In the present study, we targeted an aequorin chimera with reduced Ca2+ affinity to the ER membrane and facing the ER lumen. To this aim, the cDNA for a low‐Ca2+‐affinity aequorin variant (AEQmut) was fused to the nucleotide sequence encoding a non‐cleavable N‐terminal ER signal peptide (fl2). The correct targeting of fl2‐AEQmut was confirmed by immunocytochemical analyses in transgenic Arabidopsis thaliana (Arabidopsis) seedlings. An experimental protocol well‐established in animal cells – consisting of ER Ca2+ depletion during photoprotein reconstitution followed by ER Ca2+ refilling – was applied to carry out ER Ca2+ measurements in planta. Rapid and transient increases of the ER luminal Ca2+ concentration ([Ca2+]ER) were recorded in response to different environmental stresses, displaying stimulus‐specific Ca2+ signatures. The comparative analysis of ER and chloroplast Ca2+ dynamics indicates a complex interplay of these organelles in shaping cytosolic Ca2+ signals during signal transduction events. Our data highlight significant differences in basal [Ca2+]ER and Ca2+ handling by plant ER compared to the animal counterpart. The set‐up of an ER‐targeted aequorin chimera extends and complements the currently available toolkit of organelle‐targeted Ca2+ indicators by adding a reporter that improves our quantitative understanding of Ca2+ homeostasis in the plant endomembrane system.Precise measurements of dynamic changes in free Ca 2+ homeostasis in the plant endoplasmic reticulum (ER) requires accurate measurements of the free Ca2+ concentration in the lumen of this compartment. We designed a specifically ER‐targeted aequorin chimera that allowed quantitative monitoring of dynamic changes in ER Ca2+ levels in Arabidopsis seedlings in response to environmental cues and we analysed the interplay of ER and chloroplasts in shaping cytosolic Ca2+ signals.A better understanding of Ca The relative contribution of ER and chloroplasts in shaping cytosolic Ca2+ signals was also investigated.In the present study, we developed a novel Ca2+]ER in plant cells paves the way for future studies aimed at unravelling the integration of the plant ER in Ca2+ transport and signalling circuits.The set‐up of a novel tool to quantitatively monitor  compartments  ethanol, 1 s Figure . To alloR Brini, . Differes Figure . Use of s Figure  and was y Figure .2+ assays  did not result in increased basal ER Ca2+ levels , ruling out the possibility that the reached [Ca2+] steady‐state was dependent on limited external Ca2+ supply . Application of a touch stimulus  did not affect resting [Ca2+]ER levels  led to a rapid (after 10.4 ± 1.4 sec) and transient, approximately 10‐fold increase in [Ca2+]ER  was found to induce a less pronounced and much slower Ca2+ elevation (after 89.0 ± 4.3 sec)  Figure ,l. Inter2+ assays were also carried out in Arabidopsis seedlings stably expressing aequorin in either the cytosol or chloroplast stroma  compared to the cytosolic one  or after  the ER Ca2+ peaks , derived from bacterial flagellin, did not evoke any evident [Ca2+]ER change, at least in the observation time frame of 700 sec  were applied to mimic the symbiotic signal released by arbuscular mycorrhizal (AM) fungi during the establishment of AM symbiosis with host plants  , comprising pectic fragments of the plant cell wall originating after pathogen attack  Figure . On the k Figure .2+] in the cytosol and in the ER, the participation of active Ca2+ transporters located at ER membranes in the observed stimulus‐induced [Ca2+]ER increases seemed likely. To obtain insights into putative ER membrane‐localized Ca2+ transporters responsible for the observed ER Ca2+ increases, a pharmacological approach based on the use of specific inhibitors of plant Ca2+‐ATPases  or the ACAs (Autoinhibited Ca2+‐ATPases) blockers eosin Y (1 μm) and erythrosin B (1 μm) before stimulus application . In line with the successful use of CPA in the coelenterazine reconstitution protocol, 51.0 ± 8.8% inhibition of the stimulus‐induced ER Ca2+ increase evoked by 300 mm NaCl was observed, whereas eosin Y and erythrosine B did not induce any significant change in the [Ca2+] elevation with respect to the control   Figure , whereasd Figure .2+ responses in the ER were likewise highly reduced by EGTA pre‐treatment. In particular, 1 mm EGTA caused 70.8 ± 4.6 and 46.6 ± 18.8% inhibition of salt stress‐ and osmotic stress‐induced [Ca2+]ER increases, respectively; an even higher inhibition (94.4 ± 2.0 and 90.0 ± 5.3%) was determined by 5 mm EGTA  in the ER Ca2+ uptake, although this was less dramatic than that recorded for the other two abiotic stimuli  was not found to effectively block either the cytosolic or ER Ca2+ rises  and the corresponding free [Ca2+] was determined and plotted   in response to the tested abiotic stimuli  specifically targeted to the ER has renewed interest in better understanding the role of the ER in Ca2+‐mediated signal transduction pathways. The targeting of fluorescent GECIs, i.e. the FRET‐based Ca2+ indicator cameleon  of a maize mutant isolated in the mid‐to‐late 1990s , commonly employed for [Ca2+]ER measurements in the animal field . Although [Ca2+] in the plant ER was found to be approximately 10 to 100 times lower than that generally reported in the ER of animal cells (i.e. from 50 to 500 µm) , rendering this intracellular compartment the second main intracellular Ca2+ store of the plant cell, after the vacuole  were used to visualize ER Ca2+ signals in Arabidopsis  in response to salt, osmotic and oxidative stresses. Notably, the [Ca2+] response curve of fl2‐AEQmut exhibits a linear relationship between 10−6 and 10−4 m. This is in good agreement with the range of physiological [Ca2+]ER obtained in our experiments, providing evidence for the reliability of the [Ca2+]ER measurements performed with this aequorin chimera. Interestingly, no changes in [Ca2+]ER were observed in response to bacterial flg22, whereas OGs were found to trigger a modest, but detectable, [Ca2+]ER elevation. These data suggest a differential contribution of the ER as the Ca2+ store responsible for the modulation of cytosolic Ca2+ fluxes evoked by microbe‐ and damage‐associated molecular patterns  was expected because COs have been demonstrated to represent a fungal symbiotic signal for the arbuscular mycorrhizal (AM) symbiosis that is established between AM fungi and most land plants, but not Arabidopsis   in response to distinct environmental stimulations. Moreover, the progressive decrease in the stimulus‐triggered [Ca2+]ER transients when Arabidopsis seedlings were pre‐treated with higher doses of EGTA suggests that the depletion of the extracellular Ca2+ pool gradually leads to an increasing Ca2+ leakage from the ER and affects its ability to take up Ca2+ released in the cytoplasm upon stimulus application. Pre‐treatment with BAPTA‐AM was found to be ineffective in the chelation of intracellular Ca2+ fluxes, possibly because of cleavage of AM groups by extracellular esterases in the apoplast  was cloned into the 35SCaMV cassette (677 bp) of the plasmid p35SCaMV. The entire cassette 35SCaMV‐fl2‐AEQmut was then amplified to create additional restriction sites (NotI and XhoI) and moved into the binary vector pGreen 0029. To obtain the fl2‐YFP construct, the sequence encoding the YFP was amplified by PCR using XbaI_fl2_Yfp and Yfp_rev as primers  of the 24‐kDa α‐zein of the maize mutant er Table . After drs Table . The ampN. benthamiana and A. thaliana (Arabidopsis) Col‐0 ecotype. Standard agroinfiltration procedures were applied to fully‐expanded leaves of 4‐week‐old N. benthamiana  half‐strength MS medium, pH 5.5 containing 50 μg ml−1 kanamycin (seedlings were grown at 21°C under a 16:8 h light/dark cycle). Plants that survived were transferred into single pots and grown on soil so that F2 generation seeds could be collected separately: the progeny of each F1 seedling was subsequently screened for aequorin expression both at the RNA and protein levels.Arabidopsis Col‐0 plants were transformed by the floral dip technique  and flash‐frozen in liquid nitrogen. Total RNA was extracted using the RNeasy Plant Mini Kit  and then reverse transcribed with SuperScript III  in accordance with the manufacturer’s instructions. Primers designed on the cDNA sequence of fl2‐AEQmut  that revealed the highest probe expression were used to establish cell suspension cultures, as described recently  after 72 h. Confocal microscopy observations of Arabidopsis (after transient and stable transformation with the fl2‐YFP construct) were performed with a TCS SP5 II confocal laser scanning system  mounted on a DMI6000 inverted microscope (Leica). Samples were excited with a 488 nm Argon laser (for YFP and chlorophyll), a 543 nm Helium/Neon laser  and a 561 nm diode laser for RFP, whereas fluorescence emissions were collected at 505–540 nm for YFP, 580–610 nm for RFP, 680–720 nm for chlorophyll and 600–633 nm for ER‐Tracker Red. Immunofluorescence experiments were conducted on cell suspension cultures stably expressing fl2‐AEQmut, as described by Zonin et al.  operating at 100 kV and equipped with an Osis Veleta camera .TEM analyses and immunogold labelling (dilution 1:500) of 2‐week‐old Arabidopsis transgenic plants were carried out as described previously , the seedlings were exposed to an actinic light of 650 µE m−2 sec−1, whereas, for the last 5 min (D1–D5), the seedlings were kept in the dark. Maximum fluorescence, non‐photochemical quenching and PSII quantum yield were analyzed.Quenching analyses were performed on 2‐week‐old seedlings grown under sterility conditions on agarized medium . Seedlings were exposed to 16 h of light followed by 20 min of dark adaptation prior to subsequent analyses. Fluorescence was detected by PAM imaging using FluorCam7 : each min for 10 min, a saturation pulse of 2000 µE mm Tris‐HCl, 10 mm EGTA, 0.8 mm phenylmethylsulfonyl fluoride, pH 8.0. Proteins were resuspended at 1 μg μl−1 in reconstitution buffer and incubated with 1 mm β‐mercaptoethanol and 5 μm coelenterazine  for 4 h at 4°C in the dark. Aequorin luminescence was detected from 50 μl of the in vitro aequorin reconstitution mixture and integrated for 200 sec after the addition of an equal volume of 100 mm CaCl2.Protein crude extracts were obtained from Arabidopsis transgenic and wild‐type cell suspension cultures (4 days old) using as reconstitution buffer 150 m2+] calibration curve of the aequorin probe at 22°C, protein crude extracts were likewise obtained from both fl2‐AEQmut #6 and #10 transgenic lines using a similar buffer with a reduced EGTA content (1 mm) and reconstituted according to the same protocol with the sole exception of a 10‐fold concentration of β‐mercaptoethanol (10 mm). At the end of the reconstitution, proteins were further diluted to 0.5 μg μl−1 using an EGTA‐free equivalent buffer to lower the total EGTA concentration to 0.5 mm. Reconstituted extracts  were placed in a 96‐well microplates  to which a BackSeal  was applied: using an EnVision 2105 XCite multimode plate reader (Perkin Elmer), emitted luminescence at 22°C