Vibrio tasmaniensis LGP32, a pathogen associated with mortality events affecting juvenile oysters. The vsr217 gene is located within the 5′ untranslated region (UTR) of malK, encoding the ATPase component of the maltose importer, and is conserved within the genus Vibrio. In the presence of maltose, vsr217 is regulated by MalT, the positive regulator of the maltose regulon. vsr217 is required in cis for the full expression of malK. In addition, Vsr217 acts in trans to downregulate the expression of fbp encoding fructose-1,6-bisphosphatase, an enzyme involved in gluconeogenesis. Thus, in the presence of maltose, the induction of Vsr217 is expected to promote glycolysis by negatively regulating the expression of a key enzyme of gluconeogenesis.Vsr217 is a small RNA from IMPORTANCE Juvenile pacific oysters have been subject in recent years to summer mortality episodes with deep economic consequences. The pathogen Vibrio tasmaniensis has been associated with such mortality events. For bacterial pathogens, survival within the host requires profound metabolic adaptations according to available resources. All kinds of regulatory elements, including noncoding RNAs, orchestrate this response. Oysters are rich in glycogen, a precursor of maltose, and we previously reported that V. tasmaniensis maltose-regulated genes are strongly induced during oyster infection. Here, we report the dual mechanism by which a small regulatory RNA, generated from the 5′ untranslated region of a gene belonging to the maltose regulon, acts both in cis and trans. In cis, it stimulates growth on maltose, and in trans, it downregulates the expression of a gene associated with gluconeogenesis, thus coordinating maltose utilization with central carbon metabolism. A genome-wide search of small regulatory RNAs in Escherichia coli led to the identification of several sRNAs expressed from 5′ or 3′ UTRs  in bacterial regulatory networks has become widely recognized. The development of genome-wide transcriptomic studies has led to the exploration of transcriptomic landscapes of nonmodel bacterial organisms, either pathogens or those of environmental interest, leading to the discovery of many sRNAs  riboswitches, SreA and SreB, not only regulate their downstream gene expression in cis, but also act in trans to target the 5′ UTR of the prfA mRNA  is a member of the V. splendidus clade (Crassostrea gigas (–V. tasmaniensis LGP32 by transcriptome sequencing (RNA-seq) led us to identify 250 potential sRNAs, among which, several were confirmed by Northern blots and reverse transcription-PCRs (RT-PCRs)  . More revsr217 is a member of the maltose regulon and is positively controlled by the transcriptional regulator MalT. While, within the 5′ UTR of malK mRNA, the Vsr217 sequence acts in cis to stabilize malK mRNA, it also generates an sRNA that acts in trans to repress the expression of a key gene involved in gluconeogenesis.Here, we show that V. tasmaniensis expressed from the malK 5′ UTR. We investigated its conservation within the Vibrio genus by sequence comparison of malK upstream sequences from species representative of different Vibrio clades (E. coli and other Enterobacteriaceae (vsr217. Region V is the largest conserved sequence and contains a second predicted σ70 promoter (P2) as well as the ribosome binding site for malK. No Rho-independent terminator was predicted between the 3′ end of vsr217 and malK. This analysis highlights the conservation of a large putative regulatory region upstream of malK in Vibrio.Vsr217 is an sRNA from o clades  Fig. 1)V. tasmaneriaceae . Region vsr217 upstream of malK and the presence of a MalT box overlapping the putative P1 promoter of vsr217  mappings  corresponded to a transcription start site from the P2 promoter. Other 5′ end sequences mapped upstream of P2, some of them within vsr217. Among them, 6% had a 5′ end corresponding to a transcription initiation from P1, hence including the whole Vsr217 sequence  and Δvsr217 strains , whereas malK and vsr217-malK transcripts were unstable . Deletion of vsr217 resulted in an even more unstable malK transcript .The amount of f vsr217 , possibl strains  and B. VmalK mRNA in the Δvsr217 mutant was determined by 5′ RACE. In contrast to the parental strain, the majority of the malK transcripts (64.2%) in the Δvsr217 mutant had a 5′ end generated from the P1 promoter, whereas only 21.4% corresponded to transcripts generated from the P2 promoter  Growth of LGP32 WT and Δvsr217 strains in minimal medium supplemented by glucose or maltose. (B) Growth of WT and Δvsr217 strains in minimal medium supplemented by maltose in presence of plasmids expressing either Vsr217  and checked by Northern blotting how the deletion would affect the amounts of Vsr217, malK mRNA, and their precursor (Vsr217 contains a conserved U-rich motif at its 3′ end . We hyporecursor .polyT mutant) led to a 100-fold reduction of Vsr217 compared to that in the WT strain, whereas the amount of malK mRNA was not affected in contrast to what was observed in the Δvsr217 mutant. Consistent with this result, we observed that malK mRNA stability did not decrease in the ΔpolyT mutant compared to that in the WT strain, contrary to Vsr217 stability  harbored by the V. tasmaniensis genome were identified. In glucose, the protein contents were the same between the two strains. However, in maltose, the amounts of 22 proteins were significantly altered  in the mutant, with 11 increasing and 11 decreasing  upstream of the fbp initiation codon (trans activity on fbp expression was tested in E. coli using a two-plasmid-based reporter system (vsr217 gene cloned under a PLlacO promoter (pPLac-Vsr217). The second plasmid contained an in-frame gene fusion between the predicted pairing region of fbp mRNA including fbp initiation codon and the second codon of the gfp CDS .A biocomputing analysis using IntaRNA identified a potential base-pairing region between Vsr217 and on codon . To confr system , 26. The gfp CDS . The repfbp-gfp fusion compared to that in the strain containing a control empty plasmid  was added to fully induce vsr217 expression.The presence of pPlac-Vsr217 plasmid led to a 20% reduction in the expression of the  plasmid . ExpressVibrio , is generated by termination of transcription at its unique 3′ end. However, no Rho-independent terminator was detected to support this hypothesis. In addition, we found that deleting the conserved 13 last nucleotides of vsr217  led to a nearly complete absence of Vsr217 but had no effect on vsr217-malK or malK mRNA levels. These results suggest that the polyT motif is important for Vsr217 stability but not for vsr217-malK processing.An original feature of Vsr217 is that it is generated from the 5′ UTR of cis, it affects the stability of the malK mRNA, which decreases in the Δvsr217 deletion strain, leading to a reduction of MalK and a decreased ability to use maltose as a carbon source, which is suppressed by the expression in trans of malK but not of vsr217. In addition, Vsr217 acts as a bona fide sRNA to control the expression of fbp, a gene involved in the neoglucogenesis/glycolytic pathway. A decrease of fbp mRNA was observed in the presence of maltose, when Vsr217 is strongly induced, whereas no change was observed in glucose. This decrease was abolished in the ΔpolyT mutant, confirming that it is Vsr217 dependent in the WT strain. Direct interaction between Vsr217 and fbp was further supported by Vsr217-dependent downregulation of a fbp-gfp reporter fusion in E. coli  and Cra   –32. Whert of CRP . Both CRulations .trans-acting sRNAs derived from a 5′ UTR, may contribute to the fitness of V. tasmaniensis by optimizing maltose utilization through inhibition of gluconeogenesis.In the presence of maltose and absence of glucose, the subunit EIIA of the glucose phosphotransferase system (PTS) transporter is phosphorylated and activates adenylate cyclase: cAMP accumulates in the cell, activating CRP, which upregulates glycolysis. Maltose is transformed into glucose-6-phosphate and enters the glycolytic pathway. However, upregulation of glycolysis is counteracted by Cra, which drives the carbon flux toward gluconeogenesis. On the other hand, induction of the maltose regulon leads to a strong increase of Vsr217 that will inhibit the expression of the gluconeogenic enzyme Fbp, thus favoring glycolysis. Hence, in oysters, which are rich in the maltose precursor glycogen, Vsr217, a member of the growing family of V. tasmaniensis LGP32 mutants were constructed by allele replacement through homologous recombination as described previously  Genetic structure of the vsr217-malK locus in V. tasmaniensis LGP32. vsr217, red and capital letters; malK 5′ UTR (26 nt), green and lowercase letters; malK open reading frame (ORF) , green and capital letters; predicted −35 and −10 sequences of P1 and P2 promoters, bold and underlined letters; putative MalT box, yellow letters. The 13 nucleotides deleted in the ΔpolyT mutant are in italics. The shown sequence was inserted in pGEB12 to generate the pP1-VSR217-P2-MalK plasmid, comprising the totality of the locus. The pP1-P2-MalK and pP1-Vsr217 plasmids were then generated from this plasmid by, deleting the vsr217 sequence  and the malK sequence (green), respectively, keeping the P2 promoter and the transcription terminator (highlighted by a green box). (Middle) Structure of pLac-Vsr217. The vsr217-malK locus including its terminator  was inserted under the control of the pLacO promoter present on the pZE12 plasmid  Structure of the pTet-FBP-GFP plasmid. The 5′ UTR of the V. tasmaniensis fbp gene  and ATG in boldface font) was inserted in the pXG-10 plasmid (gfp gene present on the plasmid (in green). The putative target region in fbp 5′ UTR is in italics. Download FIG S2, PDF file, 0.1 MB.Locus K 5′ UTR  nt, greeK 5′ UTR  nt, greeCopyright © 2021 Luo et al.2021Luo et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli strains were cultured at 37°C in Luria-Bertani (LB) broth. V. tasmaniensis LGP32 and its derivative were grown at 20°C in Zobell medium  or minimal medium  supplemented with 2 g/liter d-glucose or d-maltose, where indicated. Antibiotics were used as follows: chloramphenicol (Cm) at 20 μg/ml and ampicillin (Amp) at 100 μg/ml for E. coli and Cm at 2 μg/ml for LGP32. IPTG was used at 0.5 mM.d-glucose or d-maltose. If needed, Cm was added. Overnight cultures were diluted to an optical density at 600 nm (OD600) of ∼0.05 in the same medium, and growth was monitored in a 96-well plate, by measuring cell density at OD600 at regular time intervals in a CLARIOstar  microplate reader.To measure growth of LGP32, bacteria (3 replicate cultures in each case) were cultured overnight in minimal medium supplemented with 2 g/liter 600 of ∼0.05 in minimal medium, supplemented by glucose or maltose where indicated, except for the results presented in malT mutant does not grow in minimal medium plus maltose. Cells were grown with continuous shaking at 180 rpm at 20°C. When the OD600 reached ∼0.4, bacteria were collected by centrifugation and resuspended in TRI reagent (Sigma) before total RNA extraction and purification, which was carried out using Direct-zol MiniPrep Plus (Zymo research). DNA was removed by TURBO DNase (Ambion) treatment. RNA was further purified by phenol-chloroform extraction and RNA precipitation. Ten micrograms  was used as a loading control. Membranes were scanned using an Amersham Typhoon biomolecular imager  in the phosphorimager mode. Transcript signals were quantified by ImageJ  and primers Vsr217-RT-F and MalK-RT-R . Amplification of the alK-RT-R .vsr217 strains were diluted to an OD600 of ∼0.05 and grown in minimal medium supplemented with 2 g/liter d-maltose. Bacteria were collected by centrifugation when the OD600 reached ∼0.4 and were resuspended in TRI reagent followed by RNA extraction, DNase treatment, and RNA purification. 