThe success of TFR is compromised by the persistence of LSCs in CML patients on TKI therapy, which re-expand upon TKI discontinuation. Based on the expression analysis of primary patient-derived MSCs indicating LSC-supporting potential, we investigated the impact of MF on TFR. A minority of CML patients underwent TKI discontinuation. In our cohort of patients with available MF grading at diagnosis, 12 patients discontinued TKI therapy after at least 1.5 years of MR4 or better. The clinical parameters of these patients are described in In this study, we suggest a potential link between the presence of BM fibrosis in CML patients at diagnosis and persistence of LSCs that are capable to re-expand upon TKI discontinuation, thereby resulting clinically in TFR failure.First, we could observe that in our cohort around 40% of the patients presented with MF at diagnosis and MF grade 2 correlated with higher blast counts in the blood and BM. Although the impact of BM fibrosis on prognosis was considered adverse in the pre-TKI era , it seem− MPNs, MKs and MSCs are known to play key roles in the development of fibrosis  and further studies on larger cohorts are necessary to confirm this association.Nevertheless, it is important to underline that this correlation was not statistically significant (et al (Moreover, another important aspect to discuss is that according to the literature, the correlation between the common risk scores and TRF success is not always evident. In some studies, patients with low-to intermediate-risk Sokal score had a significantly higher probability of persisting complete molecular response compared to those with high-risk score . In our et al , no signIn conclusion, in our hypothesis the BM fibrosis in CML patients likely recedes upon TKI treatment, to a macroscopically undetectable level. Nevertheless, such a process fundamentally impacts on the BM niche and one could hypothesize that specific changes might remain on a molecular level, which could drive LSC re-expansion. However, this needs further comparative studies, ideally at the single cell level, of the LSC niche in patients who either relapse after discontinuation of the TKI or who do not.However, as the grading of MF seems to vary significantly depending on the individual assessment, another, possibly technology-aided and centralized approach to grade fibrosis in the BM would help to reduce the variations seen in different studies . One maj