was recorded and integrated for 120 sec, following the administration at 3 sec of 200 μl of a variable [CaCl2] (ranging from 5.86 to 250 μm) – which further reduced the total EGTA concentration to 0.1 mm – and the addition at 60 sec of a discharge‐like solution (1 m CaCl2). The collected data were analyzed and expressed as L/Lmax  for each applied [CaCl2].To determine the [Ca2+] corresponding to each applied [CaCl2] under these experimental conditions, 50 µl of reconstitution buffer (either without EGTA or containing 0.5 mm EGTA) was complemented with 1 µm Calcium Green™‐5N and 10 µmN,N,N′,N′‐tetrakis(2‐pyridinylmethyl)‐1,2‐ethanediamine and used instead of the reconstituted protein extracts. By following an equivalent protocol, fluorescence collected with a Green Fluo filter , allowing the determination of the Calcium Green™‐5N affinity curve for Ca2+ , which was then used to calculate the exact free [Ca2+] plotted against L/Lmax values. The calibration curve, representing the best fit for the experimental data, was obtained as described by Brini et al.  were transferred in a microplate well and incubated in 600 μm EGTA solution supplemented with 5 μm coelenterazine. To set up an efficient in vivo reconstitution protocol, different procedures were experimented  with the ionophore A23187 (10 μm) or the ER Ca2+‐ATPase blocker CPA (50 μm) or both). Seedlings were floated on 1 ml H2O (six seedlings per microplate) and treated with A23187/CPA 10 min before the addition of coelenterazine. Afterwards, seedlings were extensively washed with 100 μm EGTA (10 to 20 ml per seedling) and allowed to recover for 10 min, before being singly transferred in the chamber (1 ml) of the luminometer  and subjected to challenge with stimuli and/or in vivo discharge   was applied to all subsequent Ca2+ measurements carried out in the luminometer. Each experiment started with one Arabidopsis seedling incubated in 150 µl of 100 μm EGTA, to which an equal volume of a two‐fold (2 mm) CaCl2 solution was added after 100 sec to restore resting [Ca2+]ER. ER refill trials with higher [CaCl2] followed the same procedure but with appropriate [CaCl2] solutions. At 300 sec, 300 µl of a two‐fold concentrated stock solution for each tested stimulus was injected in the luminometer chamber. Oligogalacturonides  ethanol , 5 min before stimulus application. Arabidopsis seedlings stably expressing YFP‐aequorin targeted to the cytosol (Cyt‐YA) or plastid stroma (Str‐YA) were also used in Ca2+ assays, as described previously (Sello et al., 2+ chelators, seedlings were pre‐treated either for 10 min with 1 or 5 mm EGTA or for 1 h with 50 µm BAPTA‐AM. Luminescence data were converted off‐line into [Ca2+]ER values using a computer algorithm based on the previously determined Ca2+ response curve.The best reconstitution protocol (pre‐treatment with 50 μ2+ measurement assays. RM designed plasmids and performed gene expression analyses. LC and EC performed PAM analyses. LC and LF carried out plant transformation and confocal microscopy observations. BB performed TEM analyses. DDS and EC constructed the Ca2+ calibration curve. MB, LF, DDS, IS and UCV contributed to the discussion of the results and editing of the manuscript. LN conceived the research and designed the experiments. EC and LN analysed the data and wrote the article.EC and FP set up and carried out CaThe authors declare no conflict of interest.Figure S1. Cloning strategy for the creation of the expression vector targeting the AEQmut probe to the plant ER and analysis of aequorin expression in Arabidopsis transgenic lines.Click here for additional data file.Figure S2. Phenotype, photosynthetic efficiency and ultrastructure of Arabidopsis transgenic lines stably expressing fl2‐AEQmut.Click here for additional data file.Figure S3. Confocal microscopy analyses demonstrate the ER localization of fl2‐YFP in Arabidopsis.Click here for additional data file.Figure S4. Immunofluorescence analyses of Arabidopsis cell suspension cultures stably expressing fl2‐AEQmut.Click here for additional data file.Figure S5. In vitro reconstitution assays in Arabidopsis fl2‐AEQmut transgenic lines.Click here for additional data file.Figure S6. In vivo reconstitution assays in Arabidopsis fl2‐AEQmut transgenic lines.Click here for additional data file.Figure S7. Effect of the fl2‐AEQmut reconstitution protocol on Arabidopsis cell viability.Click here for additional data file.Figure S8. Steady‐state [Ca2+]ER is independent of the concentration of CaCl2 used in the refilling step.Click here for additional data file.Figure S9. Monitoring of [Ca2+]ER dynamics in response to stimuli of biotic nature.Click here for additional data file.Table S1. List of primers used to target the fl2‐fused probes to the ER.Click here for additional data file.Movie S1. Time‐lapse confocal microscopy of a cortical sector of an Arabidopsis leaf epidermal cell stably expressing fl2‐YFP.Click here for additional data file. Click here for additional data file.


One sentence summary: Bi-directional-dual-flow-RootChip to track calcium signatures in Arabidopsis primary roots responding to osmotic stress.2+) is a key messenger in signal transduction pathways associated with a variety of stresses, including mechanical, osmotic stress and the plants’ innate immune system. These stresses trigger an increase in cytosolic Ca2+ and thus initiate a signal transduction cascade, contributing to plant stress adaptation. Here we combine fluorescent G-CaMP3 Arabidopsis thaliana sensor lines to visualise Ca2+ signals in the primary root of 9-day old plants with an optimised dual-flow RootChip (dfRC). The enhanced polydimethylsiloxane (PDMS) bi-directional-dual-flow-RootChip (bi-dfRC) reported here adds two adjacent inlet channels at the base of the observation chamber, allowing independent or asymmetric chemical stimulation at either the root differentiation zone or tip. Observations confirm distinct early spatio-temporal patterns of salinity  and drought -induced Ca2+ signals throughout different cell types dependent on the first contact site. Furthermore, we show that the primary signal always dissociates away from initially stimulated cells. The observed early signaling events induced by NaCl and PEG are surprisingly complex and differ from long-term changes in cytosolic Ca2+ reported in roots. Bi-dfRC microfluidic devices will provide a novel approach to challenge plant roots with different conditions simultaneously, while observing bi-directionality of signals. Future applications include combining the bi-dfRC with H2O2 and redox sensor lines to test root systemic signaling responses to biotic and abiotic factors.Plant growth and survival is fundamentally linked with the ability to detect and respond to abiotic and biotic factors. Cytosolic free calcium (Ca Ca2+-Dependent Protein Kinases (CPKs) and Calcineurin-B Like proteins (CBLs)  .2+]cyt measurements and experiments have been accomplished in Arabidopsis thaliana (Arabidopsis). In recent years a variety of approaches have been used for this, including the luminescent Ca2+-interacting aequorin protein,  at 100 W for 10 min. After cleaning, dry-film, negative-tone photoresist  was laminated (Sky-335R6) onto the wafer. The chip design was then transferred into the photoresist by exposure of the prepared photomask in a mask aligner . After exposure to UV light, the wafer was baked on a hot plate  for 5 min at 65°C and 20 min at 95°C. Transferred chip structures were developed in propylene glycol methyl ether acetate (PGMEA) developer for 30 min and rinsed with isopropanol for 5 min. Lastly, the wafer was hard-baked on the hot plate for 1 hour at 125°C, yielding a mold ready for replica-molding (Microfluidic chips were fabricated using photolithography and replica molding . In brie-molding .2-plasma activated (PIE Scientific Tergeo) at 15 W power for 1 minute and bonded to pre-cleaned microscope slides  by lightly pressing the exposed surfaces of the PDMS and glass together. Bonded chips were then baked for 2 hours on a hot plate at 80°C to strengthen the bond.Prior to molding, the silicon wafer was pre-treated with an anti-adhesion agent  silane, Sigma-Aldrich) to prevent sticking of the elastomer cast during the subsequent replica-molding. This step was repeated after 10 subsequent uses of the wafer as mold master. For replica-molding, PDMS  pre-polymer silicone elastomer base was mixed thoroughly with silicone elastomer curing agent at a 10:1 (w/w) ratio and degassed to remove air bubbles. Once cast onto the wafer mold, the PDMS was degassed again and placed on a hot plate at 80°C for 2 hours to cure. Subsequently, the PDMS was carefully peeled off from the wafer and cured on the hot plate for a further 2 hours at 80°C. Following the second curing bake, inlet and outlet holes were core-punched  and the PDMS was cut into individual bi-dfRC chips using a guillotine or scalpel. Chips were then Ovia passive pumping (® S2000).To permanently retain the hydrophilicity of microfluidic channels  and redu pumping  and was 2.2A dual-pressure syringe pump system  was used to deliver singular or dual treatment into the bi-dfRC observation chamber (OC). The pump system holds syringes  connected to the chip via 1/16” OD ethylene tetrafluoroethylene (ETFE) tubing (Kinesis), primed with a silicone tubing sleeve (Darwin Microfluidics) and short 1.5 mm OD metal tubes. Flow rate was set to 20 μL per minute.2.3Arabidopsis lines  for 2 min, subsequently washed 4 times with sterile Milli-Q® water (Merck). Pre-sterilised seeds were vernalised in water at 4°C for approximately 12 hours. Seeds were cultured onto half-strength Murashige and Skoog (½ MS) medium (Duchefa), 0.31 mM MES (Sigma-Aldrich) and 8% agarose. Plants on plates were grown for 4-days at a short-day cycle  with 65% humidity and 150 µmol m-2 s-1 per µA light intensity. On chip sub-culturing was achieved by transferring 4-day old Arabidopsis plants directly onto the pre-sterilised bi-dfRCs. Prior to sub-culturing, microchannels and inlets were pre-sterilised with 70% ethanol, and then washed with ½ MS/0.31 mM MES liquid medium. Agarose squares (4×4 mm) were oriented above the root inlet channel. Plants were aligned with the agarose squares and primary roots were carefully situated into the root inlet channel. Chips were stored in plastic incubation chambers , surrounded by sterile Milli-Q water, for humidity. The set-up was incubated for 5-days under the pre-alluded short-day cycle . The set up was tilted at a 45-degree angle to promote root growth into the bi-dfRC microchannel.In this study, Col-0 and G-CaMP3 2.42+ fluorescence was observed with an eGFP filter (38 HE Green Fluorescent Prot BP 450-590) and bright field (BF) was set to 2.7 ms exposure, TL lamp 30%. Images and videos were analysed either in in Fiji-ImageJ (Arabidopsis root tip quantification was accomplished utilising the freehand selection tool. Treatments were applied as either a control solution (½ Murashige and Skoog (MS) medium, plus 0.31 mM 2-(N-morpholino) ethanesulfonic acid (MES) dissolved in Milli-Q® water) or stress solution (100 