5′ and 3′ ends of Vsr217 and malK transcripts were determined using the 5′/3′ RACE system for rapid amplification of cDNA ends (Invitrogen). RNA primers used are listed in Overnight cultures of LGP32 WT and Δvsr217, and ΔpolyT strains were grown overnight in minimal medium supplemented with 2 g/liter d-maltose at 20°C. Bacteria were diluted to an OD600 of ∼0.05 and grown in the same medium until an OD600 of ∼0.4 and rifampicin was added to a final concentration of 500 μg/ml. Bacteria were collected for RNA extraction at 0, 0.5, 1.0, 2.0, and 3.0 min after the addition of rifampicin. Northern blot analysis was performed to determine the amount of Vsr217 and malK transcripts.LGP32 WT, ΔE. coli MG1655) were grown overnight in LB supplemented with Cm and Amp. Five microliters of the overnight culture was spotted onto LB agar supplemented with Cm, Amp, and IPTG where indicated, and the plates were incubated at 37°C. Plates were then scanned with an Amersham Typhoon model 5 biomolecular imager , using the fluorescence mode . Signal intensity of the bacterial spots was determined using ImageJ and the formula , where the background signal was the signal obtained for cells containing the non-GFP-expressing plasmid, to remove the intrinsic fluorescence of bacterial cells. Significance of the relative differences was determined by a Student’s unpaired t test, with four biological replicates.To monitor green fluorescent protein (GFP) fluorescence, strains to be assayed . After centrifugation of 2 ml, cells were washed in Tris/EDTA (pH 7.5) plus 20% sucrose and then resuspended in NuPAGE LDS buffer (Thermo Fisher Scientific) to a final concentration equivalent to an OD600 of 20. Samples were heated at 95°C for 10 min before loading 10 μl of each sample on a 4% to 12% polyacrylamide gradient gel (Invitrogen). After a 5-min migration to let the samples enter the gels, they were stained in Coomassie blue and then destained.P values were calculated using a Welch’s t test.For tandem mass spectrometry (MS/MS), proteins were subjected to an in-gel enzymatic digestion with trypsin. Nanoscale liquid chromatography-electrospray ionization-tandem mass spectrometry  was carried out (gradient of 120 min), and the peptides were identified by Mascot and Andromeda and quantified by MaxQuant. Statistical analyses were carried out using Perseus . P valueVibrio malK upstream sequences were aligned using Muscle at the EBI (fbp mRNA upstream region was predicted using IntaRNA (Promoters were predicted using the BPROM software, and Rho-independent terminators were predicted using the FindTerm software . Vibrio  the EBI . Putativ IntaRNA .


Ca) channels are exquisitively Ca2+ sensitive, intracellular Ca2+ regulating channel activity by complexing with calmodulin (CaM), which is bound to the cytosolic carboxyl tail. Although CaM antagonists might be expected to decrease IKCa channel activity, the effect of W-7 in human T lymphocytes are conflicting. We therefore evaluated the effect of W-7 on basolateral IKCa channels in human colonic crypt cells. Intact crypts obtained from normal human colonic biopsies by Ca2+ chelation were used for patch clamp studies of basolateral IKCa channels in the cell-attached configuration. IKCa channel activity was studied when the bath Ca2+ concentration was changed from 1.2 mmol/L to 100 μmol/L and back to 1.2 mmol/L, as well as from 100 μmol/L to 1.2 mmol/L and back to 100 μmol/L, both in the absence and presence of 25 μmol/L W-7. Decreasing bath Ca2+ from 1.2 mmol/L to 100 μmol/L decreased IKCa channel activity reversibly in the absence of W-7, whereas there was a uniformly high level of channel activity at both bath Ca2+ concentrations in the presence of W-7. In separate experiments, increasing bath Ca2+ from 100 μmol/L to 1.2 mmol/L increased IKCa channel activity reversibly in the absence of W-7, whereas there was again a uniformly high level of channel activity at both bath Ca2+ concentrations in the presence of W-7. We, therefore, propose that W-7 has a specific stimulatory effect on basolateral IKCa channel activity, despite its ability to inhibit Ca2+/CaM-mediated, IKCa channel-dependent Cl− secretion in human colonic epithelial cells.Intermediate conductance potassium (IK Routine histology was normal in all cases. The study was approved by the Leeds Health Authority Ethics Committee. Intact crypts were isolated by a modification of a previously described Ca2, 1.2 MgCl2, 5 glucose, 10 HEPES, 5 Na+ butyrate, and titrated to pH 7.4 with NaOH. Patch pipettes fabricated from borosilicate glass were filled with a solution containing (mmol/L): 145 KCl, 1.2 CaCl2, 1.2 MgCl2, 5 glucose, 10 HEPES, 5 Na+ butyrate, and had resistances of 5-6MΩ. Single channel currents were recorded with a patch clamp amplifier  at a holding voltage of − 40 mV, referenced to the pipette interior. Currents were stored on videotape after pulse code modulation . Stored currents were low pass filtered (750 Hz) and loaded into computer memory via a DigiData 1200 interface system using pClamp software  for off-line analysis. Single channel open probability (PO) was calculated using an analysis programme written in Quick Basic 4.0 , as previously described . Statistical analyses were performed using the Student’s 2+ concentration of 1.2 mmol/L, However, W-7 stimulated channel activity at a bath Ca2+ concentration of 100 μmol/L (data not shown). Subsequent experiments were, therefore, done to test the effect of W-7 when the bath Ca2+ concentration was switched from 1.2 mmol/L to 100 μmol/L, and vice versa.In preliminary experiments, 25 μmol/L W-7 had no effect on channel activity at a bath CaCa channel activity to lowering bath Ca2+ from 1.2 mmol/L to 100 μmol/L are shown in Fig. Ca channel activity when the bath solution initially contained 1.2 mmol/L Ca2+. Within 2 min of lowering bath Ca2+ to 100 μmol/L, there was a sustained decrease in channel activity, consistent with a decrease in intracellular Ca2+ concentration. Channel activity was restored to its original high level by raising bath Ca2+ to 1.2 mmol/L. When this protocol was repeated in the presence of 25 μmol/L W-7, the high level of channel activity at a bath concentration of 1.2 mmol/L was similar to that in the absence of W-7. However, a striking finding was that in the presence of W-7, lowering bath Ca2+ concentration to 100 μmol/L failed to decrease channel activity, and the subsequent raising of bath Ca2+ to 1.2 mmol/L had no additional effect.Representative recordings showing the effect of W-7 on the response of IK2+ was changed from 1.2 mmol/L to 100 μmol/L and then back to 1.2 mmol/L, either in the absence of W-7 , or in the presence of W-7 (n = 6), are summarized in Table Ca channel activity at a bath Ca2+ concentration of 1.2 mmol/L. Whereas channel activity decreased by 49% (P < 0.005) when bath Ca2+ was lowered from 1.2 mmol/L to 100 μmol/L in the absence of W-7, there was no change in channel activity when bath Ca2+ was lowered in the presence of W-7. These data suggest that W-7 may have ‘clamped’ IKCa channel activity at a level  when the bath solution contained 1.2 mmol/L Ca2+. To explore this possibility, additional experiments were done at an initial bath Ca2+ concentration of 100 μmol/L.Data obtained from experiments when bath CaCa channel activity to raising bath Ca2+ from 100 μmol/L to 1.2 mmol/L are shown in Fig. Ca channel activity when the bath solution initially contained 100 μmol/L. Within 2 min of raising bath Ca2+ to 1.2 mmol/L, there was a marked increase in channel activity, consistent with an increase in intracellular Ca2+ concentration, and channel activity decreased to the basal level when bath Ca2+ was lowered to 100 μmol/L. Repeating this protocol in the presence of W-7, the high level of channel activity at an initial bath Ca2+ concentration of 100 μmol/L was similar to that seen at an initial bath Ca2+ concentration of 1.2 mmol/L in the absence of W-7. Channel activity did not increase further when bath Ca2+ concentration was raised to 1.2 mmol/L, and was unchanged when bath Ca2+ was subsequently lowered to 100 μmol/L.Representative recordings showing the effect of W-7 on the response of IK2+ was changed from 100 μmol/L to 1.2 mmol/L and then back to 100 μmol/L, either in the absence of W-7 , or in the presence of W-7 (n = 4), are summarized in Table Ca channel activity at a bath Ca2+ concentration of 100 μmol/L. Whereas channel activity increased by 84% (P < 0.001) on raising bath Ca2+ from 100 μmol/L to 1.2 mmol/L in the absence of W-7, channel activity was unaffected when the same change in bath Ca2+ was performed in the presence of W-7. Thus, these observations suggest that W-7 stimulates basolateral IKCa channels in human colonic crypt cells by a mechanism that appears to be largely (if not entirely) independent of the intracellular Ca2+ concentration, rather than by ‘clamping’ channel activity at a high level commensurate with the intracellular Ca2+ concentration that prevailed at a bath Ca2+ concentration of 1.2 mmol/L.Data obtained from experiments when bath CaCa channels by Ca2+-mediated agonists in human colonic crypt cells. However, in preliminary experiments, rather than producing little or no change in basal channel activity , W-7 prevented the decrease in channel activity that occurred when bath Ca2+ concentration was lowered from 1.2 mmol/L to 100 μmol/L. We therefore focused on evaluating the effect of W-7 on human colonic crypt cells in the absence of Ca2+-mediated agonists. The data in Table 2+ concentration elicited the expected changes in IKCa channel activity, which presumably reflected corresponding decreases or increases in intracellular Ca2+ concentration. In the absence of W-7, PO was 0.5–0.6 at a bath Ca2+ concentration of 1.2 mmol/L, and decreased by ~ 50% when bath Ca2+ was lowered to 100 μmol/L. By contrast, PO was invariably higher (~ 0.7) in the presence of W-7, irrespective of the bath Ca2+ concentration. This novel and entirely unexpected observation suggested that amongst the CaM inhibitors, W-7 at least has the ability to stimulate IKCa channels, which raises questions about its suitability in studies designed to evaluate the role of CaM in IKCa channel regulation. Coincidentally, it may be that the level of basolateral IKCa channel activity seen in human colonic crypt cells in the presence of W-7 is the highest achievable, since previous studies have always indicated lower levels of activity, even after using thapsigargin to increase intracellular Ca2+ concentration , and calmidazolium, either had no effect or inhibited the activity of native and cloned IKds Ikura . CaM antCa channels in human colonocytes to changes in bath Ca2+ concentration differs from studies in human erythrocytes, which showed that Ca2+-dependent IKCa channel activity in cell-attached and excised inside-out patches was unaffected by 10–100 μmol/L W-7  has previously been shown to increase 86Rb (K+) influx and efflux in the human salivary epithelial cells line HSG-PA in a partially Ca2+-dependent manner  stimulation or in response to the Ca2+ ionophore A23187  . Consistent with this view, the inclusion of PS produced a negative electrostatic potential in the aqueous phase close to the vesicle surface which decreased exponentially with distance, the zeta potentlal (ζ) being the electrostatic potential ~ 2 Å from the membrane surface, and the addition of 30 μmol/L W-7 to the LUVs decreased ζ by ~ 23 mV , where basolateral IKCa channels are a critical component (Tabcharani et al. − secretion.While the precise mechanism by which W-7 might interact with K


