Their relative tissue- and organelle-selective distribution, together with agonist-selective ion permeabilities provides a rich palette from which extracellular stimuli can choose. TPCs are emerging as mediators of immunity, cancer, metabolism, viral infectivity and neurodegeneration as this short review attests.In recent years, our understanding of the structure, mechanisms and functions of the endo-lysosomal TPC (two-pore channel) family have grown apace. Gated by the second messengers, NAADP and PIP That is, in addition to their roles of trafficking cargo, repairing membranes, degrading macromolecules and nutrient sensing, acidic vesicles generate Ca2+ signals. The purpose of this article is to update our previous overview [2+-permeable channels found on such acidic vesicles — the TPCs (two-pore channels) — and we confine our remarks to the mammalian channels.These Hoverview  with mor2+] within endosomes is tens of micromolar, whereas in lysosomes it is ∼300–600 µM  is ∼12 mM, the lysosomal luminal [Na+] is reported from 21 mM  and TPC2-A1-P [PIP2 mimetic]), and are selective for TPC2 over TPC1 and TRPMLs [Historically, activation of TPCs in intact-cell populations has been limited to NAADP delivery via liposomes ,72 or ce of TPC2 . Each mid TRPMLs . TPC2-A1d TRPMLs   [Interestingly, photo-release of another lipid, sphingosine, acutely evoked Caot TPC2) ; is thisot TPC2) , but the2+ transients in myocytes [2 [2 [Of broad interest, the mTOR inhibitor, rapamycin, evokes TPC2-dependent Camyocytes  and promcytes [2 . Likewises [2 [2 ,78. Whiles [2 [2 ,76,79.Other signalling inputs may interact with TPCs including protein kinases such as LRKK , JNK/p382 [2+ [+ (ΔΨ) [In the global population, TPC2 naturally occurs with a spectrum of different polymorphisms  and some2 ,76,83 or2 . However2 [2+ , Na+ (ΔΨ [+ (ΔΨ) ,83 and p [+ (ΔΨ) ,56,83 ha2+ signals converted (decoded) into downstream responses? A common theme is that TPCs affect vesicle formation, trafficking, maturation and movement, many of which are sensitive to Ca2+ and lysosomal membrane potential. Being small Ca2+ stores, endo-lysosomes are designed to generate local rather than global Ca2+ signals, and TPCs couple to downstream physiology via local Ca2+ signals for which other Ca2+ sources cannot substitute, as exemplified by phagocytosis [2+-decoders include Ca2+-dependent channels, protein kinases/phosphatases and membrane-fusion machinery.How are TPC-dependent Caocytosis ,42, exococytosis , membranocytosis , receptoocytosis ,87, deveocytosis , vesiculocytosis ,89. Ca2+2+ release, TPCs trans-activate Ca2+-regulated ion channels on other membranes that are closely apposed, probably at MCSs. Via Ca2+-induced Ca2+ release (CICR), IP3Rs or RyRs on the ER can amplify the small Ca2+ release from endo-lysosomal TPCs to evoke global Ca2+ signals [2+-sensitive channels  depolargnalling  under thgnalling . Althougtivation .2+ signals are otherwise decoded is underexplored. Privileged TPC-coupling to downstream processes implies that Ca2+-sensitive decoding proteins are intimately associated with TPCs and sense these high Ca2+ nanodomains. Several TPC interactomes have been published . Phagocytosis is uniquely driven by local Ca2+ from TPCs  [2+-dependent phosphatase, calcineurin, may be the Ca2+ decoder [How local TPC Caiewed in ), but susignals) , where t decoder .2+-binding decoders are growing, and the GTPase, dynamin, has been linked to the NAADP/TPC axis during ‘inward’ trafficking at phagocytosis [2+-binding proteins. In neuronal cells, the NAADP/TPC axis activates AMPK during autophagy [The molecular switches downstream of the immediate Caocytosis  and endoocytosis . Regardiocytosis , althougutophagy . In melautophagy . Affirmiutophagy .Our appreciation of the importance of TPCs is expanding, but the following, recent examples incidentally reinforce that the precise molecular details of the circuitry are often