mM sodium chloride (NaCl) or 20% polyethylene glycol dissolved in ½ MS/0.31mM MES media). For raw data collection fluorescence intensity was measured in the original raw video frames. Linear section 1 was sampled at the root tip columella cells, while linear sections 2 and 3 represent meristematic and elongation zone 1 and 2(ME1 and ME2) of the root, respectively, and linear sections 4 and 5 represent elongation and differentiation zone 1 and 2 (ED1 and ED2) of the root, respectively  5x lens (EC Plan-Neofluar 5x/0.15 M27) (i-ImageJ  or ZEN B2.52+) of transverse pixel sections was summed up for each spatio-temporal position.Kymographs were used to show spatial intensity over time for exemplified roots. For this, original.czi files were converted into.avg video formats using Zen blue software, without compression. Every picture frame was defined as a time-point. The fluorescence intensity (corresponds to cytosolic Cawww.jetbrains.com) in Python ver. 3.6 (https://www.python.org/downloads/release/python-360/) to generate kymographs. The main functionalities were provided by the open-source computer vision library OpenCV (https://pypi.org/project/opencv-python/). The script reads out every picture within the video and applies a summation longitudinal or across the root in a manually predefining region of interest (ROI). Operation of the script is controlled via command line interface using the standard library argparse (https://docs.python.org/3/library/argparse.html).An in-house python script was developed with PyCharm  were used to connect the stiffer tubing to pre-made 1 mm × 1 cm metal tubes with a 90-degree bend , as shown in r bubble . Lastly,Arabidopsis root  and buffering the Ca2+ response  into the channel. Hence, the solution moves through the bi-dfRC with a speed of ~6.84*103 µm/s. In contrast, the observed Ca2+ signal in the root induced by NaCl or PEG move at a speed of 4-14 µm/s, which is far slower than the solution. Therefore, it can be excluded that the flow of the solution in the channel of the chip is responsible for the movement of the Ca2+ signal within the root. NaCl-induced salinity stress and PEG-induced drought stress both reduce water absorption and obstruct water movement, inducing downstream osmotic and oxidative stress pathways . The signal transferred longitudinally through the cortical and stele tissues, at a speed of 6.8 μm/s   . This was followed by a Ca2+ wave that traveled shoot-ward throughout the stele tissue of the elongation zone to the differentiation zone at a speed of 5.9 µm/s   and the inducing stressor. Early studies showed Ca2+ release, and corresponding increase in G-CaMP3 fluorescence, initiated in the root system upon immersion of entire 6 to 7-day old plantlets in NaCl   of 64.8 nM  . A much  64.8 nM , compare 64.8 nM , which m2+ release, and corresponding increase in G-CaMP3 fluorescence, and longitudinal dispersion of Ca2+ following primary root tip exposure to NaCl and PEG in G-CaMP3 transgenic roots reported here was slower, yet comparable to observations in 2+ through varying tissues following initial Ca2+ release, and corresponding increase in G-CaMP3 fluorescence, at both the differentiation zone or tip treatment sites showed a similar tissue-specific dispersion pattern.Interestingly, the initial Ca3.4.22+ signals initiated on the treated side of the root. Observations showed varying cell types and tissues were involved in the spatio-temporal dispersion of Ca2+, in dependence on the initial stress site. A one-sided NaCl or PEG treatment at the differentiation zone always led to a transversely moving signal, from the contact to the non-contact side. The signal then moved longitudinally to the tip. Upon NaCl treatment at the differentiation zone, the Ca2+ burst emanated from epidermal cells spanning the elongation and differentiation zone on the treatment side of the root, in addition to the root tip. The signal increased 1.5-fold in intensity and transversely moved from the treated to untreated side of the root at a speed of 9.4 μm/s. A secondary Ca2+ wave was observed, which moved longitudinally to the root tip through stele tissue at a speed of 5.2 μm/s   through cortical and stele tissue.   and root cap to the epidermal and cortical cells of the elongation zone, causing the root to bend away from high salt concentrations  hydrogen peroxide (H2O2), and intracellular Ca2+ for systemic propagation of the signal between varying cells and tissue  and lipids, while observing and tracking RNA and protein movement within root cells. We believe that this technology will position us better for future studies that will potentially lead to novel insights into mechanisms of molecular adaptations that underlie improved tolerance and survival of crop plants to challenges imposed by climate and pathogens.Many Caely vary . The G-Cely vary . Howeversitivity . ConsequThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.2+ quantification and Kymogram analysis. VN designed and prepared the wafer for the chips. AT provided technical assistance to CA. C-NM and VN conceived the project. CA wrote the article with contributions of all the authors. C-NM and VN supervised and completed the writing. C-NM agrees to serve as the author responsible for contact and ensures communication. All authors contributed to the article and approved the submitted version.C-NM and VN supervised the experiments. CA performed and designed the experiments and analysed the data. R.H. wrote the script for Ca


IDH2 and FLT3 mutations, whereas non-responders were associated with PTPN11 mutations. The combination of PI3K/mTOR dual pathway inhibition with bimiralisib and BCL2 inhibition with venetoclax has emerged as a candidate treatment in IDH2- and FLT3-mutated AML.In October 2020, the FDA granted regular approval to venetoclax (ABT-199) in combination with hypomethylating agents for newly-diagnosed acute myeloid leukemia (AML) in adults 75 years or older, or in patients with comorbidities precluding intensive chemotherapy. The treatment response to venetoclax combination treatment, however, may be short-lived, and leukemia relapse is the major cause of treatment failure. Multiple studies have confirmed the upregulation of the anti-apoptotic proteins of the B-cell lymphoma 2 (BCL2) family and the activation of intracellular signaling pathways associated with resistance to venetoclax. To improve treatment outcome, compounds targeting anti-apoptotic proteins and signaling pathways have been evaluated in combination with venetoclax. In this study, the BCL-XL inhibitor A1331852, MCL1-inhibitor S63845, dual PI3K-mTOR inhibitor bimiralisib (PQR309), BMI-1 inhibitor unesbulin (PTC596), MEK-inhibitor trametinib (GSK1120212), and STAT3 inhibitor C-188-9 were assessed as single agents and in combination with venetoclax, for their ability to induce apoptosis and cell death in leukemic cells grown in the absence or presence of bone marrow stroma. Enhanced cytotoxic effects were present in all combination treatments with venetoclax in AML cell lines and AML patient samples. Elevated in vitro efficacies were observed for the combination treatment of venetoclax with A1331852, S63845 and bimiralisib, with differing response markers for each combination. For the venetoclax and bimiralisib combination treatment, responders were enriched for  The combination of venetoclax and the HMA azacitidine results in a remission rate of approximately 70% [With a median age of 68 years at diagnosis, acute myeloid leukemia (AML) is predominantly a disease of the elderly. The majority of these patients are not eligible for intensive chemotherapy with curative intent, and the standard of care has been treatment with hypomethylating agents (HMA) ,2. The ately 70% . HoweverNPM1, DNMT3A, or IDH2 mutations, while resistance was associated with mutations in FLT3, RAS, or TP53 genes [TET2, IDH1, and IDH2 mutations, while non-responders were associated with FLT3 and RAS mutations [RUNX1 and SRSF2 mutations may also be associated with venetoclax response [High response rates and durable remissions to venetoclax treatment were found to be associated with 53 genes ,5,6. In utations . RUNX1 aresponse ,9. Differesponse .RAS-mutated AML [FLT3- or TET2-mutated AML [Hematological cells of various origins, including AML, exhibit specific dependencies on either BCL-2, BCL-XL, or MCL-1 for survival ,12. Thisated AML , combinaated AML . In vivoated AML , and a pated AML , while vated AML ,21.FLT3 gene mutations lead to abnormal activation of the pathway in AML [In myeloid cells, FLT3 is a growth factor receptor signaling via PI3K-AKT-mTOR, and y in AML ,23. Abouy in AML . Pan PI3y in AML . PQR309 y in AML . Bimiraly in AML  and in ay in AML . Unesbuly in AML . The comy in AML . Chemothy in AML .In the current study, the BCL-XL inhibitor A1331852, the MCL1-inhibitor S63845, the PI3K inhibitor bimiralisib (PQR309), the BMI-1 inhibitor unesbulin (PTC596), the MEK-inhibitor trametinib (GSK1120212), and the STAT3 inhibitor C-188-9 were assessed as single agents and in combination with venetoclax, for their ability to induce apoptosis and cell death in leukemic cells grown in the absence or presence of bone marrow stroma. Several combinations were found to be effective in vitro: venetoclax and S63845, venetoclax and A1331825, and venetoclax and PQR308.FLT3-ITD and FLT3 wild type, NPM1 mutant and wild type, as well as TP53 wild type, mutant, hemizygous, and null cells , trametinib (p = 0.003), S63845 (p = 0.005), A1331825 (p = 0.01), PTC596 (p = 0.01), or C-188-9 (p = 0.04), Combination indexes were calculated according to Chou Talalay [Cell viability was determined in seven AML cell lines and one BCL2-driven DLBCL cell line treated with increasing dosages of single compounds and in combination using a variety of targeted therapies including the BCL-XL inhibitor A1331825, the PI3K inhibitor bimiralisib (PCR309), the STAT3 inhibitor C-188-9, the BMI-1 inhibitor PTC596, the MCL1 inhibitor S63845, and the MEK inhibitor trametinib. Drug concentrations in the combination studies were chosen to correspond to minimally effective concentrations in single compound assays determined in initial titration. Five AML cell lines and OCI-Ly1 were susceptible to 100 nM venetoclax and multiple combination treatments, while PL-21 and MOLM-16 cells were resistant to 100 nM venetoclax and most combination treatments, with the exception of venetoclax and A1331825 . Across To investigate the efficacy of venetoclax combination treatments in the bone marrow environment, cell viability was determined in the susceptible AML cell lines MOLM-13, ML-2, SKM-1, and OCI-AML3, grown in the absence or presence of bone marrow stroma cells. Stroma cells secrete granulocyte and macrophage colony-stimulating factors  and a variety of cytokines, which can induce STAT signaling in leukemic cells ,40,41,42Effects of venetoclax combination treatments on cell cycle and apoptosis were studied in the susceptible AML cell lines MOLM-13, ML-2, SKM-1, and OCI-AML3. Treatment with bimiralisib and venetoclax lead to the induction of