Trainees and investigators from underrepresented minority (URM) backgrounds face unique challenges to establishing successful careers in clinical and translational research. Structured training for mentors is an important mechanism to increase the diversity of the research workforce. This article presents data from an evaluation of the University of California, San Francisco (UCSF) Center for AIDS Research (CFAR) Mentoring the Mentors program aimed at improving mentors’ competency in working with diverse mentees in HIV research.Mentors from around the USA who had in one of seven separate 2-day training workshops conducted from 2013 to 2020 were invited to participate in an online evaluation survey of their experiences with the training and their subsequent mentoring activities.There was a high response rate (80%) among the 226 mentors invited to complete the survey. The 180 respondents were diverse in demographics, professional disciplines, and geographic distribution. Quantitative and qualitative data indicate a lasting positive impact of the training, with sustained improvements documented on a validated measure of self-appraised mentoring competency. Respondents also endorsed high interest in future, follow-up training with continued focus on topics related to mentoring in the context of diversity.The evaluation of the UCSF CFAR Mentoring the Mentors program showed lasting impact in improving mentoring practices, coupled with high interest in continued in-depth training in areas focused on diversity, equity, and inclusion. Researchers who are underrepresented minorities (URMs) in health-related sciences often face unique barriers in the development of independent research careers, including the absence or inadequacy of mentoring and research collaborations –8. HistoThe heightened challenges faced by trainees and academicians from underrepresented backgrounds are particularly pronounced in HIV research, as documented by an inadequate and fragile pipeline of diverse investigators focused on HIV priority areas .Given thad hoc methods and approaches that vary in consistency, intensity, and effectiveness [So, how do we develop a well-trained and diverse scientific research workforce? One way is through training mentors to more effectively mentor. The importance of effective and dedicated mentoring of early-stage investigators (ESIs) in academic research has been well documented, with growing evidence of the benefits of mentoring on productivity, job satisfaction, and quality of life ,29. Howetiveness . With intiveness ,32.The literature on mentoring offers some effective strategies and perspectives in designing effective mentoring training programs, including aligning mentor and mentee expectations and specifying roles early in the mentoring relationship –34. Of pSince 2012, the University of California, San Francisco (UCSF) Mentoring the Mentors program has conducted annual 2-day, in-person intensive mentoring training workshops to systematically build mentoring competency with an emphasis on training in techniques to mentor URM and otherwise diverse mentees in HIV research. The workshops are sponsored by the UCSF Center for AIDS Research (CFAR) with co-sponsorship from the CFAR Network of Integrated Clinical Systems (CNICS). The Mentoring the Mentors program focuses on HIV investigators based in the USA, as the program includes a focus on NIH and other funding opportunities available only to US investigators. The program curriculum, spanning two full days and led by faculty from a range of clinical and translational research disciplines and expert consultants, includes a combination of interactive, didactic, and case-based sessions that address pertinent issues in mentoring. These include an overview of principles and evidence for best mentoring practices, time and priority management, communication skills, how to mentor grant and paper writing, the role of individual differences  on mentoring, and resources for mentoring more effectively. A prominent focus throughout the training is content and discussion related to mentoring across differences, with activities addressing implicit bias, microaggressions, and strategies to address barriers that uniquely affect research funding for URM ESIs. The operationalization of diversity largely followed the guidance from the NIH, which provides categories of individuals from racial and ethnic groups not well represented in science, persons with disabilities, and those from disadvantaged backgrounds. We extended this definition to include diversity along other dimensions, including gender identity, sexual identity, and religious or cultural views. Toward the end of each workshop, we implement a Mentor Consultation Clinic, in which the participants break into groups of 5–6 and provide input on one member’s current mentoring challenge. In this exercise, participants are instructed to apply the training content  to a specific mentoring situation before offering advice and recommendations. See Table To date, we have trained over 226 HIV researcher mentors from across the USA, and the program and short-term evaluation results have previously been described –37. ThosThe purpose of this paper is to report longer-term mentoring outcomes from training program participants by assessing their perspectives once they have returned to their professional roles at their home institutions. In essence, we sought to answer the question of how successful was the program in enhancing participants’ ability to mentor diverse investigators in HIV research.To be eligible for this evaluation, individuals must have participated in one of seven in-person 2-day workshops between October 2012 and January 2020. From November to December 2020, a total of 226 unique participants who had attended at least one workshop were invited by email to complete a web-based survey via Qualtrics . Following initial invitations, reminders were sent weekly to those who had not responded and those who had started, but not completed, the survey. To encourage participation, mentors who completed the survey received a US$10 gift code for an online retailer and were entered into a raffle for an additional one of five randomly awarded US$100 gift codes.The primary objective of the survey was to solicit data on the long-term impact of the program. The survey included questions about demographics, professional disciplines, current positions, and allocation of professional effort across domains . We then assessed perceived impact of the training on the participants’ mentoring using quantitative items  using 5-point Strongly Agree to Strongly Disagree Likert scale. These items were supplemented with an open-ended item that asked them to explain how the training impacted their mentoring using text entry responses.As with prior reports of short-term impacts of the individual years’ workshops –37, the Finally, we queried interest in a potential follow-up “Mentoring 201” advanced workshop and solicited interest in specific topics that were presented to respondents as well as open-text fields, so that respondents could share their ideas for follow-up trainings.Of 226 mentors invited, 180 (80%) completed the survey. Of those who did not complete the survey, two (<1%) responded that they were now retired or were no longer in a mentoring role; 32 (14%) never responded to multiple invitations; 3 (1%) opened but did not complete the survey; 9 (4%) opted out of completing the survey ; and one (<1%) had a non-functioning email address, and we were unable to locate more current contact information.Respondents’ primary work locations (based on zip code of their primary work institution) demonstrated geographic diversity throughout the US see Fig. a that coApproximately two-thirds reported currently being in a leadership role related to mentoring or training. These included directing training programs, leadership of mentoring or developmental cores for research centers, and positions in academic affairs. Twenty-two percent reported having changed institutions or positions since participating in the training. Of those, 71% reported that their new position offered greater opportunities for mentoring. Since participating in the training, one-third reported having received an award related to their mentoring and an additional 15% reported that they were aware of being nominated for such an award. Mean number of current primary and secondary mentees were 4.6 and 5.0, respectively.Mentors reported working with diverse mentees. Specifically, the following percentage of mentors reported working with mentees from the following groups: 98% with mentees who are female; 96% with mentees from racial or ethnic minority backgrounds; 79% with mentees from socioeconomically disadvantaged backgrounds; 46% with mentees who identify as gender minorities; 83% with mentees who identify as sexual minorities; 27% with mentees with disabilities; and 74% with mentees who are the first in their immediate families to attend college.Scores on all six subscales of the MCA showed increases from pre-workshop to immediate post-workshop levels, with sustained increases at long-term follow-up Fig. . While l“Being a good mentor is not something we are taught in medical school. It was a very powerful experience to have the chance to reflect on my mentorship style and learn some practical tips for improving my skills.” Woman, White, Medicine, Associate Professor.Narrative comments reinforce these findings. Some referred to the unique opportunity to learn mentoring skills, noted as a gap in their prior training.“It was a tremendous opportunity to focus on a critical area of professional development that is a rare focus. Academics are expected to ‘know’ how to effectively mentor and support mentees in their own career development but rarely in our own education and career development do we get support, knowledge and training on ‘how’ to most effectively mentor others. This is why this program is invaluable.” Woman, Middle Eastern/North African, Social/Behavioral Science, Professor.and“I consider mentorship to be one of the most important aspects of my job. But in early-career I felt thrown into these relationships with minimal instruction, oversight, and constructive feedback. The Mentoring the Mentors program provided a sense of community of a group of professionals who are striving to do mentorship better and the encouragement of leaders and experts who desire to foster equity and excellence in mentorship.” Woman, White, Public Health, Associate Professor.and“I learned that there is a science in mentoring and that as a mentor I need to be more systematic.” Woman, Black/Latinx, Social/Behavioral Science, Professor.Some identified the value of specific tools covered in the training.