lacking and we do not know the messenger, permeant ion or the decoders. Our understanding of the roles of TPCs is still in its infancy.2+ signals [TPCs contribute to neuronal homeostasis . The neu signals  which in signals . TPCs ar signals ,101, neu signals  and axon signals ,102. AbeIn the vasculature, the role of TPCs is growing and, in particular, at vasculogenesis. The proliferation of endothelial precursors cells is dependent upon NAADP and TPC1 ,103, andMetabolically, the nutrient-sensing kinase complex of mTOR inhibits TPC2 which thereby responds to nutrient status . Recipro2+ circuitry to regulate vesicular trafficking events in an immune context [TPCs are abundantly expressed in immune cells and are involved in often complex Ca context . Extrace context  and macr context  in macro context , of cyto context  and the  context . Thus, TGiven their physiological roles — particularly of membrane and protein trafficking — TPCs are implicated in a wide range of diseases that are a significant health burden . For neu2+ signals at MCSs [2+ release [TPCs contribute to cardiovascular complications. In blood vessels, TPCs exacerbate hypoxia-induced hypertension ,118 or m at MCSs , and TPC at MCSs . Similar release  that mig release ; therefo release ,122.Pathogens deliver toxins and/or enter the host cells to replicate, often gaining access via the endocytic pathway where they traffic through the endo-lysosomal system by co-opting host pathways. Since TPCs regulate vesicular uptake pathways  ,42,123 aAn increasing field is that of TPCs in cancer  where, rIn this brief overview, we have highlighted the diversity of both the ionic nature of the TPC signals and the breadth of the (patho)physiological processes in which TPCs play an important role, and this is only set to grow. A common theme is the involvement of TPCs in vesicular trafficking. With still so many unknowns, and the likely intersection with hitherto unsuspected pathways, the field of endo-lysosomal ionic signalling will continue to be a rich source to mine.2+-permeable channels that are emerging as important signal transducers across biology and phyla.TPCs are endo-lysosomal CaUnusually, their ion conductances depend on the stimulus: they are plastic channels.2+, electrical, lysosomal pH).Different conductances confer the ability of TPCs to signal in different modalities (e.g. CaNew TPC protein regulators have recently emerged.


Methods: Electromechanical analyses of rabbit atrial tissues were conducted. Intracellular Ca2+ monitoring using Fluo-3, the patch-clamp method for ionic current recordings, and a fluorescence study for the detection of reactive oxygen species and intracellular Na+ levels were conducted in enzymatically dissociated atrial myocytes. Results: Ziprasidone-treated atriums showed sustained triggered activities after rapid pacing, which were inhibited by KN-93 and ranolazine. A reduced peak L-type Ca2+ channel current and enhanced late Na+ current were observed in ziprasidone-treated atrial myocytes, together with an increased cytosolic Na+ level. KN-93 suppressed the enhanced late Na+ current in ziprasidone-treated atrial myocytes. Atrial myocytes treated with ziprasidone showed reduced Ca2+ transient amplitudes and sarcoplasmic reticulum (SR) Ca2+ stores, and increased SR Ca2+ leakage. Cytosolic and mitochondrial reactive oxygen species production was increased in atrial myocytes treated with ziprasidone. TNF-α and NLRP3 were upregulated in ziprasidone-treated myocytes, and the level of phosphorylated calcium/calmodulin-dependent protein kinase II protein was increased. Conclusions: Our results suggest that ziprasidone increases the occurrence of atrial triggered activity and causes intracellular Ca2+ and Na+ dysregulation, which may result from enhanced oxidative stress and activation of the TNF-α/NLRP3 inflammasome pathway in ziprasidone-treated myocytes.Background: Atypical antipsychotics