apoptosis, cell cycle arrest, and cell death in MOLM-13 cells , as wellAfter initial studies in AML cell lines, the treatment combinations of venetoclax with A1331825, S63845, bimiralisib, trametinib, C-188-9, or PTC596 were applied to patient-derived mononuclear cells isolated from peripheral blood (PBMC) or bone marrow (BMMC) . A totalIDH2 mutation, as well as elevated blast cell percentage in peripheral blood or bone marrow associated with response, presence of PTPN11, TET2 or ASXL1 mutation indicated lack of response, while FLT3, NPM1, RUNX1, and TP53 status as well as CD34 levels were not associated with treatment response  family and the activation of intracellular signaling pathways were the major factors leading to resistance to venetoclax ,5,7. AccIn this study, we describe a panel of venetoclax combination treatments with enhanced cytotoxic effects on AML cells grown in the absence or presence of bone marrow stroma, including the BCL-XL inhibitor A133825, the MCL1 inhibitor S63845, the BMI1 inhibitor PTC596, the dual PI3K-mTOR inhibitor bimiralisib, the STAT3 inhibitor C-188-9, and the MEK inhibitor trametinib. The in vitro IC50 concentrations of the tested inhibitors were determined to be in the range of physiologically relevant concentrations for all compounds, except C-188-9, where in vitro IC50 levels exceeded the plasma concentrations present in PDX mice. AML cells grown in the presence of bone marrow stroma were generally less affected by the combination treatments than AML cells grown in the absence of stroma, indicating a protective effect of the bone marrow environment on AML cells. MOLM-13 cells were protected toward venetoclax and combination treatments when grown on stroma, ML-2 cells were protected toward trametinib, OCI-AML3 cells were protected toward all venetoclax combination treatments, and SKM-1 cells were more susceptible to venetoclax and A1331825, and protected toward bimiralisib and trametinib, when grown on stroma. SKM-1 cells have been found to be more susceptible to venetoclax and to the STAT5 inhibitor AC-4-130 when grown on stroma . A cell-IDH2, NPM1, FLT3, DNMT3A, PTPN11, ASXL1, TET2, KRAS, RUNX1, and TP53 genes have been described as response markers to venetoclax treatment [IDH2 mutation was the single most significant and consistent biomarker associated with response to venetoclax and multiple venetoclax combination treatments, while TP53 mutation was not associated with response. Notably, the TP53 double mutant cell line OCI-Ly1 was most sensitive to venetoclax, while the TP53 double mutant cell line MOLM-16 was resistant.To validate the findings in a translational setting, venetoclax combination treatments were applied to mononuclear cells isolated from the peripheral blood or bone marrow of primary AML patients. The addition of the BCL-XL inhibitor A133825, the MCL1 inhibitor S63845 or the PI3K inhibitor bimiralisib to venetoclax induced substantial reductions of cell viability in 50–70% of the tested AML samples, while the addition of the MEK inhibitor trametinib, the BMI-1 inhibitor PTC596 or the STAT3 inhibitor C-188-9 to venetoclax was less effective, with substantial reductions of cell viability in 20–40% of tested AML samples. In order to define the patient subgroups who may profit from novel targeted combination therapies, potential response markers were deduced from the correlation analysis of cell viabilities grouped according to diagnostic parameters, including gene mutation status of prevalent tumor suppressors and oncogenes, peripheral blood and bone marrow blast cell percentage, and levels of CD34 positive cells. A significant association between venetoclax response and elevated blast cell percentage has been previously described , and wasFLT3 mutations were associated with response to venetoclax and bimiralisib, but not to venetoclax monotherapy or in combination with A1331825 or S63845, indicating that FLT3-mutated cells may be specifically susceptible to the combined inhibition of BCL2, PI3K, and mTOR. The TET2 gene mutation has been associated with response to venetoclax and HMA combination treatment [TET2 mutation apparently associated with resistance to venetoclax in combination with A1331825 or bimiralisib, possibly due to presence of concurrent mutations in KRAS, PTPN11, or TP53 genes in the primary AML samples. PTPN11 mutations were associated with lack of response to venetoclax and A1331825 or bimiralisib, but not to venetoclax and S63845, indicating differential target cell specificity and differential mechanisms of action for S63845 in combination with venetoclax. Activating mutations of the SHP2 protein, encoded by the PTPN11 gene, leads to hyper-activation of the downstream RAS-MAPK signaling pathway and confer resistance to venetoclax and multiple venetoclax combinations. The combination of venetoclax and the MCL1 inhibitor AZD5991 was proposed to overcome this resistance [PTPN11 status. ASXL1 mutations have been associated with distinct epigenomic alterations that lead to sensitivity to venetoclax and azacytidine [ASXL1 mutation apparently associated with resistance to venetoclax in combination with A1331825, but not in combination with S63845 or bimiralisib, possibly due to the presence of concurrent mutations in KRAS, PTPN11, RUNX1, or TP53 genes in the primary AML samples. Further studies in larger cohorts may be required to validate the relevance of TET2, TP53, and ASXL1 mutation status as response markers to venetoclax combination treatments. Additional biomarkers of response to venetoclax combination treatments may arise from studies on expression levels of pro-apoptotic BCL-2 family proteins and anti-apoptotic proteins  as well as the components of the upstream signaling pathways in larger cohorts.The mutation status of other genes was relevant to response, however, with differential indicators in specific venetoclax combination treatments. sistance . In accocytidine . In our IDH2-mutated AML in the absence of PTPN11 mutations, while the combination of venetoclax and S63845 may be effective in AML in the presence of PTPN11 mutations. The combination treatment of venetoclax and bimiralisib may be effective in AML with IDH2, NPM1, and FLT3 mutations. We propose the combination of PI3K/mTOR dual pathway inhibition with bimiralisib and BCL2 inhibition with venetoclax as a candidate treatment in clinical trials for IDH2- or FLT3-mutated AML.In conclusion, different combinations of targeted therapies emerge that are suitable in the treatment of specific subsets of AML patients. In this study, elevated in vitro efficacies were detected in the combination treatments of venetoclax with BH3 mimetics A1331852 and S63845 and the dual PI3K-mTOR inhibitor bimiralisib, in AML with elevated blast cell percentage, at drug concentrations that can be reached in vivo in the plasma, with different response markers in each combination. The combination treatment of venetoclax and A1331825 may be effective in the treatment of FLT3, NPM1, TP53, and conventional karyotype analysis of at least 20 metaphases were performed in all samples. In addition, all samples were analyzed by NGS sequencing of the myeloid panel genes. The genes tested within the NGS panel can be categorized into several major functional categories, including the spliceosome , epigenetic modifiers , cohesions , transcription factors , signaling molecules , and chromatin modifiers (EZH2 and ASXL1).Mononuclear cells of AML patients diagnosed and treated at the University Hospital, Bern, Switzerland, between 2018 and 2022, were included in this study. Informed consent from all patients was obtained according to the Declaration of Helsinki, and the studies were approved by decisions of the local ethics committee of Bern, Switzerland, decision number 221/15. Peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) were collected at the time of diagnosis before initiation of treatment. The AML cells were analyzed at the central hematology laboratory of the University Hospital Bern according to state of the art techniques . Mutatio2. Human bone marrow stroma cell line HS-5 (ATCC® CRL-11882™) was supplied by the American tissue culture collection . HS-5 cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  in standard tissue culture flasks . HS-5 cells secrete granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), macrophage-CSF (M-CSF), Kit ligand (KL), macrophage-inhibitory protein-1 alpha, interleukin-1 alpha , IL-1beta, IL-1RA, IL-6, IL-8, IL-11, and leukemia inhibitory factor (LIF) [Human AML cells lines OCI-AML3 , MOLM-13 , FLT3-ITD, and TP53wt), MOLM-16 , ML-2 , FLT3wt, TP53mut) and HL-60  as well as B-cell lymphoma cell line OCI-Ly1 , PTEN del, TP53 mut) were supplied by the German Collection of Micro-organisms and Cell Cultures . AML cells were grown in RPMI-1640 medium , OCI-Ly1 in Iscove’s modified Dulbecco medium , supplemented with 20% fetal bovine serum , in tissue-culture flasks  in a standard cell culture incubator at 37 °C with 5% COor (LIF) ,42. For 5/mL on tissue culture plates suspension 96 well , and treated with targeted compounds. For assays with patient-derived mononuclear cells, the cells were cultured for 24 h prior to treatment. The BMI1 inhibitor PTC596 (HY-112041), the MCL1 inhibitor S63845 (HY-100741), the MEK inhibitor trametinib (HY-10999), the STAT3 inhibitor C-188-9 (HY-112288) and the PI3K inhibitor PQR309 (HY-12868) were purchased at MedChemExpress . PQR309  is a novel brain-penetrant dual PI3K/mTOR inhibitor with in vitro and in vivo anti-lymphoma activity as single agent and in combination. A stock solution of Venetoclax was prepared by dissolving a tablet in DMSO . Cell viability was determined 20 h after the start of treatment using the MTT-based cell proliferation kit I . This time point was selected because the cellular responses were effectual for the calculation of combination indexes after 20 h of treatment with two compounds in leukemic cells. For AML cell lines, four independent assays  with four measurements  per dosage were performed. For hematological patient samples, two independent assays with three technical replicates per dosage were performed. For the calculation of combination indexes, two dosages of venetoclax and two dosages of the other compounds were applied alone and in combination. Combination indexes were calculated on Compusyn software . Data are depicted as scatter plots with median values and SD. In grouped analysis, significance of differences in median values was calculated by Mann–Whitney test.For assays with AML cell lines, cells were plated at a density of 5 × 10Imaging cytometry was carried out on the NC-3000 cell analyzer  with reagents supplied by ChemoMetec. To determine the induction of cell death apoptotic cells were stained with AnnexinV-CF488A conjugate  in AnnexinV buffer and Hoechst 33,342 (10 μg/mL) for 15 min at 37 °C, followed by several washes. Propidium iodide was added shortly before imaging. For cell cycle analysis, cells were incubated in lysis buffer with DAPI (10 μg/mL) for 5 min at 37 °C before imaging on the NC-3000 cell analyzer.