“The Mentoring the Mentors workshop helped me better understand my own communication style and how to effectively work with mentees who have different backgrounds from me. I also got some excellent concrete tools to use in my own mentoring, such as mentoring agreements and worksheets to guide weekly meetings with mentees.” Woman, White, Medicine, Associate Professor.and“I appreciated the way the workshop approached the complex role of mentoring into a smaller subset of skills and tools that seemed easier to engage with. The instructors were all extremely engaged and engaging, and shared specific examples of mentoring challenges and strategies that were immediately applicable to my own work with mentees.” Man, White, Public Health, Professor.and“Above all, the course is conducted in a supportive, creative, and fun environment that helps attendees feel ‘safe’ in expressing their opinions in order to learn from each other.” Man, White, Medicine, Associate Professor.Some referred to the value of the safe environment cultivated in the workshops“The Mentoring the Mentors facilitators created a supportive, dynamic, thought-provoking environment in which to explore my own pre-conceptions and think about different learning styles. It was energizing to share ideas with other people who want to be better at supporting the next wave of researchers and really recognizing and developing their talents and strengths.” Woman, White, Social/Behavioral Science, Research Scientist.and“This workshop helped me to think about the challenges that diverse mentees face and to come up with strategies to help them succeed.” Man, White, Public Health, Professor.Others noted the importance of the training focus on diversity, equity, and inclusion“Mentoring diverse trainees requires specific skills, self-awareness, and dedication. The Mentoring the Mentors program was a critical step in my journey towards becoming a better and more effective mentor for trainees of diverse backgrounds.” Woman, White, Social/Behavioral Science, Professor, first in family to attend college.and“The UCSF Mentoring the Mentors program expands beyond stressing the importance of increasing diversity in mentors and mentees to effectively teach mentors how to continuously be mindful of and address biases that are inherent in the mentor-mentee relationship. This is a skill that is critical in fostering a productive and successful mentor-mentee relationship.” Woman, Black, Epidemiology, Associate Professor, first in family to attend college.andWhen asked whether respondents would be interested in a follow-up advanced training (Mentoring 201), a majority reported being definitely (68%) or probably (26%) interested. When asked to rate interest in specific topics, those that received the highest ratings were 1) mentoring during times of stress and uncertainty; 2) best practices for distance mentoring; and 3) racism/sexism/xenophobia/discrimination as they relate to mentoring . Other topics offered by survey respondents included a focus on mentor self-care , co-mentoring best practices, training mentees to be effective mentors, working with challenging or stuck mentees, mentoring mentees from different disciplines, mentoring mentees from low- and middle-income countries, and improving one’s institutional culture and commitment to mentoring.Effective mentoring is crucial to the success of ESIs; training programs in mentoring are limited and enhancing mentoring skills via training may be particularly impactful for mentees from URM groups. We report on combined outcomes from 7 years of a designated mentoring training program with a specific focus on diversity. The results of this evaluation show sustained effects on mentoring competency among mentors who participated in the program. Of note is that the mentors are now working with a diverse range of mentees, including racial, ethnic, sexual, gender minorities; mentees with disabilities; and mentees who were the first in their immediate families to attend college. This is highly encouraging given the need to increase the diversity of the pipeline of investigators focused on research to end the HIV epidemic. It is also encouraging that the mentors trained through the Mentoring the Mentors program are generally based in geographic areas that harbor the highest prevalence and incidence of HIV in the USA Fig. a and b.Results suggest that the impact of the mentor training may extend beyond the immediate orbit of each participant’s direct mentoring pool, as a substantial proportion of workshop participants went on to hold leadership positions related to mentoring, to replicate what they learned in the training, or otherwise help others improve their mentoring effectiveness. In addition, we anticipate that improvements in participants’ mentoring practices will allow them to serve as role models to their mentees who will likely emulate their mentors as they develop their own mentoring practices. This formal and informal dissemination of the training offers promise for extending the reach of the program well beyond the pool of mentors who directly participated in the workshops.The emphasis on mentoring across differences is central to the UCSF CFAR’s Mentoring the Mentors program. Addressing diversity is one of two areas, however, that showed a less sustained impact in mentoring practices over time. There are several speculative explanations for this finding. First, it may be that there has been heightened awareness of issues of diversity, equity, and inclusion as they relate to mentors in the decade since the program started. Second, the long-term survey occurred at the end of 2020, a year in which social, political, and public health issues brought multiple dimensions of disparities and discrimination into the public awareness. This salience may have resulted in respondents being more aware of their need to better support mentees from underrepresented backgrounds in their mentoring. Finally, it may be that our curriculum must continue to evolve to have greater impact on the capacity of mentors to address diversity in their mentoring practices. Indeed, we are refining our current program and developing an advanced mentor training program in which we expand our focus on diversity, including bringing in content related to intersectionality in the mentoring relationship as well as working with mentees during times of stress and uncertainty. In response to survey feedback, we are also developing a module on remote mentoring best practices, a topic of necessity during the COVID-19 pandemic but which has relevance beyond that point as more mentors take on mentees who are geographically distant.There are limitations that should be considered when generalizing the results of this evaluation. The mentors surveyed in this study had self-selected to both previously participate in an intensive in-person mentor training workshop and also agreed to complete this survey when invited. Therefore, the current sample may represent a group that is particularly dedicated to mentoring ESIs in HIV research. We also note that there was disproportionate representation among female mentors  in our trainings and in this evaluation. It is unclear whether this suggests a pattern of higher interest among female mentors in seeking help in improving their mentoring skills over the level of interest among their male counterparts; this warrants future investigation. Likewise, although we had a high response rate (80%), those who did not respond may have different perspectives or may have been particularly affected by the COVID-19 pandemic to the extent that they did not have the time or bandwidth to participate . Similarly, the pool of mentors surveyed reflected investigators who, at least at the time of their participation in the Mentoring the Mentors program, were based at US institutions, and thus we have not captured the perspectives of investigators working primarily in low- and middle-income countries. While the MCA is a validated instrument, all other items were developed for this evaluation only and therefore do not have prior validation. The online survey format is well suited for quantitative data collection but may not be as effective in soliciting qualitative responses; the relatively brief responses provided in response to the open-ended inquiries may not have fully captured the rich and nuanced underlying themes that might have emerged through in-depth interviews with structured probing of relevant content. Our evaluation did not include the perspectives of mentees who work with the mentors trained in our program; including their perspectives and objective metrics of mentee successes would be valuable in future work to document the impact of the training. Finally, the data represent a narrow slice in time during an active phase of the unprecedented COVID-19 crisis, which may have altered the perceptions of respondents in a way that may not reflect their longer-term perspectives. In spite of these constraints, we contend that the findings presented in this study offer a unique and valuable contribution to the mentoring literature and illuminate strategies and opportunities for future mentor training programs.In conclusion, our designated mentoring program for HIV researchers had long-term effects on mentoring competency. The data from this study support the impact and ongoing need for specialized training in tools and techniques of effective mentoring among researchers. Our results also support an increased focus on dynamics related to diversity, equity, and inclusion in mentoring trainings to help cultivate and maintain a diverse pipeline of future researchers committed to clinical and translational research. Although this paper focuses on the perspectives of mentors working primarily in HIV research, our findings are not confined to that context. Rather, the results are generalizable to the challenges and opportunities for mentoring across a wider range of academic research settings. Given the interest in subsequent and more advanced workshops, it is also apparent that growth in mentoring skills is an ongoing process, and that systematic support and evidence-based training for mentors should be provided throughout mentors’ careers.