increase the risk of atrial arrhythmias and sudden cardiac death. This study investigated whether ziprasidone, a second-generation antipsychotic, affected intracellular Ca Ziprasidone is a second-generation antipsychotic (SGA) used to treat schizophrenia and bipolar disorder . In clin2+ cycling, remains poorly understood, and further investigation is needed [While antipsychotics’ involvement in atrial arrhythmias has not been well investigated, a national database analysis demonstrated that exposure to antipsychotics, especially SGAs, increased the occurrence of AF . While as needed .2+ and Na+ homeostasis, and levels of oxidative stress/inflammasomes under ziprasidone treatment. We hypothesized that ziprasidone caused intracellular Ca2+ and Na+ dysregulation and oxidative stress, leading to atrial arrhythmogenesis. Our results suggest that ziprasidone impaired homeostasis of intracellular Ca2+ and Na+ homeostasis, enhanced reactive oxygen species (ROS) production, and activated the tumor necrosis factor alpha/NOD-, LRR-, and pyrin domain-containing protein 3 (TNF-α/NLRP3) inflammasome signaling pathway in atrial myocytes, which provides proarrhythmogenic substrates.The objective of the present study was to evaluate the atrial electrophysiological characteristics, cardiomyocyte CaAll the animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the National Defense Medical Center, Taipei, Taiwan (IACUC-22-061), and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals guidelines of the National Institute of Health.Male New Zealand white rabbits  were used in the present study. The rabbits were kept in stainless steel cages in a controlled environment  under a 12:12 h light–dark cycle with ad libitum access to standard food and deionized drinking water.3, 0.5 NaH2PO4, 0.5 MgCl2, 2.7 CaCl2, and 11 dextrose gases, with a mixture of 95% O2/5% CO2. The dissected LA tissue was pinned with needles onto the bottom of a tissue bath. The other end of the LA tissue was connected to a Grass FT03C force transducer  with silk thread. The epicardial side of the LA preparations faced upward. The preparations were superfused with normal Tyrode’s solution at a constant rate (3 mL/min) and saturated with a 95% O2/5% CO2 gas mixture. The bath temperature was maintained at 37 °C. Before the electrophysiological assessments, the preparations were allowed to equilibrate for 1 h in the bath.The rabbits were anaesthetized using an intramuscular injection of a mixture of zoletil 50 (10 mg/kg) and xylazine (5 mg/kg), with an overdose of inhaled isoflurane (5% oxygen) in a vaporizer, after which they were killed. The anesthetic dose was confirmed as adequate after the rabbits did not exhibit corneal reflexes and motor responses to pain stimuli induced with a scalpel tip. After heparin (1000 units/kg) was administered intravenously, the hearts were harvested through midline thoracotomy, as described previously . For the20, APD50, and APD90, respectively. Atrial preparations were acutely perfused with ziprasidone (100 μg/mL) at a constant rate to assess the treatment responses. The rapid atrial pacing (RAP) protocol (a 20 Hz pacing rate for 1 s) was performed with or without ziprasidone to evaluate the triggered electric activity evoked by ziprasidone treatment. Triggered activity was defined as the occurrence of spontaneous APs in the absence of electrical stimuli. Burst firing was defined as the occurrence of accelerated spontaneous APs. The occurrence rate, sustained frequency, and duration of the triggered activity after RAP were recorded.The transmembrane action potentials were recorded using glass microelectrodes (filled with 3M KCL) connected to a WPI Duo 773 electrometer , as described previously . The sig2, 0.5 mmol/L MgCl2, 5.4 mmol/L KCl, 10 mmol/L glucose, and 10 mmol/L 4-(2 Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), pH adjusted to 7.4 with NaOH) for 10 min, and