Acute myeloid leukemia (AML) is a hematological malignancy commonly found in adult patients. Low overall survival and resistance to therapy are the main issues in AML. The first line of treatment for AML chemotherapy is the induction phase, namely, the phase to induce remission by administering a combination of daunorubicin (DNR) for three days followed by administration of cytarabine (Ara-C) with continuous infusion for seven days, which is referred to as "3 + 7." Such induction therapy has been the standard therapy for AML for the last four decades. This review article is made to discuss daunorubicin and cytarabine from their chemical structure, pharmacodynamics, pharmacokinetics, and mechanisms of resistance in AML. Acute myeloid leukemia (AML) is a hematological malignancy characterized by blockade in the differentiation process of hematopoietic progenitor cells of myeloid series and rapid proliferation of immature myeloid cells, resulting in the accumulation of blast cells in the bone marrow . The con2 and up to 3000 mg/m2 body surface area) or allogeneic hematopoietic stem cell transplant depending on cytogenetic and molecular classification [Standard chemotherapy as the first line of treatment for AML is induction therapy by giving daunorubicin (DNR) or idarubicin (IDA) combined with cytosine arabinoside . Following induction therapy, patients are often given consolidation therapy, generally conducted by giving high dose cytarabine  for seven days, together with either idarubicin (12 mg/m2 for three days) or daunorubicin (60-90 mg/m2 for three days) [Many clinical guidelines also still recommend chemotherapies as the mainstay treatment with occasional combination with targeted therapies and/or immunotherapies based on molecular analysis of cancer. For AML, induction chemotherapy consisting of cytarabine and anthracycline is recommended by many clinical guidelines and the most used anthracycline is daunorubicin with a dose of 45 to 60 mg/mree days ,10. The ee days) . GuideliThe chemical structure of daunorubicin and cytarabineStreptomyces peucetiusvar. caesius [Daunorubicin (DNR) is an antineoplastic agent of the anthracycline class of antibiotics, derived from the mutant isolate  caesius . These a caesius . Daunoru caesius . The str caesius .Cytarabine with another name cytosine arabinoside or Ara-C is an antineoplastic agent in the form of an analog of a pyrimidine nucleoside . CytarabPharmacodynamics and pharmacokinetics of daunorubicinThere are several mechanisms of daunorubicin as an antineoplastic agent. First, daunorubicin acts as a DNA intercalating agent ,16. The The third mechanism is the formation of free radicals by daunorubicin. Free radicals are formed from the reduction of the quinone group in daunorubicin by the cytochrome P450 reductase enzyme. The reduction of daunorubicin causes the addition of one electron. This electron quickly binds with oxygen to produce superoxide anion, which then produces hydrogen peroxide (H2O2). An increase in excess levels of reactive oxygen species (ROS) causes damage to important components in cells, causing cell death ,19.Daunorubicin is given by intravenous injection, but plasma levels of daunorubicin decrease rapidly due to tissue absorption. Thus, daunorubicin will be distributed in various organs of the body, especially in the kidneys, liver, lungs, and heart, then it will enter into cell components, especially nucleic acids. Additionally, it can pass through the bloodstream to the placenta . DaunoruPharmacodynamics and pharmacokinetics of cytarabineCytarabine enters cells through nucleoside transport proteins from the solute carrier (SLC) family, namely, human equilibrative nucleoside transporter (hENT) 1 and 2, and hCNT3 (human concentrative nucleoside transporter 3). In addition, cytarabine uptake is also regulated by the transmembrane proteins ABCC10 (MRP7) and ABCC11 (MRP8)  into cytarabine monophosphate in cells. Furthermore, cytarabine is phosphorylated into cytarabine diphosphate and cytarabine triphosphate by the enzymes deoxycytidine monophosphate kinase (dCMK) and nucleoside diphosphate kinase (NDK). The form of cytarabine triphosphate (Ara-CTP) is an active form that can inhibit the action of DNA polymerase. Ara-CTP competes with deoxycytidine triphosphate (dCTP) in the process of DNA transcription. After cooperating with the DNA strand, Ara-CTP acts as a terminator chain thereby inhibiting DNA elongation, DNA synthesis, and DNA repair. The consequence of this is the blockade of the cell cycle from the G1 phase to the S phase, causing cell death ,20.After administration of cytarabine intravenously, distribution occurs in two phases: the first phase with a half-life of about 10 minutes, followed by a second phase with a half-life of one to three hours. After the distribution phase, more than 80% of the drug is in the plasma in an inactive form (1-βD-arabinofuranosyluracil). Within 24 hours, about 80% of the drug can be found in the urine in the non-toxic form of 1-βD-arabinofuranosyluracil (the inactive form of cytarabine) .Role of gene mutations in AML chemotherapy resistanceThe mechanism of AML cell resistance to chemotherapy can be caused by gene mutations. Among them are the FMS-like tyrosine kinase (FLT3) gene and DNA methyltransferase 3A (DNMT3A) gene mutations. The FLT3 gene is located on chromosome 13q12; it is a gene encoding the FLT3 tyrosine kinase receptor, which is expressed on the membrane of hematopoietic stem cells (CD34+) and other hematopoietic progenitor cells . FLT3 isFLT3 receptor activation occurs due to the presence of FLT3 ligands, but mutations that occur in the FLT3 gene can cause FLT3 receptor activation without the presence of ligands, resulting in uncontrolled cell proliferation, survival, and anti-apoptosis. A common mutation in the FLT3 gene is internal tandem duplication (ITD), with base insertions varying from three to >1,000 bases in exons 14 and 15 in the juxtamembrane domain region . The freFLT3-ITD mutations in AML are known to be frequently found in patients with normal cytogenetics and are associated with a poor prognosis, especially in patients aged <60 years, with a low incidence of relapse (relapse) and overall survival (OS) ,25. In cFLT3-ITD mutations in leukemic cells have been shown to cause resistance to cytarabine ,28. Jin According to several studies, DNMT3A is one of the most commonly mutated genes in AML ,30. HoweBoth FLT3-ITD and DNMT3A are common mutations encountered in AML. FLT3-ITD is routinely checked in developed countries since it is used as risk stratification in clinical guidelines to determine treatments ,10. OtheMechanism of AML resistance to daunorubicinThe mechanism of AML resistance to daunorubicin can occur in several ways, namely, increasing the expression of the drug efflux transporter ATP-binding cassette (ABC), decreasing the activity of the target enzyme (DNA topoisomerase II), and the mechanism of resistance through the apoptotic pathway . The druStopping the activity of the DNA topoisomerase II enzyme is the target of therapy with a daunorubicin regimen. Therefore, reduced activity of the DNA topoisomerase II enzyme can cause AML cell resistance to daunorubicin. It was found that the activity of the DNA topoisomerase II enzyme was reduced in AML cells that were resistant to anthracyclines compared to AML cells that were sensitive to anthracyclines . The mecMechanism of AML resistance to cytarabineA preliminary study of the pathways that play a role in the mechanism of transport, activation, and degradation of cytarabine in cells helps us understand the mechanism of resistance of AML cells to cytarabine. The first mechanism of resistance is caused by a deficiency of cellular uptake of cytarabine in cells ,37. ThisThe second mechanism of AML cell resistance to cytarabine is the overexpression of enzymes that inactivate cytarabine such as cytidine deaminase (CDA), CMPD, and NT5C2 . MeanwhiThe third mechanism is the formation of free radicals by daunorubicin. Free radicals are formed from the reduction of the quinone group in daunorubicin by the cytochrome P450 reductase enzyme. The reduction of daunorubicin causes the addition of one electron. This electron quickly binds with oxygen to produce a superoxide anion, which then produces H2O2. An increase in excess levels of ROS causes damage to important components in cells, causing cell death ,19.Clinical studies on chemotherapies2 versus 60 mg/m2 for AML induction [Currently, chemotherapies remain the main treatment of AML, especially in developing countries. Thus, many trials on chemotherapies are still ongoing such as trials involved in evaluating induction doses of daunorubicin or idarubicin. For example, the trial by Burnett et al. compared a daunorubicin dose of 90 mg/mnduction . The resnduction .It is actually recommended to combine chemotherapies with other treatments nowadays to prevent relapse from the minimal residual disease . The comIn AML with an FLT3-negative profile, gemtuzumab ozogamicin (GO) is combined with standard chemotherapies. A meta-analysis by Hills et al. showed that the addition of GO increases OS . HoweverRelapsed and refractory AML (R/R AML) is a major issue today for clinicians. Up to 40% of AML patients are unable to achieve complete remission even with a combination of intensive chemotherapy and targeted therapies ,46. ThesImprovement in complete remission outcome is needed for R/R AML patients. One of the pathways that can be done is by further research into new chemotherapies. A trial by Ravandi et al. in refractory/early relapsed AML patients aged ≥60 years old compared vosaroxin plus cytarabine (versus placebo plus cytarabine) in 711 patients . VoreloxThe addition of hypomethylating (HMA) therapy with FLAG-Ida can also be used for a salvage regimen. Decitabine combined with aclarubicin and cytarabine (DAC) showed better efficacy than DAC alone. In some studies -52, noveChemotherapies whether used as a single therapy or in combination with other therapies remain the mainstay treatment for AML currently. However, AML cells have several mechanisms such as drug efflux transporter, enzyme inactivation, gene mutations, and alteration of signaling pathways that can cause resistance to chemotherapies. The most common gene mutations in AML include FLT3-ITD and DNMT3A mutations. Additional studies should be conducted to improve the effectiveness of chemotherapy from pharmacological and pharmacokinetic perspectives, as it may improve the medical outcomes of AML patients.