Given the controversial roles of FMS-like tyrosine kinase 3 inhibitors (FLT3i) in various treatment stages of acute myeloid leukemia (AML), this study was designed to assess this problem and further explored which FLT3i worked more effectively.FLT3(+) patients, or demonstrating the efficiency of allogeneic hematopoietic stem cell transplantation  in FLT3(+) AML. Relative risk (RR) with 95% confidence intervals (CI) was used for estimating complete remission (CR), early death and toxicity. Hazard ratio (HR) was used to assess overall survival (OS), event-free survival (EFS), relapse-free survival (RFS) and cumulative incidence of relapse (CIR).A systematic review, meta-analysis and network meta-analysis (NMA) were conducted by filtering PubMed, Embase, Cochrane library, and Chinese databases. We included studies comparing therapeutic effects between FLT3i and non-FLT3i group in AML, particularly p = 0.04) and refractory and relapsed FLT3(+) AML (rrAML)  compared to non-FLT3i arm, followed by improved survival  rrAML: OS, HR 0.60, p < 0.01; RFS, HR 0.40, p = 0.01). In addition, allo-HSCT improved survival in FLT3(+) AML , which was further prolonged by FLT3i administrated after allo-HSCT . Additionally, FLT3i consistently improved OS (p < 0.05) regardless of FLT3-ITD ratio, when compared to non-FLT3i group. Besides, FLT3i showed significantly increased risk of thrombocytopenia, neutropenia, anemia, skin- and cardiac-related adverse effects, increased alanine aminotransferase, and increased risk of cough and dyspnea (p < 0.05). In NMA, gilteritinib showed the highest probability for improved prognosis.After addressing all criteria, 39 studies were eventually analyzed. Better CR was accomplished by FLT3i in untreated AML  AML and further boosted survival benefits from allo-HSCT as maintenance therapy, suggesting better prognosis if FLT3i is combined before and after allo-HSCT. In NMA, gilteritinib potentially achieved the best prognosis, which should be identified in direct trials.FLT3i safely improved prognosis in induction/reinduction stage of  FLT3) mutations are detected in approximately one-third of patients (FLT3(+) patients with internal tandem duplication (FLT3-ITD) ranging from 3 to more than 100 amino acids located in the juxtamembrane region and a FLT3 point mutation in the tyrosine kinase domain (TKD) in approximately 8% of newly diagnosed AML  patients always manifest poor prognosis, distinguished by high resistance frequency to induction chemotherapy and relapse, decreased response to salvage therapy, and shorter survival. However, TKD influence on prognosis remains contradictory (Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by a maturation block and accumulation of myeloid progenitor cells . Among tpatients , comprispoptosis . FLT3-ITadictory .FLT3 inhibitors (FLT3i) are a type of tyrosine kinase inhibitors with FLT3 inhibitory activity that may be particularly utilized to treat FLT3(+) AML with likely improved prognosis. According to current clinical studies, they proved that AML patients benefited from FLT3i  patients were also summarized to observe the effects of FLT3i as the maintenance therapy after allo-HSCT. We further performed network meta-analyses (NMA) and ranked the prognostic effects of various FLT3i based on phase 2 and 3 RCT to check the most effective FLT3i.These FLT3i are used in various stages, including induction with/without consolidation therapy in newly diagnosed AML, maintenance treatment after allogeneic hematopoietic stem cell transplantation  and salvage therapy in refractory and relapsed AML (rrAML). Up to now, no extensive study has been found to comprehensively explore the role of FLT3i in various AML treatment stages and explore which FLT3i probably works the best. Herein, we conducted a meta-analysis in an attempt to clarify the clinical benefit and safety of FLT3i. Data from allo-HSCT in This study was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)  and was FLT3”, “acute myeloid leukemia”, “AML”, “hematopoietic stem cell transplant”, “HSCT”, “sorafenib”, “lestaurtinib”, “midostaurin”, “quizartinib”, and “gilteritinib”. The included reports were: (i) published in English or Chinese, (ii) limited to retrospective cohort studies or RCT reporting the therapeutic effects of FLT3i on AML, especially for FLT3(+) patients, or restricted to studies demonstrating prognostic effects of allo-HSCT on FLT3(+) AML, (iii) designed to contain two arms or more for comparing prognostic influence of FLT3i or allo-HSCT with controls. Studies were excluded if they: (i) reported unavailable or insufficient data, (ii) were reviews, case reports, editorials and letters, (iii) had overlapping cohorts and (iv) were sing-arm studies.A literature search was conducted through databases of PubMed, Embase, Cochrane library, China National Knowledge Infrastructure, and Wanfang since inception until September 30th, 2020, following keywords “FMS-like tyrosine kinase 3”, “Study selection was conducted in two stages. Initially, abstracts and titles of potential literature were independently browsed and screened by QX and SH according to inclusion and exclusion criteria. Both reviewers then evaluated the candidate articles and decided on their inclusion. Any discrepancy was discussed and, if required, settled through discussion or consultation with a third reviewer (LY). After selecting the candidate studies, full texts were checked to identify final eligible ones.P-value < 0.05 implied publication bias existence.The methodologic quality of primary studies was assessed separately by two reviewers (QX and SH), based on Newcastle-Ottawa-Scale (NOS)  and CochClinical information from the included studies was extracted independently by two authors (QX and SH), and any reported disagreement was settled by discussion or consultation with the third author (LY). The extracted data were comprised of the first author, study characteristics, patients’ baseline and prognostic information. The endpoints included overall survival (OS), event-free survival (EFS), relapse-free survival (RFS), cumulative incidence of relapse (CIR), early death , adverse events and complete remission (CR), defined by revised International Working Group Criteria , withoutp-value of < 0.05. The χ2-based Q statistic was used to assess the heterogeneity among studies. Low, moderate, substantial and considerable heterogeneity showed 2I < 30%, 30%–50%, 50%–75% and > 75%, respectively. A P-value ≥ 0.10 implied no heterogeneity or slight heterogeneity, whereas P < 0.10 meant significant heterogeneity existence  for survival were calculated with the inverse variance method, and pooled RR and 95% CI for CR, early death and adverse events were produced from the Mantel-Haenszel method . Analysexistence . When P-via packages of “gemtc” and “rjags” in RCT. We also calculated HR or risk ratios (RR) regarding non-FLT3i group as the baseline to act as the effect measure, displayed in forest plots, where RR and HR with 95% credible intervals (95% Crl) were utilized to explain the extent of effects in CR and survival, respectively. The range of 95% Crl without covering 1.00 implied statistical significance. To estimate relative HR and RR, a Markov Chain Monte Carlo simulation was finished with 10000 adaptations and 100000 iterations of each of the three automatically generated Markov chains. After finishing all simulations, NMA determined the probability that each treatment would be best by calculating the probability of simulations in which a certain treatment ranked best. For each iteration, therapies were ranked according to the evaluated log HR or log RR. The data from Bayesian NMA were compared with data from pairwise meta-analyses to assess inconsistency using the node splitting method  AML, except for Roellig et al. (FLT3(+) AML patients given sorafenib-related therapies as induction strategy, with or without consolidation and maintenance therapy following allo-HSCT. Six studies (n = 951) only focused on the maintenance therapy of sorafenib after allo-HSCT, and two studies (n = 235) regarded sorafenib as part of salvage therapy following relapse. Midostaurin was used in the stages of induction, consolidation and maintenance treatment in CR in four studies (n = 1604) and one article (n = 60) reported results of midostaurin-related maintenance therapy after allo-HSCT. For lestaurtinib studies, two (n = 500) focused on the induction stage in untreated FLT3(+) AML and one (n = 224) involved salvage therapy in FLT3(+) rrAML. In addition, the clinical efficacy of gilteritinib (n = 371) and quizartinib (n = 367) was evaluated in RCT for rrAML, and two studies (n = 178) combined several FLT3i. Finally, fourteen studies (n = 1797) compared prognostic discordance between allo-HSCT and non-HSCT in FLT3(+) AML.Totally, 6859 AML patients were included, and their studies were conducted in g et al.  and Servg et al.   in FLT3i groups but inconsistently in prolonged EFS  and better RFS   and better RFS    and Xuan= 0.025) , the comp = 0.01; heterogeneity: 2I = 13.3%, P = 0.330) and RFS . Besides, midostaurin consistently prolonged OS  but lestaurtinib did not provide better survival (p > 0.05).The subgroup analyses were further evaluated among various FLT3i (FLT3(+) AML were then investigated in 14 studies . OS and RFS were improved by allo-HSCT but with high heterogeneity . After sensitivity analyses, combined HR of OS and RFS became consistent, demonstrating pooled HR of 0.53 for OS  and pooled HR of 0.57 for RFS , respectively .We next analyzed FLT3i role as the maintenance treatment after allo-HSCT in eight studies, consistently showing better survival , and only one study was involved in gilteritinib, quizartinib, and lestaurtinib, respectively. Better OS was observed in gilteritinib  and quizartinib . However, lestaurtinib did not support better survival (p > 0.05).Prognostic effects of FLT3i in salvage regimen in rrAML were estimated among seven studies. After excluding Levis et al.  to decrep = 0.04; heterogeneity: 2I = 63.9%, P = 0.005) and rrAML . After sensitive analysis, combined RR of CR in rrAML became consistent, demonstrating pooled RR of 0.61 . In the subgroup analyses for newly diagnosed AML , 0.85  and 1.36 , respectively. In the subgroup analyses for salvage regimen in rrAML (p = 0.01), 0.79 , 0.45 , and 0.56 , respectively.CR from 15 studies was assessed  and low ratio  when compared to non-FLT3i group. However, FLT3i significantly prolonged RFS in patients with high-ratio FLT3-ITD  rather than those with low-ratio FLT3-ITD mutations , when compared to non-FLT3i arm. Finally, there was only one study involved in EFS, showing no prolonged survival in FLT3i group in both of high and low FLT3-ITD ratio.The treatment effectiveness of FLT3i was then analyzed in patients with low and high vs. non-FLT3i: 28.96% vs. 18.36%; RR, 2.20; 95% CI, 1.39–3.48, p = 0.0008; 2I = 55.2%, P = 0.108), neutropenia , as well as anemia  regardless of grades.Totally, early death and 21 types of toxic effects were analyzed , especially for grades ≥ 3  with low heterogeneity . FLT3i also consistently showed a tendency of increased risk for all-grade gastrointestinal-related toxicities . Additionally, significantly increased alanine aminotransferase was found in FLT3i group at all grades  and grade ≥ 3 . Cardiac-related adverse events were mainly reported in sorafenib in all grades  and grade ≥ 3 . Higher risk of all-grade cough  and dyspnea  was reported in FLT3i group.Besides, FLT3i were clearly related to higher risk of skin-related adverse effects at all grades .P < 0.10) existed. The heterogeneity sources were listed in FLT3(+) AML, as well as OS of FLT3i in salvage regimen. The funnel plots were illustrated in Sensitivity analyses were conducted if high heterogeneity  and OS   and ServFLT3(+) AML, and which FLT3i might be the best, which were investigated in this study. FLT3i possibly supported better CR in induction and salvage therapy that further boosted the improved survival of FLT3i. Allo-HSCT improved survival in FLT3(+) AML and FLT3i as the maintenance therapy after allo-HSCT might further enhance survival benefit gained from allo-HSCT. FLT3i also improved OS regardless of stratification of FLT3-ITD ratio, when compared to non-FLT3i group. Additionally, FLT3i consistently showed significantly increased risk of thrombocytopenia, neutropenia, anemia, skin-related adverse effects, increased alanine