were then digested using a Ca2+-free solution ) containing 300 units/mL of collagenase  and 0.25 units/mL of proteinase  for 8 to 12 min. After perfusion, the hearts were removed from the cannulas, and the left atriums were excised. The left atriums were cut into small pieces, gently triturated using a plastic transfer pipette in 50 mL of Ca2+-free solution and filtered through a nylon mesh until single cardiomyocytes were obtained. The solution for the dissociated cells was then gradually changed to NT solution. The isolated cells were stored in NT solution at 20–22 °C and were allowed to stabilize in the solution for at least 30 min before the experiments. Rod-shaped myocytes with clear striation and without granulation were used within 6–8 h. To test the effects of ziprasidone, the cells were incubated with ziprasidone (100 μg/mL) for 30 min before intracellular Ca2+ monitoring, electrophysiological measurements, assessing intracellular Na+ levels, and detecting reactive oxygen species (ROS).Atrial myocytes were enzymatically dissociated, as previously described, with some modifications . BrieflyCa,L was composed (in mM) of 130 CsCl, 1 MgCl2, 5 MgATP, 10 HEPES, 0.1 NaGTP, and 5 Na2-phosphocreatine (pH adjusted to 7.2 with CsOH). The filling solution of microelectrodes for INa,L was composed (in mM) of 130 CsCl, 4 Na2ATP, 1 MgCl2, 10 EGTA, and 5 HEPES (pH adjusted to 7.3 with NaOH). The ICa,L was determined as inward currents during voltage-clamp steps from a holding potential of –50 mV to potentials from –40 to +60 mV in steps of 10 mV for 300 ms at a frequency of 0.1 Hz using a perforated patch clamp with amphotericin B as described previously [Ca,L was assessed between 5 and 15 min after formation of perforated whole-cell patch in each cardiomyocyte, to avoid ‘run-down’ effects [Na,L, a step/ramp protocol  was used. The INa,L was determined as the tetrodotoxin (30 µM TTX)-sensitive current, obtained when the voltage was ramped back to –100 mV as described previously [The ionic currents of the atrial myocytes were measured using whole-cell configuration patch-clamp techniques with an Axopatch 1D amplifier , as described previously . A smalleviously . The ICa effects . To meas2+ indicator (10 μM Fluo-3 AM) at room temperature for 30 min, and fluorescent imaging was performed as previously described [F) against the baseline fluorescence (F0) to obtain reliable data about transient intracellular Ca2+ changes, denoted as (F − F0)/F0, and to exclude variations in the fluorescence intensity caused by different volumes of injected dye. Ca2+ transients were elicited using field stimulation at 2 Hz. SR Ca2+ stores were measured by fast application of 20 mM caffeine following a pulse stimulation train at 2 Hz for 30 s. The SR Ca2+ stores were determined from the peak amplitudes of the caffeine-elicited Ca2+ transient. The SR Ca2+ leak was measured as the tetracaine (1 mM)-induced reduction in intracellular Ca2+, as previously described [2+ transients with repeated pulses (2 Hz for 15 s), the superfusate was rapidly changed to a 0 Na+/0 Ca2+ solution, composed of (in mM) 140 LiCl, 0.5 MgCl2, 5.4 KCl, 10 glucose, 10 EGTA, and 10 HEPES ) containing 1 mM tetracaine, which was applied for a minimum of 20 s to produce reduction in intracellular Ca2+Atrial cardiomyocytes were incubated with Caescribed ,18. Brieescribed . Briefly+ level, respectively. The measurements were carried out using an inverted laser-scanning confocal microscope  as previously described [Atrial myocytes were incubated with 10 μM CellROX green, 2 μM MitoSOX Red , and 5 µM Asante NaTRIUM Green-2 AM  in NT solution to assess the cytosolic and mitochondrial ROS production and the cytosolic Naescribed . Light wescribed .2+ regulatory proteins, including NF-κB (p65), TNF-α, NLRP3, sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a), and calcium/calmodulin-dependent protein kinase II (CaMKII). Whole-cell