Multivariate stepwise regression found the best predictive model included AML-BS, AML length to aortic valve diameter ratio, AML length to LV width ratio, and midventricular septal thickness metrics (AUC 0.84). Excluding AML-BS, metrics grouped according to septal hypertrophy, LV geometry, and AML anatomy each had similar associations with LVOTO , significantly less than their combination . Anatomical metrics derived from a standard three-chamber CMR cine acquisition can be used to highlight risk of LVOTO, and suggest further investigation if necessary. A combination of geometric factors is required to provide the best risk prediction.Left ventricular outflow tract obstruction (LVOTO) is common in hypertrophic cardiomyopathy (HCM), but relationships between anatomical metrics and obstruction are poorly understood. We aimed to develop machine learning methods to evaluate LVOTO in HCM patients and quantify relationships between anatomical metrics and obstruction. This retrospective analysis of 1905 participants of the HCM Registry quantified 11 anatomical metrics derived from 14 landmarks automatically detected on the three-chamber long axis cine CMR images. Linear and logistic regression was used to quantify strengths of relationships with the presence of LVOTO (defined by resting Doppler pressure drop of > 30 mmHg), using the area under the receiver operating characteristic (AUC). Intraclass correlation coefficients between the network predictions and three independent observers showed similar agreement to that between observers. The distance from anterior mitral valve leaflet tip to basal septum (AML-BS) was most highly correlated with Doppler pressure drop (RThe online version contains supplementary material available at 10.1007/s10554-022-02724-7. Hypertrophic cardiomyopathy (HCM) is a highly complex genetic disorder, characterised by the presence of left ventricular hypertrophy (LVH) without ventricular dilatation, that cannot be otherwise explained by abnormal loading. It is typically inherited as an autosomal dominant trait  with an gradient”) ≥ 30 mmHg is indicative of LVOTO [Typically, Doppler transthoracic echocardiography is used for LVOTO evaluation, where a resting peak pressure drop  LV hypertrophy, including basal septal thickness, (ii) mitral valve interplay, including its systolic anterior motion (SAM) and and/or elongation of the anterior mitral leaflet (AML), and (iii) LV chamber morphology, including anterior displacement of the papillary muscles –18.In this study we developed deep learning algorithms to provide fast and accurate detection of anatomical landmarks in 3CH long-axis CMR images to assess the extent to which landmark distances can be reliably estimated. We sought to evaluate the relationships between landmark dimensions and Doppler echocardiographic pressure drop in a large cohort of HCM patients drawn from the HCM Registry study , 20. We Figure The HCM Registry Study is a prospective registry consisting of 2750 patients recruited from 44 European and North American sites. Exclusion and inclusion criteria have been detailed previously , 20. BriCMR image acquisition was performed on General Electric, Philips and Siemens scanners, at either 1.5 or 3.0 Tesla, using multi-channel, phased-array chest coils and electrocardiographic gating. Short-axis cine steady-state free precession (SSFP) imaging was performed in slices of 8 mm thickness to cover the entire heart followed by long axis SSFP cine imaging. Cine SSFP parameters comprised TR/TE 3.1/1.2 ms, in-plane resolution of 2–2.5 mm and temporal resolution of 40–50 ms. Parasternal long axis 3CH view CMR cine images were considered in this study.Echocardiographic data was obtained from routine clinical care closest to the CMR acquisitions , and included continuous-wave Doppler acquisitions to determine the LVOT pressure drop, which was estimated through the simplified Bernoulli formulation . A restiA standard operating procedure (SOP) was developed to standardise the manual labelling of anatomical landmarks in CMR images using ITK-Snap version 3.8.0 . Five frIn total, 14 landmarks were identified, as summarised in Fig. Selected anatomical metrics were grouped according to three mechanisms identified in the literature: (i) septal hypertrophy, (ii) AML anatomy, and (iii) LV cavity morphology. Septal hypertrophy was captured by thickness at the base and at mid-ventricle. AML anatomy included AML length, length to LV width ratio, and AML length to aortic valve diameter ratio. Since the distance from AML tip to the basal septum quantifies the obstruction gap directly, this was not included in the individual mechanism groupings. LV cavity morphology was captured by LV length, and LV width at mid-ventricle . Also, the distance between the anterior papillary muscle and the septum was included in parallel with the LV width. Finally, the basal diameter was captured by two landmarks at the PML hinge and the basal septum.Overall, 192 randomly selected cases were annotated with all 14 landmarks by three observers , taking 30–35 min per case on average.Details of the network design and training scheme are given in the Supplementary Material. Briefly, landmarks were detected in two stages. Firstly, a region-of-interest (ROI) network was trained to detect four landmarks . Typically, ICC < 0.4 is interpreted as poor agreement, 0.4–0.59 fair, 0.6–0.74 good, and 0.75–1.0 excellent . Analysi2 coefficient of determination served as a measure to explain the proportion of variance in the LVOT pressure drops that could be explained by the different metrics.Univariate linear regression analysis was performed to quantify the association between each individual metric and the LVOT Doppler pressure drop. The RWe then evaluated the ability of all anatomical distances to predict the obstruction. Stepwise multivariate linear regression was performed using the Statistical Package for Social Sciences (SPSS) for Windows . The SPSS stepwise regression model started with zero predictors and the most influential predictors of LVOTO were added sequentially with a significance of p < 0.001 and removed if p > 0.100. Then a logistic regression model (Model 1) was developed using the variables identified by the stepwise regression, with a tenfold cross validation method to evaluate the goodness of fit.Furthermore, to investigate the mechanisms of LVOTO, selected anatomical metrics were grouped together according to three possible mechanisms: septal wall thickness, AML anatomy and LV cavity morphology. Accordingly, four additional logistic regression models were evaluated: Model 2 with all anatomical metrics excluding the AML tip to basal septum distance, and then one for each individual group.Receiver Operating Characteristic (ROC) curve analysis was performed and the Area Under the Curve  was computed which served as a baseline to compare the relative strength of association with LVOTO. The fast DeLong’s algorithm was used to estimate the AUC confidence intervals at 95%  and optiThe study population characteristics are summarised in Table Intraclass correlation coefficients (ICC) for intra- and inter-observer errors are shown in Fig. The agreement between the network prediction and observers is shown in Fig. Average anatomical metrics are compared between oHCM and nHCM groups in the Supplementary Material Table S3. Taking a conservative Bonferroni correction for 55 multiple comparisons (p < 9.0E − 04), the oHCM group showed greater basal septal thickness, greater midventricular septal thickness during systole, smaller AML tip to basal septum distance, longer LV length, across all frames measured. AML length and AML length/aortic valve diameter ratio were larger at ED, and aortic valve diameter was smaller during systole, in the oHCM group.The univariate regression analysis for prediction of LVOT pressure drop as a continuous variable  showed significant relationships (p < 9.0E − 04) with basal septal thickness, midventricular septal thickness, AML tip to basal septum, Papillary muscle to midventricular septum, LV length, aortic valve diameter, basal diameter and AML length/LV width ratio at mid-systole and end-diastole. In addition, AML length and LV width was significant at mid-systole but not end-diastole.The AUC for univariate predictors showed the highest AUC for AML tip to basal septum distance . Next highest was basal septal thickness  and LV length .Results of AUC for multivariate models are shown in Supplementary Table S6. In Model 1, all metrics in all frames were considered in the multivariate stepwise regression. The 8 significant anatomical metrics found were entered into the logistic regression model including: AML tip to basal septum in mid-systole, mid-systole − 2 and mid-systole + 2 frames; LV length in mid-systole + 2; basal diameter in mid-systole + 2; AML length to aortic valve diameter ratio in end-diastole; AML length to LV width ratio in mid-systole + 2; midventricular septal thickness in end-systole.In model 2, all metrics in all frames were considered in multivariate stepwise regression, excluding AML tip to basal septum distance in all frames as it directly impacts on pressure drop. The 9 significant anatomical metrics found were entered into the logistic regression model including: basal septal thickness in end-diastole; AML length to aortic valve diameter ratio in end-diastole; AML length to LV width ratio in mid-systole + 2; LV width in mid-systole − 2; LV length in mid-systole + 2; basal diameter in mid-systole + 2; papillary muscle to IVS in end-diastole and mid-systole − 2; aortic valve diameter in mid-systole − 2.In order to compare different possible mechanisms of LVOTO, the anatomical metrics were grouped into three main types: (a) septal hypertrophy , (b) AML anatomy ; (c) cavity morphology . Anatomical metrics associated with each of these mechanisms were evaluated in separate logistical regression models and shown in Fig. Figure www.cardiacatlas.org. The strongest relationships with Doppler pressure drop were exhibited by the mid-systolic AML to basal septum distance, as expected since this distance is highly related to the vena contracta of the outflow tract and hence the pressure drop estimated from the Bernoulli method. However, other metrics grouped according to the geometric mechanisms of septal hypertrophy, LV chamber geometry and AML anatomy were also highly associated, to a similar extent, with the presence of obstruction. In particular, basal septal thickness and LV length were strong predictors of obstruction. Also, a combination of mechanisms gave significantly stronger associations with Doppler pressure drop than each of the septal hypertrophy, LV cavity or AML leaflet anatomy mechanisms alone, indicating that each of these mechanisms contribute independent information.A better understanding of the mechanisms underlying obstruction in HCM will enable better criteria to plan and monitor therapies that aim to decrease the obstruction. Here, we defined and automatically quantified anatomical metrics related to LVOTO from 3CH CMR images. The methods can be applied automatically to standard acquisitions and are available from The distances measured between landmarks were consistent across different observers in 9 out of 11 metrics. All 4 metrics of the LV bulk anatomy  reported ICC values > 0.90 that are considered to be of excellent reliability . The metThe main reason for the low reliability of valve metrics is attributed to the relatively poor definition of the anatomy of the valve leaflet in the image. The resultant turbulent flow and movement of the AML can cause a distorted appearance on CMR. This is often exacerbated by an elongated AML, which is also a common morphological finding in HCM that contributes to SAM .A more detailed analysis of the individual landmarks in CMR images revealed a reasonable consistency between the three independent observers with an average absolute error of 2.7 mm ± 0.6 mm. There were nevertheless differences across them: landmark 12, the apex of the heart, exhibited the least variation (1.6 mm), whereas landmark 10, at the lateral wall as one of the points to define LV width, exhibited the greatest variation (4.1 mm). These differences are caused by the room for subjectivity when identifying the landmarks defined in the SOP: whereas the apex is typically clearly visualised on 3-chamber long-axis cardiac CMR, and thus is an unambiguous landmark, the landmark 10 is not uniquely defined along the myocardium . Similar difficulties are encountered with the basal septal thickness labels. Despite this ambiguity, the distances between pairs of landmarks reported good to excellent reliability: thicknesses and diameters are not heavily affected by their manual assessment at a variable location along the length of anatomical structures.The performance of the landmark detection network is instrumental to this study, as it allows to study morphological determinants of LVOTO in a large cohort in a systematic manner. The machine reproduced the human performance in 5 out of 9 metrics: the excellent reliability of 2 out of 4 LV bulk metrics (LV length and PM to IVS distance) and the good reliability of 3 out of 5 metrics close to the LVOT . One reason for the robustness of the LV length measurement is that it was calculated from three separate landmarks: the more data that is provided to the model, the better its performance will be .By contrast, machine performance showed a sharp decline in two metrics  and a small decline in LV width and midventricular septal thickness. This is likely due to the difficulty in identifying the AML length in CMR 3CH images.2 value of 0.189, p < 0.001).In a 3CH view the anatomy of the LVOT obstruction can be assessed by the AML to basal septum distance. In our study this is the distance that reported to be the most powerful independent predictor of LVOTO (AUC of 0.796 at mid-systole) and to negatively correlate between the with the degree of obstruction . Clinically, and in previous studies, basal septal thickness has been known to be among the best predictors for LVOTO since obstruction tends to be more severe in patients with focal basal septal hypertrophy [A second mechanistic factor in the LVOTO is the basal septal thickness, that is reported to be a significant predictor and positively correlated with the obstruction degree . A three-dimensional analysis of LV shape may offer additional insights into anatomical features influencing obstruction.Although Doppler echo pressure drop is the preferred method for identification of LVOTO, many patients have poor characterization due to inadequate windows and limitations in the Bernoulli estimation. A 3CH CMR evaluation, able to identify patients with higher likelihood of LVOTO from anatomical metrics, may become a valid alternative providing valuable insights about the mechanisms causing the obstruction.In general, among metrics with significant differences during systole, the mid-systolic frame was representative of the differences so could be used if a single frame metric is required. We computed two additional logistic regression models, Model 3 and Model 4, which were similar to Model 1 and Model 2 respectively, except that metrics at frames ± 2 from mid-systole were ignored. Model 1 and Model 3 were not significantly different, and also Model 2 and Model 4 were not significantly different.Other mechanisms of LVOTO include regional wall motion abnormalities, papillary muscle function, presence of apical HCM, malcoaptation of the mitral valve leaflets leading to mitral regurgitation, or conduction abnormalities. The landmarks captured were limited to the 3CH view, which despite being ideal in terms of providing a simple single view prediction of oHCM, meant that certain information could not be captured, including the aortoseptal angle which has been shown to correlate with LVOTO . MoreoveWhile the 3-chamber LAX view was ideal for visualising the AML and septal hypertrophy, it often provided poor visualisation of the anterior papillary muscle. Incorporating the short axis view of the heart into the study would provide better representation of the papillary muscle morphology, which is thought to play an important role in LVOTO .Phase contrast flow images were not investigated in this study. Also, the presence of flow artefacts in the CMR images often made the identification of the AML tip challenging. Flow artefacts commonly occur during systole due to fast and turbulent blood flow in the LVOT. This results in an increased signal phase shift, leading to intra-voxel phase dispersion and a loss of signal; hence the black appearance of the flow artefacts . Thus, tA further practical limitation of the study was that the findings were compared with resting LVOT pressure drops, obtained from Doppler TTE which were generally not measured on the same day as when the CMR acquisitions were performed, and with potential medical interventions between measurements. Due to the transient nature of LVOTO, the time interval between when the CMR and echocardiographic acquisitions were completed, as well as the pressure drop during physiological stress, could have significant implications on the predictive value of the distance measurements. Future work should investigate relationships between resting geometry and stress induced obstruction, using a more comprehensive analysis of 3D shape, which can be obtained from multiple cine views. Since appropriate echo and CMR data were only available for 1095 out of 2667 cases, selection bias cannot be ruled out.Finally, studies have identified SAM of the MV as a significant morphological determinant of LVOTO , which iwww.cardiacatlas.org3CH CMR cine slices, typically acquired in all CMR exams, can be used to obtain anatomical metrics which identify patients with higher risk of LVOTO, suggesting further investigation if necessary in patients with high scores. Furthermore, metrics grouped into septal hypertrophy, LV geometry, and AML anatomy mechanisms each show strong associations, with the combination providing significant improvement, indicating that LVOTO is related to a combination of geometric factors. Network code and executables, as well as protocols for annotations, are available from Supplementary file1 (MP4 88 KB)Supplementary file2 (MP4 110 KB)Supplementary file3 (DOCX 105 KB)Below is the link to the electronic supplementary material.