aminotransferase, cardiac-related adverse events, cough and dyspnea, but were not associated with high early death and increased risk of GVHD. NMA showed that gilteritinib probably favored the highest possibility toward better prognosis, which should be identified in more direct head-to-head RCT.Until now, no consensus was present on FLT3i role in various AML treatment stages, especially for FLT3 was the antileukemic activity of sorafenib in inhibiting other kinases such as RAF  (FLT3(+) patients aged 18-59 years, midostaurin plus chemotherapy achieved prolonged survival, regardless of FLT3-TKD and different levels of mutant FLT3-ITD ratio, probably resulting from enough exposure to this inhibitor. Given a benefit among patients with low allelic FLT3-ITD mutation and a large disease burden, the benefit of this multitargeted kinase inhibitor might lie beyond its ability to inhibit FLT3, like inhibiting KIT in FLT3 (–) AML (FLT3(+) AML, possibly due to the rising level of FLT3 ligand induced by chemotherapy, which could interfere with activity of FLT3 inhibition, including lestaurtinib  AML  confirmeaurtinib .FLT3(+) AML. However, some variables greatly affect the effectiveness of allo-HSCT, including disease status (CR or not), FLT3 variables , and using FLT3i before and/or after allo-HSCT. Unfortunately, no RCT assessed the most suitable post-remission treatment in FLT3(+) AML, considering diverse combinations (FLT3(+) AML (30%-59%) (FLT3-ITD(+) patients undergoing allo‐HSCT (FLT3-ITD(+) AML. In the RADIUS phase 2 RCT (FLT3(+) AML patients may still benefit from sorafenib after allo-HSCT because of the less use of other FLT3i  . Indeed,30%-59%)  showed ello‐HSCT , probabise 2 RCT , midostaer FLT3i .FLT3(+) AML (FLT3(+) rrAML, we also explored consistent or different therapeutic efficiency between various FLT3i in such patients. Except for lestaurtinib, the combined OS could be consistently improved by sorafenib, gilteritinib, and quizartinib, showing the potential of FLT3i in treating FLT3(+) rrAML. A phase 3 RCT randomized 371 FLT3(+) rrAML to either gilteritinib or salvage chemotherapy (FLT3(+) rrAML patients also confirmed quizartinib availability in improving prognosis (p < 0.05). Until now, the promising efficiency of gilteritinib and quizartinib could be observed in FLT3(+) rrAML in the two large RCT. However, a lack of evidence is present to add the two FLT3i to induction, consolidation, and maintenance therapy after allo-HSCT. Several phase 2 or 3 RCT are being processed . Additionally, two retrospective studies of sorafenib from Bazarbachi et al. (FLT3(+) rrAML, which should be further confirmed in RCT. Finally, midostaurin might be specifically effective in the untreated AML rather than rrAML, since in vitro studies, midostaurin had broader activity and might achieve greater clinical utility in newly diagnosed AML with blasts tending to be less addicted to FLT3-mediated signaling than rrAML (Due to high refractory and relapse incidence in 3(+) AML  and limiotherapy , showingi et al.  and Xuani et al.  also revan rrAML .FLT3-ITD(+) AML stratified by ITD allelic ratios, showing that FLT3i consistently achieved significantly improved OS in both of high and low ratio crossing sorafenib, quizartinib, midostaurin and even lestaurtinib, further suggesting the benefits of FLT3i in FLT3-ITD(+) AML. However, there was no information involved in assessing clinical efficiency of gilteritinib in patients with different levels of FLT3-ITD ratio, which needs to be further explored in more studies.We also explored the effectiveness of FLT3i on For adverse events, FLT3i were significantly associated with increased risk of thrombocytopenia, neutropenia and anemia regardless of grades, high risk of skin- and cardiac-related adverse effects, especially for grades ≥ 3, all-grade increased alanine aminotransferase, high risk of all-grade cough and dyspnea, as well as a tendency of increased risk for all-grade gastrointestinal-related toxicities. However, these adverse events were generally manageable based on treatment interruptions or dose reductions . MoreoveFinally, due to co-existence of several FLT3i, it would also be meaningful to explore which inhibitor might be the best. Herein, we finished an NMA based on all RCT to settle this problem. The results displayed that gilteritinib probably tended to favor the highest probability of improving prognosis compared with other FLT3i, standard of care and standard chemotherapy. The relevant RCT involved in gilteritinib for rrAML treatment also illustrated improved survival as mentioned above . HoweverFLT3(+) AML and therapeutic efficiency of FLT3i as maintenance therapy after allo-HSCT and as salvage regimens in rrAML were summarized and explored, showing that combining FLT3i, especially sorafenib, into the treatment before and after allo-HSCT might be more beneficial in improving prognosis, which should be further explored in RCT. Our study also primitively proposed NMA to compare various FLT3i, but with limitations as mentioned above. There were also several limitations in this study. Of 39 studies, 28 studies were retrospective, making it difficult to precisely control selection, attrition, information and confounding bias. In particular, due to RCT lack, limited results and conclusion were obtained from NMA. Besides, some data were extracted from Kaplan-Meier survival curves and numeric reports, probably resulting in slight disparity with the fact. Finally, this study did not make subgroup analyses based on age, co-existing mutations with FLT3 mutations, and cytogenetic stratifications due to limited relevant data in primary studies. As a consequence, we will continue updating this study to explore suitable treatment of FLT3i in AML patients.Overall, our study was up to now the biggest and the most comprehensive meta-analysis involved in various FLT3i in AML, containing all RCT and retrospective cohort studies. In addition to FLT3i function in induction stage in untreated AML, the role of allo-HSCT in FLT3(+) AML and salvage therapy of FLT3(+) rrAML and further enhanced survival advantages from allo-HSCT as the maintenance therapy. This probably indicates that better prognosis could be achieved if FLT3i is added into both treatments before and after allo-HSCT. Besides, FLT3i probably improved OS regardless of FLT3-ITD ratio. Additionally, FLT3i were significantly linked to increased risk of all-grade thrombocytopenia, neutropenia, anemia, all-grade skin- and cardiac-related adverse effects, especially for grade ≥ 3, as well as all-grade increased alanine aminotransferase, enhanced risk of all-grade cough and dyspnea, and a tendency of increased risk for all-grade gastrointestinal-related toxicities, but not related to higher incidence of early death and GVHD compared to non-FLT3i group. In NMA, gilteritinib potentially accomplished the best prognosis, which should be identified in direct head-to-head RCT.In this work, FLT3i reinforced better prognosis in the induction stage of newly diagnosed The original contributions presented in the study are included in the article/QX and LY contributed to conception and design of the study. QX and SH collected data from database. QX and SH performed the statistical analyses. QX and LY wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by Chinese National Major Project for New Drug Innovation (2019ZX09201002003), National Natural Science Foundation of China , Shenzhen Science and Technology Foundation  and Shenzhen Key Laboratory Foundation (ZDSYS20200811143757022).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


OPA1 mutations. OPA1, dependent on its GTPase and GED domains, governs inner mitochondrial membrane (IMM) fusion and cristae organization, which are central to oxidative metabolism. Mitochondrial dynamics and IMM organization have also been implicated in Ca2+ homeostasis and signaling but the specific involvements of OPA1 in Ca2+ dynamics remain to be established. Here we studied the possible outcomes of OPA1 and its ADOA-linked mutations in Ca2+ homeostasis using rescue and overexpression strategies in Opa1-deficient and wild-type murine embryonic fibroblasts (MEFs), respectively and in human ADOA-derived fibroblasts. MEFs lacking Opa1 required less Ca2+ mobilization from the endoplasmic reticulum (ER) to induce a mitochondrial matrix [Ca2+] rise ([Ca2+]mito). This was associated with closer ER-mitochondria contacts and no significant changes in the mitochondrial calcium uniporter complex. Patient cells carrying OPA1 GTPase or GED domain mutations also exhibited altered Ca2+ homeostasis, and the mutations associated with lower OPA1 levels displayed closer ER-mitochondria gaps. Furthermore, in Opa1−/− MEF background, we found that acute expression of OPA1 GTPase mutants but no GED mutants, partially restored cytosolic [Ca2+] ([Ca2+]cyto) needed for a prompt [Ca2+]mito rise. Finally, OPA1 mutants’ overexpression in WT MEFs disrupted Ca2+ homeostasis, partially recapitulating the observations in ADOA patient cells. Thus, OPA1 modulates functional ER-mitochondria coupling likely through the OPA1 GED domain in Opa1−/− MEFs. However, the co-existence of WT and mutant forms of OPA1 in patients promotes an imbalance of Ca2+ homeostasis without a domain-specific effect, likely contributing to the overall ADOA progress.Autosomal Dominant Optic Atrophy (ADOA), a disease that causes blindness and other neurological disorders, is linked to  Mitochondria are compartmentalized and dynamic organelles that undergo multiple membrane reshaping processes . Mitocho2+ controls OXPHOS and in turn, ATP production by activating different dehydrogenases and supporting pyruvate supply inside the mitochondria , and its release to the cytosol is mediated through the activation of the IP3 receptor (IP3R) or the Ryanodine receptor  clearance (2+ (Ca2+mito) uptake is determined by: 1) the Voltage-dependent Anion Selective Channels (VDACs) and Mitochondrial Calcium Uniporter complex (mtCU), located at the outer mitochondrial membrane (OMM) and IMM, respectively; and 2) Δψm, which is the main component of the electrochemical gradient acting as the driving force  which causes blindness due to Retinal Ganglion Cells (RGCs) death (Pathogenic mutations in s) death . OPA1 dys) death . The axos) death .2+ homeostasis has been described in the literature with many discrepancies between different groups. Knockdown of Opa1 in RGCs caused altered Ca2+cyto clearance upon ER Ca2+ (Ca2+ER) release and excitotoxicity, suggesting that OPA1 may have a role in Ca2+ homeostasis and disease progress , the essential MCU regulator (EMRE), and the dominant-negative MCU subunit (MCUb)  (2+ homeostasis may involve mechanisms beyond its fusion activity.Cat (MCUb) . The MCUCU1/2/3) . An inteCU1/2/3) . Also, OCU1/2/3) . These d2+ transfer  forms of OPA1 indistinctly disrupts Ca2+ homeostasis, potentially contributing to ADOA disease progression.In this work, we studied the role of OPA1 and its disease causing mutations in Ca2+ homeostasis, we first used MEFs in which the mitochondrial matrix-targeted Ca2+-sensitive protein, mtRCaMP was expressed to monitor the [Ca2+] mito simultaneously with [Ca2+] cyto tracked by a Ca2+ sensing dye, Fura2-AM. The cells were also transfected with the muscarinic type 3 receptor (M3R) to allow Carbachol (CCh)-induced [Ca2+] cyto transients through activation of IP3R-mediated Ca2+ mobilization from the ER  in Opa1−/− cells compared with WT cells (127.8 ± 7.8 nM), and upon the acute rescue with the human OPA1 isoform 1 (OPA1), we found a partial restoration of the baseline  compared with WT cells (775.9 ± 47.6 nM). Moreover, the exogenous expression of OPA1 partially rescued the [Ca2+]cyto signal  to discharge ER Ca2+ by blocking the ER-Ca2+ re-uptake through the Sarcoendoplasmic Reticulum Calcium-ATPase (SERCA). To follow the Ca2+ that the mitochondria face upon Ca2+ER release, the cells were transfected with an outer mitochondrial membrane-targeted Ca2+ sensor (OMMRCaMP). Both [Ca2+]cyto and [Ca2+]OMM responses showed a decreased Ca2+ release from ER  was induced by adding 1 mM CaCl2 after the ER Ca2+ depletion (Opa1−/− cells showed an augmented [Ca2+]cyto transient amplitude (790.7 ± 44.2 nM) compared with WT cells (629.4 ± 44.5 nM), and this effect was completely rescued upon OPA1 expression  by a potentiometric dye, TMRE. Consistent with Fülop et al . Our results showed IMM topology perturbation and cristae compartmentalization in the Opa1−/− MEFs, as previously described  (Opa1−/− + OPA1 WT stable MEF cells (Opa1−/− cells (Opa1−/− + OPA1: 17.81 ± 0.67 nm vs. Opa1−/−: 14.98 ± 0.72)  . To testEF cells  and test ± 0.72) .2+cyto and Ca2+mito homeostasis including an attenuated ER Ca2+ storage and enhanced SOCE. Still, the Ca2+mito uptake response to IP3R-mediated Ca2+ release is enhanced, likely because of closer physical proximity between mitochondria and the ER.Thus, we conclude that the lack of Opa1 causes multiple changes in the CaOPA1 mutations replicate the defects in Ca2+ homeostasis and ER-mitochondria distance Opa1−/− cells exhibited. Fibroblasts carrying specific mutations at the GTPase domain included OPA1 c.870+5G>A, with a deletion of the Exon8, and c.889C>T with an early stop codon at GTPase domain. Both patients presented a severe form of the disease, involving other symptoms besides optic nerve atrophy, known as ADOA+ (MIM# 125250). Also, we studied patients carrying specific mutations in GED: OPA1 c.2713C>T leading to a stop codon and loss of the GED domain, and c.2818+5G>A with a deletion of GED coding exon 27. These patients presented symptoms restricted to the eye or plain ADOA (MIM#165500) (We wanted to address if ADOA patient-derived fibroblasts carrying domain-specific heterozygous #165500) . Western#165500) .2+ signaling, we first measured resting [Ca2+]. Our results showed that patients’ cells have unaltered resting [Ca2+]cyto . Yet, we found no differences between control and GED mutant c.2713C>T carrying cells . The inclusion of OPA1 GTPase or GED mutants in the presence of WT Opa1 in MEF cells, slightly diminished the Ca2+mito uptake without alteration of Mcu levels. A leftward shift in [Ca2+]cyto dependence to promote [Ca2+]mito rise was a particular characteristic observed in the Opa1−/− cells. Moreover, ADOA patient cells exhibiting low levels of OPA1 displayed a closer ER-mitochondria distance, suggesting that OPA1 integrity and levels are necessary for physiological intracellular Ca2+ regulation and ER-mitochondria Ca2+ transfer. Thus, the cellular models studied here, with patient cells and the acute expression of OPA1 mutants in a WT background, all point towards a dominant-negative Ca2+ homeostasis phenotype, independent of the involved domain.Finally, ADOA is a dominant disease, most of the patients are heterozygous 2+ homeostasis and in determining functional ER-mitochondrial coupling, with the GED region playing a fundamental role in stabilizing the efficiency of inter-organelle Ca2+ transfer. The co-existence of WT and ADOA-related mutants could perturb ER-to-mitochondrial communication, providing a mechanism for disease progression in patients affected with ADOA disease.In conclusion, our study provides new evidence on a central role for OPA1 in Ca2) at 37°C. Given that human myoblasts grow slowly, skeletal muscle-derived fibroblasts were generated from myoblast samples. Briefly, the patient’s derived myoblasts were cultured in Skeletal Muscle Cell Growth Medium (PromoCell #CatC-23160) and plated in collagen I-coated dishes. Cells were trypsinized and pre-plated in an uncoated dish for 30 min to separate fibroblasts from myoblasts, where the former adhere to the uncoated dish while the latter were resuspended and placed aside. The patient’s fibroblasts were used between passages 3–9. All the cells were tested for mycoplasma contamination regularly using the following protocol  (provided by David Chan). Patient-derived cells were provided by Newcastle Research Biobank for Rare and Neuromuscular Diseases, based on a Material Transference Agreement with Pontificia Universidad Católica de Chile, and were maintained following the institutional biosecurity and bioethics protocols. All the cells were cultured in high glucose Dulbecco-Eagle modified medium containing sodium pyruvate  and supplemented with 10% of fetal bovine serum (FBS), 2 mM glutamine, and 100 U/ml penicillin, and 100 μg/ml streptomycin in humidified air (5% COprotocol .2+-sensitive proteins mtRCaMP (Kd ∼ 1 µM) and CEPIA3mt (Kd ∼ 10 µM). The transfection was performed with OPTI-mem (Thermofisher) or Transfectagro (Corning) and lipofectamine 2,000 or 3,000  according to the manufacturer´s protocol. For optimal expression, the cells were grown for 48 h after transfection. One μg of DNA per plasmid per 35 mm dish was used for each experiment.Cells were plated on glass 25 mm coverslips and then transfected with specific constructs using Lipofectamine 2,000–3,000 (Invitrogen). MEF cells were transfected pCCEY plasmid encoding for human OPA1 isoform 1. OPA1 GTPase or GED mutants were built in the same backbone. For mitochondrial matrix calcium analysis, we used mitochondrial matrix-targeted Ca2+ release from the ER. For SOCE, after Fura2-AM incubation, cells were washed once with a Ca2+ free buffer. Imaging was performed in a Ca2+-free ECM containing 0.25% BSA. Cells were stimulated by CCh 200 nM to activate the IP3R and induce Ca2+ release from the ER and recorded for 150s, then the cells were stimulated with Thapsigargin 2 µg to deplete the ER of Ca2+ and recorded for 300s, and finally, 1 mM of Ca2+ was added to induce SOCE. Images were acquired using an ImagEM EM-CCD camera (Hamamatsu) fitted to an Olympus IX81 microscope with LED source . The configuration for each fluorescent probe or protein was as follow: Fura2-AM was recorded using 340–380 nm excitation; mtRCaMP was recorded with a 577 nm excitation filter; CEPIA3mt was recorded using 485 nm excitation filter , using dual-band dichroic and emission filters . Image collection frequency, 1 Hz. Calibration of the Fura2-AM signal was carried out at the end of each measurement, adding 1 mM CaCl2, followed by 10 mM EGTA/Tris, pH 8.5. The Fura2-AM ratios were calibrated in terms of nM [Ca2+]cyto using the following formula:d was 224 nM, R is ratio 340/380 nm, Rmin is the ratio 340/380 after EGTA addition, Rmax is the ratio 340/380 nm after 1 mM CaCl2 addition, Fmax is the maximum 380 nm fluorescence upon EGTA addition. Fmin is the minimum 380 nm fluorescence upon 1 mM CaCl2 addition. Maximal [Ca2+]mito uptake was calculated as Fmax/F0 where Fmax is the maximum fluorescence intensity and F0 is the mean resting fluorescence intensity before IP3R stimulation or SOCE protocol induction.MEFs were transfected with the mtRCaMP, the muscarinic receptor type 3 (M3R), and OPA1 mutants. Forty-eight hours after transfection the cells were incubated in a serum-free extracellular medium  containing 2%BSA and loaded with Fura2-AM (2 µM) in presence of 0.0003% Pluronic F-127 and 100 μM sulfinpyrazone for 15 min at room temperature. Cells were washed once with ECM containing 2% BSA, and recorded in ECM containing 0.25% BSA, and transferred to the thermostated stage (37°C) of the microscope. Cells were stimulated by Carbachol (CCh) 200 nM to activate the M3R, and subsequently, the IP3R, to induce Ca2+]mito vs. [Ca2+]cyto analysis we plotted the mean trace curve from basal to maximal [Ca2+]cyto in X-axis, with regards to the same time points corresponding to [Ca2+]mito relative fluorescence from basal to maximal [Ca2+]cyto, in the Y-axis. For maximal IP3R-induced [Ca2+]cyto vs maximal [Ca2+]mito uptake we used the maximal [Ca2+]cyto ± SEM in the X-axis, and the maximal [Ca2+]mito uptake ±SEM, in the Y-axis. To quantify the changes in the [Ca2+]mito vs. [Ca2+]cyto we consider considered the [Ca2+]cyto at the moment of a 2-fold increase in [Ca2+]mito upon CCh stimulation. For SOCE, we considered the [Ca2+]cyto at the moment of a 1.5-fold increase in [Ca2+]mito for CCh stimulation.For mito vs. [Ca2+]cyto we considered the [Ca2+]cyto at the moment of a 1.4-fold increase in [Ca2+]mito upon Histamine stimulation.For ADOA-derived fibroblasts experiments, the cells plated on glass coverslips were transfected with mtRCaMP and loaded with Fura2-AM (2 µM). Cells were evaluated in 0.25% BSA-ECM containing and transferred to the thermostated stage (37°C) of the microscope. Live-cell image series was performed every 1.5s, in a Nikon Eclipse Ti inverted microscope with a Lambda DG4 wavelength-switch xenon light source (Sutter Instruments) and attached by iXon3 897 Andor EMCCD camera controlled by NIS software (Nikon). Cells were stimulated by Histamine to activate the IP3R and induce ER Cabasal—FFCCP using absolute fluorescence values.Cells plated on glass coverslips were washed with ECM and loaded 10 min at room temperature with Tetramethyl rhodamine methyl ester or Tetramethyl rhodamine ethyl ester probe (TMRM 20 nM for MEF cells or TMRE 10 nM for human fibroblasts) in non-quenching mode. Mitochondrial membrane potential in MEF cells was measured at the custom-built LED Olympus epifluorescence imaging system. TMRM was recorded using a 577 nm excitation filter, using dual-band dichroic and emission filters . Cells were recorded every 1s. ADOA-derived cells experiments were performed at the Nikon Eclipse Ti microscope, using the following configuration: ex. 540 nm—em. 620/60 nm, 1 image every 1.5s. FCCP 5 µM/Oligomycin 5 μg/ml (MEF cells) or FCCP 10 µM (ADOA-derived fibroblasts) was used to induce loss of mitochondrial membrane potential. Resting membrane potential was calculated as ΔF5 cells were fixed with glutaraldehyde 2.5%. Staining was performed as previously described . For MICU1, MICU2, and EMRE, were used IR LI-COR Bioscience secondary antibodies. Densitometry was performed using ImageStudio software (LI-COR Bioscience). Antibodies used in this study: OPA1, MICU1 , MICU2 and EMRE  MCU , mtHSP70 , TIM23 , TOM20 , TUBULIN , GAPDH .OPA1 c.899G>A (G300E) and GED mutant OPA1 c.2708delTTAG was kindly donated by Guy Lenaers and used in previous studies (The pCCEY plasmid containing human OPA1 isoform 1 WT was kindly donated by Guy Lenaers. Mutagenic OPA1 variants were designed using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) and following the manufacturer´s protocol. The actual generation of specific mutations was confirmed by sequencing using the ABI PRISM 3500 xl Applied Biosystems (FONDEQUIP EQM150077) at Pontificia Universidad Católica de Chile or sequenced at Macrogene inc., Seoul, South Korea. pCCEY plasmid encoding GTPase mutant  studies . For priOpa1−/− MEFs cells were transfected as described above with a lentiviral plasmid containing the human OPA1 isoform 1 and a puromycin resistance cassette (pLenti-OPA1). Transfected cells were selected with Puromycin 2 μg/ml to obtain a polyclonal stable cell line. The cells were grown in growth media described above in presence of Puromycin 2 μg/ml. The pLenti-OPA1 was obtained from VectorBuilder.t-tests were used for all normally distributed data, whereas Mann-Whitney tests were used for nonparametric data. For multiple comparisons, One-way ANOVA followed by Dunnett’s multiple comparison test was used to determine the significance of normally distributed data. For nonparametric multiple comparisons, a Kruskal-Wallis test followed by Dunn’s multiple comparison test was used to determine significance (p < 0.05). In all cases, data not indicated as significant should be considered not statistically different.Image acquisition and analysis were performed using Fiji (ImageJ) software. The statistical analysis was carried out using the GraphPad Prism 8 Software. For pairwise comparisons, unpaired