Rabbit Haemorrhagic Disease Virus (RHDV), which is a calicivirus, is used as a biocontrol agent to suppress European wild rabbit populations in Australia. The transmission of RHDV can be influenced by social interactions of rabbits; however, there is a paucity of this knowledge about juvenile rabbits and the roles they may play in the transmission of RHDV. We aimed to quantify the social interactions of juvenile (< 900 g) and adult (> 1200 g) rabbits in a locally abundant population in the Central Tablelands of New South Wales, Australia. Twenty-six juvenile and 16 adult rabbits were fitted with VHF proximity loggers to monitor intra- and inter-group pairings. Use of multiple warrens by these rabbits was investigated using VHF base stations at nine warrens and on foot with a hand-held Yagi antenna. Juvenile rabbits were strongly interconnected with both juveniles and adults within and outside their warren of capture, and almost all juveniles were well-connected to other individuals within their own social group. Inter-group pairings were infrequent and fleeting between adults. Both juvenile and adult rabbits used multiple warrens. However, visits to warrens outside their warren of capture, particularly those within 50 m, were more common and longer in duration in juveniles than in adults. The high connectivity of juveniles within and between warrens in close proximity increases potential pathogen exchange between warrens. Therefore, juvenile rabbits could be of greater importance in lagovirus transmission than adult rabbits. The strength of juvenile rabbit inter- and intra-group pairings, and their tendency to use multiple warrens, highlight their potential to act as ‘superspreaders’ of both infection and immunity for lagoviruses and other pathogens with similar lifecycles. Confirmation of this potential is required through examination of disease progress and rabbit age-related immune responses during outbreaks. Oryctolagus cuniculus) is a keystone species and recognised as endangered in its native range [Although the European wild rabbit  strains cause RHD in rabbits, and all strains are lagoviruses of the Caliciviridae family . The traIn response to the declining efficacy of myxoma virus in Australian rabbits, two variants of the biological control agent Rabbit Haemorrhagic Disease Virus 1 (RHDV1) were released nationally: the GI.1c variant in 1996 ; and the K5 variant  in 2017 ,15–17. IThe presence of the interacting variants complicates the epidemiology of RHD in Australia and the role that juvenile rabbits play in the transmission of RHDV and RHDV GI.4c is somewhat uncertain ,23,24. TDesmodus rotundus, [Procyon lotor, [Contacts between susceptible and infected animals are key drivers in pathogen transmission ,30. Sociotundus,  and raccn lotor, ).Recently, proximity loggers and VHF devices have been used to investigate pairing rates  for pathogen transmission between adult and sub-adult 900 g–1200 g) rabbits ,33,42, b00 g rabbTherefore, juveniles potentially play a more important role in pathogen transmission between warrens than adults and could be the key player in the transmission and persistence of lagoviruses in rabbit populations. Here, we aimed to determine rates of visitation to warren of capture and other warrens by juvenile and adult rabbits, to measure the frequency and duration of inter- and intra-group pairings among adult and juvenile rabbits, and to determine the likely relative importance of juvenile rabbits in lagovirus pathogen transmission between rabbit warrens.This study was conducted on a private property located between Orange and Cargo in the Central Tablelands of NSW , partially covered with poly shade-cloth, were deployed at burrows and warrens along the gully in the centre of the site . BecauseTrapping was carried out from late September to mid-December when breeding was occurring. Traps were set between 1500 hr and 1700 hr, Monday–Thursday, and checked the following morning at 0600 hr, Tuesday–Friday. Trapping was not conducted during forecasted poor weather conditions . Captured animals were removed from traps and secured in calico bags. All newly captured animals were fitted with a microchip  for identification then weighed. Suitable animals were fitted with proximity loggers (see below). All animals were released at their point of capture. It is possible that the warren of capture (WOC) for some rabbits was not their natal warren or common social group. For this reason, rabbit locations are referred to as their ‘WOC’, rather than ‘natal warren’ .VHF encounter proximity loggers  were used to gather information on individual’s social behaviour. In general, each logger emitted a unique ultra-high frequency signal while searching for the frequency of other loggers within a distance pre-set by researchers . When twPrior to deployment, we tested each logger for faults, set the pairing range at 30–50 cm (close proximity deemed to be a pairing between two rabbits), and set the separation time to 30 seconds. Due to concerns about battery life, an additional transmitter  with its frequency matched to the corresponding logger, was glued to the VHF proximity radio-loggers (Sellys® Quick Fix™ super glue). Mesh wings were glued to the ventral side of the logger to increase the surface area for attachment to the rabbits. Given the length of antennas for juvenile loggers (32 cm), antennas were curled to 3 cm diameter, then heat-shrunk into place to limit the chance of entanglement with obstructions in the field.NSW Local Land Services Act, 2016), and tissue samples were taken for a wider study investigating the serology of wild European rabbits. To recover loggers and extract the data, adults were either re-trapped and then humanely killed by stunning with a captive bolt followed by cervical dislocation . This ensured that the data collected would be from rabbits that were still in the juvenile life stage for the duration of the study, but that were large enough for the loggers to be < 5% of their body weight. The hair between the shoulder blades of juvenile rabbits was trimmed and shaved so that the proximity logger could be glued to the animal. Proximity loggers were then secured by folding the surrounding longer guard hairs over the mesh wings . Collarsthanasia ), or shoLoggers were fitted to 77% of captured adults and 36% of captured juveniles. The low rate for juveniles was because many were too small. Of the 42 VHF proximity loggers deployed, only two were recovered and re-used after cleaning with 2% hyperchlorite solution. Redeployed loggers were re-labelled with a decimal (e.g. 2.1 and 2.2) to distinguish between the two rabbits. We recovered 40 logging devices at the completion of the study (adult logger numbers 38 and 46 could not be found). All juvenile loggers were found, either on the surface or in warrens. Six loggers  did not provide data either due to logger failure or no interactions with other logging devices, or provided a series of broken one-second pairings greater than five minutes apart (see Data cleaning below).Base stations were used to record pairing information when rabbits come within a predefined distance of an area of interest. We deployed 12 base stations over nine warrens  were excluded from this display as they only occurred as ‘encounters’. Blank spaces are used to identify loggers that failed to record data, produced no pairings following data cleaning, recorded invalid pairings or were not recovered at the completion of the study (see text).Data from all base stations were cleaned according to rabbit transponder deployment and retrieval dates, as described for VHF proximity loggers. Base station numbers were replaced with warren number, and where two base stations were located on a warren, the data were merged with information on WOC.Rabbits were located using an extended three element folding handheld Yagi antenna and receiver , Monday–Friday at 05:30 and 16:00, and once more at a random time between 22:00 and 02:00 (to eliminate observer bias [ver bias ). This pi and j to denote any pair of loggers, total pairing time with logger j was extracted from the logger i record and total pairing time with logger i was extracted from the logger j record. The two totals were never in agreement due to the operational issues described earlier. Therefore, the larger of the two values was taken to represent total time in pairing for the observation period.Total pairing duration was calculated for each logger pair. Using labels 10 transformation. The log scale durations for each group were summarised by average and 95% confidence intervals  and the distribution of duration times within each group presented graphically . A formantervals  to enabligraph package [A network representation of pairing between logger pairs was constructed using the  package  in the R package  by using-1.One hundred individual rabbits were trapped in 297 capture events over 45 trapping nights. Of these rabbits, 76% were juvenile, 22% were adults, and 2% sub-adults were captured. Nine adults were recaptured at least once during trapping events and 64% of the juveniles were trapped twice or more times. Using the total number of individuals that we trapped as a minimum number known alive at the completion of trapping, a minimum density of rabbits across the study site was 16.8 rabbits haAfter data cleaning, 26,371 pairings were recorded across all individuals . However, most (59.8%) pairings were of 1 sec in duration. There were 10,499 J-J pairings of > 1 sec duration, but only 44 such A-J and 11 such A–A pairings).Pairings between juveniles were more common and of longer duration than A-J pairings or A-A pairings .Although there were 630 possible pairings within our sample of 36 rabbits, the total number of observed contact combinations was 79, comprising 56 J–J pairs, 20 A–J and three A–A.The network analysis indicates a possible eight social pairing groups existed in the study population , as the Juvenile rabbits, unlike adult rabbits, made strong connections with individuals from outside of their social group , with moMost juveniles from the same social group were connected and shared strong bonds . However, given that these warrens had two base stations deployed, contact data from these warrens were still collected. The base stations logged encounters with 37 rabbits . Base stations were associated with rabbit’s WOC and are referred to as inter- and intra-warren detections .Of the 24 juvenile rabbits detected by base stations, 15 visited one or more warren systems Figs . InteresTen adult rabbits were detected at multiple warrens , three were only detected at their WOC Figs  and one Locations collected with the hand-held VHF antenna confirmed that adults used larger areas than juveniles. Although most frequently located in their WOC, six juveniles were occasionally detected in warrens other than their WOC . For exath and 10th of November 2017 respectively), which may have caused their death. The third rabbit (adult logger number 32: 1300 g) tested negative to virus, and the cause of death could also not be determined. We suspected that three other rabbits were taken by predators: one juvenile logger (logger number 21) was found near a red fox (Vulpes vulpes) den under a blackberry (Rubus fruticosus) shrub and two adult collars (logger numbers 39 and 45) were found with fox bite marks and evidence of chewing. Two other rabbits (logger number 38 and 46) went missing during the study and were not recovered despite searches in and adjacent to the study location.Three rabbits were known to have died during the study. One juvenile rabbit (300 g) and one adult (1000 g) without loggers were confirmed, by an independent