Plants were engineered with a reporter called CGf, a ratiometric, genetically encoded Ca2+ reporter with an mCherry reference domain fused to an intensiometric Ca2+ reporter GCaMP6f. Relative changes in [Ca2+]cyt were estimated based on CGf’s apparent KD around 220 nM. The ratiometric output provided an opportunity to compare Ca2+ dynamics between different tissues, cell types, or subcellular locations. In leaves, CGf detected heat-triggered cytosolic Ca2+ signals, comprised of three different signatures showing similarly rapid rates of Ca2+ influx followed by differing rates of efflux (50% durations ranging from 5 to 19 min). These heat-triggered Ca2+ signals were approximately 1.5-fold greater in magnitude than blue light-triggered signals in the same leaves. In contrast, growing pollen tubes showed two different heat-triggered responses. Exposure to heat caused tip-focused steady growth [Ca2+]cyt oscillations to shift to a pattern characteristic of a growth arrest (22%), or an almost undetectable [Ca2+]cyt (78%). Together, these contrasting examples of heat-triggered Ca2+ responses in leaves and pollen highlight the diversity of Ca2+ signals in plants, inviting speculations about their differing kinetic features and biological functions.Land plants evolved to quickly sense and adapt to temperature changes, such as hot days and cold nights. Given that calcium (Ca Multipl2+ signaling networks are implicated in most abiotic stress responses in plants  .Can plants . Howeverresponse . For exaoroplast . Some of7–13 μM) , which m2+ reporter to investigate heat-triggered [Ca2+]cyt changes in leaves and pollen tubes from Arabidopsis thaliana. This reporter, called CGf, was engineered with an mCherry fused to the N-terminal end of an intensiometric Ca2+ reporter GCaMP6f, and incorporates a ratio design feature similar to other ratiometric reporters comprised of two tandem fluorescent proteins  was transformed into Arabidopsis thaliana COL-0 using Agrobacterium tumefaciens (GV3101 strain) floral dip method (–2 s–1) followed by 8 h dark. For whole-plant heat stress imaging, seedlings grown in the above conditions for 10 days were transplanted to hydrated 36 mm diameter Jiffy-7 Peat pellets  and placed in Turface MVP  in Magenta vessel GA-7-3 . Plants grew additional 2 weeks (or until ready for imaging) under long day light cycles (16 h-light/8 h-dark) at 22°C.Hygromycin-resistant p method . SteriliUbiquitin10promoter:mCherry-GCaMP6f (plasmid stock 2935) was constructed through standard molecular techniques using a pGreenII vector system  and GCaMP6f (plasmid stock 3221) were cloned separately into a KanR pET28-Novagen vector (Sigma-Aldrich). Promoters used included a Ubiquitin 10 promoter .For plant expression, r system  with a hr UBQ10;  for pGrer UBQ10;  for pET2X2) unless stated otherwise. For seed set analyses, mature siliques were cleared by incubation in 70% ethanol at room temperature over 24 h.Transgene transmission was measured by scoring hygromycin resistance of F1 progeny from reciprocal outcrosses. Seeds were processed as described in section “Plant materials and Growth Conditions.” Statistical significance was determined using Pearson’s chi-squared test  and ORCA-Flash4.0 V2 Plus sCMOS digital camera . For ratio imaging, separate signals from GCaMP6f and mCherry domains were detected with filter set 38 eGFP shift free  and filter set 63 HE mRFP shift free .The same imaging equipment was used for pollen heat stress assays except with a PlanNeoFluar Z 2.3x/0.57x objective and a W-view Gemini Image Splitting Optics for simultaneous two fluorescence imaging  equipped with emission filter sets  and a 25.5 × 36 × 2 mm (W x L x H) T560lpxr-UF2 dichroic mirror .For high resolution imaging, pollen time lapse images were captured using a Leica DMi8 inverted microscope fitted with a Yokogawa CSU-W1 spinning disk confocal scanner module and a CCD camera. Images were captured with 63X/1.4 NA objective with filter switching .2+]cyt changes were monitored using wild type COL-0 plants stably expressing CGf. Analyses shown were conducted using ∼3-week-old rosettes when plants showed a minimum of 8–9 true leaves. Plants were allowed to adapt to dim light for at least one hour prior to the start of a blue light exposure at time –120 min (Ex470/40nm with 24 μmol m-2s-1 intensity). Images corresponding to GCaMP6f and mCherry were obtained every 5 sec for 5 h.Blue light- and heat-triggered  concentrations using a Calcium Calibration Buffer Kit (Invitrogen) with 30 mM MOPS pH 7.2, 100 mM KCL, and varying additions of 10 mM EGTA or 10 mM CaEGTA. Dilution series were performed as directed by the manufacturer’s protocol with absorbance measured using Shimatzu RF-6000 Spectrofluorometer. Fluorescence intensities were measured for GFP at Ex 488 nm/Em 512 nm.To measure Leaf tissue from 10-day-old seedlings, grown as described in section “Plant Materials and Growth Conditions” was collected and frozen in liquid nitrogen. Frozen tissue was ground into a frozen powder using prechilled mortar and pestle, adding homogenization buffer (HB) as needed . Homogenization buffer consists of 100 mM Tris pH 7.5, 150 mM NaCl, 290 mM sucrose, 10 mM imidazole, 10% glycerol, 0.1% Tween-20, EDTA-free protease inhibitor cocktail V , and 1 mM PMSF (phenylmethylsulfonyl fluoride). Plant extracts were filtered through cheesecloth prewet with HB. Filtered extract was spun in glass Corex tubes at 6,000 g for 15 min at 4°C to remove cellular debris. Crude plant extracts were analyzed using SDS-PAGE precast gels , followed by Western blot analysis of reporters using primary RFP monoclonal antibody (Thermo Fisher cat # 200-301-379S) and secondary F(ab’)2-Goat anti-Mouse IgG (H + L) antibody .2+ reporter was generated with an N-terminal mCherry followed by a linker and an intensiometric Ca2+ reporter GCaMP6f  (KD [Ca2+] of 220 nM) resulting from a potential Ca2+ dependent energy transfer from GCaMP6f to mCherry , the spectra for the Ca–650 nm) . An alte–650 nm) . In contTo evaluate whether the CGf fusion was proteolytically stable when expressed in plants, stable transgenic plants were generated that expressed CGf under the control of a UBQ10 promoter. Plants were chosen for analysis using fluorescence microscopy to confirm strong expression throughout the plant, including leaves and pollen. Protein extracts from leaves were subjected to SDS-PAGE. A Western blot analysis was conducted using a primary antibody recognizing mCherry. A single band was detected at a size expected for an intact mCherry-GCaMP6f fusion . This pr2+ reporters that harbor a calmodulin domain  to excite the GFP domain of CGf  to the newest  . This anTo evaluate whether there were signaling differences at the “single cell” level, images were also analyzed with smaller regions of interest (ROI)s in non-vein tissues . While t2+ signals in growing pollen tubes, two patterns of tip-focused [Ca2+]cyt oscillations were observed (KD 250 nM) (KD 450 nM) (steady growth Ca2+ (SGC) oscillations and arrested growth Ca2+ (AGC) oscillations. Time lapse images and a representative kymograph show a transition from SGC to AGC oscillations within the same pollen tube over a 18 min imaging window at room temperature   and GCaM 450 nM) . These tperature . SGC oscCa2+]cyt .2+ oscillations often displayed peak signal intensities that ranged from 70 to 100% of the Ca2+ saturated maximum. While these high signal ratios were outside the linear calibration range for CGf, they suggest that many signals reached magnitudes equal or greater than 960 nM [Ca2+]cyt, which corresponds to CGf’s estimated Ca2+ saturated maximum.It appeared that tip-focused Ca2+ oscillations, rapid [Ca2+]cyt transients of equal magnitude were also observed at dispersed locations in the pollen tube shank, as shown in time lapse images  oscillations prior to the heat exposure. Considering that a 35°C heat stress increased bursting frequency 100-fold, a milder heat stress exposure was applied by increasing the temperature from 22 to 32°C (1°C per 6 s for 1 min)  , a stresr 1 min) . In thisightfold .arrested growth Ca2+ (AGC) or a dampened Ca2+ (DC) oscillation, as shown in representative kymographs  . In addition, the aequorin reporter requires the addition of a substrate. Therefore, mixed results may be due to different efficiencies of substrate loading or other constraints on substrate/aequorin interactions.CGf-based analyses in leaves provide strong evidence that heat stress can induce cytosolic Can plants . Prior sn plants . In a reeedlings . Anotherng level , as expe channel . These m7–13 μM) , which m2+ induced Ca2+ release from internal Ca2+ stores  or a severely dampened oscillation with a nearly undetectable baseline [Ca2+]cyt (78% of cases).The CGf analyses with pollen provided an example of a heat stress response that appears very different than leaves. Unlike leaves, pollen tubes failed to show a heat-triggered increase in [Cabe shank . Instead2+]cyt oscillations occurred within a minute, correlating with a rapid and potentially thermo-protective switch to a pause in tip growth.While pollen fertility is considered highly vulnerable to heat stress, the underlying causes remain speculative and are likely different during various phases of pollen development and fertilization . It is n2+ signals. While it is not yet clear how many functionally different Ca2+ signals are generated in plants, pollen cells alone express at least 36 potential Ca2+-permeable ion channels, along with multiple kinetic-modifying Ca2+ pumps and exchangers located in various compartments including the vacuole, ER, plasma membrane and other membrane organelles  can be found in the article/in vitro spectrofluorometer studies. EM conducted effects of CGf expression to root, rosette, and seed sets development. MM conducted Western Blot analyses. GM and JH initiated the CGf reporter design for pollen. JH led the development of transgenic lines, reporter constructs, and genetic analyses. W-GC led the experiments on seedling imaging. W-GC and JH designed the experiments and supervised the overall project. All authors contributed constructive comments on the manuscript.CW led the experiments on pollen imaging and helped design all experiments. S-HK conducted whole-plant imaging in response to heat stress and data analyses. EB conducted the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.