laboratory (via serology test), to have seropositive results to GI.2  , these iet al [Adult-juvenile pairings were more frequent and longer in duration than A–A pairings, and as juveniles can shed virus for many weeks after infection , these iet al . The lowIn our study, J-J inter-group pairings were longer and more numerous than A–J and A–A. These factors are likely sufficient for the expression of behaviours that enable the direct and indirect transmission of virus between social groups. The substantially longer J–J inter-group pairing time increases the likelihood of associated susceptible juveniles becoming infected. The length of time and type of transmission behaviours are also just as important in pathogen exchange within a pathogen network . TransmiDue to particularly high connectivity, some individuals could play a larger role in lagovirus transmission than others. Highly connected individuals, or ‘superspreaders’ ,56, haveJuvenile rabbits are likely also important for spreading acquired immunity. Like RHDV, RHDV GI.4c can be shed by juveniles up to seven days after infection, providing temporary immunity against RHDV1 . This fuPairings between members of the same group are also important for pathogen transmission ,35,47. JJuvenile rabbits are naturally resistant to RHDV1s up to about six weeks of age , but resMore than half of the juvenile rabbits used multiple warrens, confirming the potential of them spreading and shedding virus outside of their WOC. Juvenile rabbits can travel up to one and a half kilometres from their natal site ,33. ThisExploration by juvenile rabbits prior to dispersal provides them with information on resource availability and hierarchical structure of neighbouring groups ,32,48. ANot all individuals within a warren system use or have access to the entire warren . AlthougSurprisingly, most adult rabbits were detected by base stations at more than one warren. Previous studies using VHF tracking found that most adult rabbits remained within the confines of their social territories . HoweverSeveral mortalities were observed in our study, and both GI.2 and predation were identified as the causes of the mortality events. The outbreak of GI.2 could have affected the pairing frequency and duration of pairings. We possibly underestimated the frequency of pairings between rabbits, and potentially overestimated the duration of pairings, e.g. the 5 hr 51 min outlier pairing between an adult and a juvenile  could haFelis catus) [Because rabbits form a significant part of introduced predator diets  ), predats catus) . Furthers catus) . Althoug-1 [It is unknown what effects that lagovirus observed clinical signs ,34,43,59-1 ) and migAs lagoviruses are spread by both direct and indirect pairing, the high connectivity of juvenile rabbits may increase their potential in transmitting the pathogens across social groups. In particular, the strength of A–J inter-group pairings and juvenile inter-warren use, confirmed the potential for juveniles to act as superspreaders of lagoviruses ,55. ThisS1 Table(XLSX)Click here for additional data file.S2 TableCarlise-may-Aug-2020 excel conver.(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


To gain insights into the e-mentoring experience, needs of the stakeholders (mentors and mentees) challenges and strategies to overcome the challenges.Qualitative exploratory study was conducted in Islamic International Medical College, from February 2021 to July 2021. The study duration was six months. Six ‘Semi-structured interviews’ of mentors and two ‘focus group discussions’ with mentees were conducted. A purposeful sampling technique was employed to select the respondents. Data were audio taped and transcribed verbatim. After that analysis of data was done by inductive content analysis. Data were coded line by line. Open codes were combined to form categories, which were combined to form themes through abstraction.Data was analyzed by using Atlas.ti. After analyzing data from mentors and mentees, 21 open codes sorted into 15 categories and abstracted to from five major themes. Participants talked about the problems of online sessions like; connectivity issues, impaired interaction, nonspecific goals, unaware of MS Teams use. They suggested strategies to make these sessions more practical; like workshops for training, face to face sessions before online mode, blended approach, careful selection of mentors and mentees. All were satisfied with security and witnessed anonymity and privacy.Online mentoring can help students feel less lonely through social contact. E-mentoring provides flexibility to those who would usually deal with discrimination to being mentored because of their gender, ethnicity, disability or geographical location. This program was shifted from face to face to electronic mode during the pandemic. That’s why this study is planned to explore the new experience and address the challenges to suggest the strategies for improving the program. Non-probability purposive sampling was done. Students were contacted through class representatives and invited voluntarily to join.The study lasted six months, from February 2021 to July 2021. This study was conducted at “Islamic international Medical college, Rawalpindi”. IRB approval was taken from the Riphah University, , data richness and volume, research design requirements and sample size guidelines.11Data of FGDs and SSI were analyzed separately. The whole transcript of each FGD was taken as a separate unit of analysis. One of the authors read the transcript multiple times, along with listening to the audios. Mistakes were corrected. Next, coding of the transcript was done line by line by highlighting and adding the comments. Then all codes were separated on another word document. All similar codes were combined to reduce the number. Similar codes were grouped into subcategories, combined to form generic categories, and lastly, generic categories were grouped under main categories/themes by abstraction.The triangulation was conducted using two data sources: FGDs and semi-structured interviews. The participants were from different medical years and of both genders.Twelve students participated in two FGDs while six faculty members were interviewed as mentors, all from Islamic International Medical College Rawalpindi. Fifty percent mentees were girls and 50% were boys. Data were analyzed by using Atlas ti. After analyzing data 21 open codes emerged, sorted into 15 categories and abstracted to from five significant themes. Major themes which emerged were; Connection: a sense of community, goal orientation, security and control, digital support, technology related problems, and last one was regarding proposed strategies like; careful selection of participants, workshops for computer literacy, meet before e-mentoring and blended approach. details are mentioned in Online mentoring, like any other innovation, is not free of critics. The much more significant potential audience can be benefited from the training of mentors, reasonable matching of mentors and mentees.Connection: A sense of community:1.  Mentorship will not always lead to establishing friendship but mutual respect and gratitude for the other person will help to foster an element of zeal to the experience that elevates the experience of mentoring.2. Goal orientation of mentors and mentees influences the mentoring program’s career-related mastery and effectiveness.193. Confidentiality and security are a critical components of an effective and successful mentoring relationship. It is thought to be doubtful in online modality.4. Digital support, few participants of study had reservations on Microsoft teams, which is one of the forums used in this institute. However, all were sure that the conversations and meetings are secure. A trustworthy relationship helps to open the discussions and allow progress towards the success.5. Information and communication technology (ICT) skills are interpersonal and exclusively one basis. Still, they are prone to interfere more than facilitating the formation and development of the online mentoring relationship. However, sometimes distance communication tools like online discussion, messaging, and video discussions, etc, supports and enhance the mentoring (coaching) relationship in educational settings.20lack of facial expressions and the inability to read another person’s body language. Previous research also said this issue of online mentoring. One of the foremost critics of online mentoring is that it prevents mentor and mentee from observing the facial expressions and body language when it is asynchronous.Confidentiality of this relationship should be respected but online security may not be perfect. So there is the possibility that someone could find out details of the confidential mentoring relationship. Our participants have high level of confidence in each other and they don’t doubt in any breech of the trust. These issues of e-mentoring are the best approach to reduce their impact is that all participants must understand the risk involved and agree on the ground rules, as with face-to-face mentoring sessions.information technology proficiency are the issues needed to be addressed. With time after training of mentors, reasonable matching of mentees and mentors, will make online mentoring programs meet the expectations of both sides as in face to face mentoring.workshop training, so that an adequate number of mentors can be trained at a time. Mentors had the opinion that responsive students were more accessible to mentor. In another study the researcher believed that students who “owned their process more and took the initiative and were more proactive in asking for support could stay on track and move through the process more quickly.blended approach, i.e. the mentoring to be online with intermittent physical sessions. Multiple means of communication can be used in the mentoring program, with the first face to face meeting; followed by a conversation on phone or skype, phone call, email, virtual classroom, Facebook etc. A blended communication model may be an ideal and realistic way for individuals to create and maintain their working and social relationship development in the weird world of current century.22E-mentoring being a new entity is not without critics. The major challenge of study participants mentioned the This study is of its kind that explored in-depth the perspective of mentors and mentees regarding online mentoring of medical students in Pakistan. This is the first study at the national level that addressed the challenges of online mentoring and proposed the strategies to improve it. We have suggested the action plan derived from our study’s qualitative data, which can help improve the online mentoring programs.It was conducted in only one institute, if it had included multiple Centers, it could have derived more themes.E-mentoring provides flexibility to those who usually face hurdles to being mentored because of their gender, ethnicity, disability or geographical location. Mentees can take responsibility for initiating contact and participating actively in online discussions. Mentors also experience personal growth through mentoring, helping others in the profession, and providing advice and support. Online mentoring is an opportunity for innovation in mentorship. It’s still in a phase of evolution and can be made more effective if people’s concerns are addressed and challenges are resolved which needs a strategic planning according to the issues.SJ: Conceived and designed the study. Data collection and manuscript writing and responsible for the integrity the manuscript.UM: Editing and critical review of the manuscript.