lysates were prepared from left atrial cardiomyocytes. The cell lysis buffer was composed of 100 mM Tris-HCl (pH 8.0), 0.1% sodium dodecyl sulfate, 1% Triton X-100, 150 mM NaCl, and a protease inhibitor cocktail . The myocytes protein extracts were separated by conventional gel electrophoresis, and were then transferred to polyvinylidene difluoride membranes . After blocking with 5% non-fat milk using TBST (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20) at room temperature for 1 h, the membranes were incubated with the following primary antibodies: NF-κB , TNF-α , NLRP3 , SERCA2a , p-SERCA2a , p-CaMKII , and GAPDH . Subsequently, the membranes were incubated with anti-mouse  or anti-rabbit  secondary IgG antibodies. Immunoreactive proteins were detected using enhanced chemiluminescence  and analyzed using the ImageJ software 1.5 .Western blotting was carried out to assess the levels of cytokines, inflammasome markers, and Cat-tests or Pearson’s chi-square tests using SigmaPlot version 12  were used to exam the differences between treatments. The ‘n’ represents the numbers of total cells from the total hearts (n = cells/hearts), and the ‘N’ is the animal number. Statistical significance is denoted as *, **, and *** for p < 0.05, p < 0.01, and p < 0.005, respectively.Student’s 20, APD50, or APD90 in the atrial tissues  in the atrial myocytes was enhanced after ziprasidone treatment  in the atrial cardiomyocytes was higher after ziprasidone treatment .We then tested if ziprasidone affected the myocyte Careatment A. The IN2+ regulation machinery was attenuated by ziprasidone. The steady-state Ca2+ transient amplitudes in atrial myocytes were smaller after treatment with ziprasidone    B. The mi < 0.05) C.While the protein levels of NF-κB (p65) did not change in ziprasidone-treated myocytes, the expression of TNF-α and NLRP3 was upregulated A. Zipras2+ and Na+ homeostasis. Our data demonstrate that ziprasidone provoked post-RAP atrial triggered activities, which were suppressed by KN-93 and ranolazine. Ziprasidone reduced the ICa,L and enhanced the INa,L, as well as increased the intracellular Na+ level. Ziprasidone decreased the Ca2+ transient and SR Ca2+ stores, increased SR Ca2+ leakage, and enhanced both cytosolic and mitochondrial ROS production in atrial cardiomyocytes. TNF-α/NLRP3 inflammasome signaling was upregulated in ziprasidone-treated myocytes. These results suggest that ziprasidone impairs the regulation of Ca2+, enhances oxidative stress, and activates inflammatory signaling, which leads to the occurrence of arrhythmic events in atriums. These findings help to elucidate the mechanisms underlying atrial arrhythmias associated with ziprasidone treatment  opening, leading to increased SR Ca2+ leakage [2+ leakage, which depletes SR Ca2+ and increases the cytosolic Ca2+ concentration [2+ leakage in the ziprasidone-treated myocytes. Furthermore, CaMKII-mediated SR Ca2+ leakage has been shown to promote AF in a mouse model [2+ in cardiomyocytes may trigger ectopic electrical activity, such as EADs/DADs, and, consequently, life-threatening arrhythmias [+ and Ca2+ regulation by phosphorylating the related proteins and channels [Na,L inhibitor ranolazine, which further confirmed the arrhythmogenic role of the upregulated CaMKII and INa,L induced by ziprasidone.Ziprasidone-treated atrial myocytes had smaller Ca+ stores . Deplete leakage ,24. The ntration ,26, whicse model . The ovehythmias ,29,30. Tchannels ,32,33,34Na,L contributes substantially to cytosolic Na+ levels [2+ into the cytosol and, as a result, depolarize the membrane potential and increase the probability of ectopic electric activities [Na,L may result from the phosphorylation of the channel Nav1.5 by upregulated CaMKII [Na,L inhibitor, ranolazine, which has been shown to reduce intracellular Na+-dependent Ca2+ overload and ameliorate arrhythmic events [Na,L, thereby stimulating arrhythmogenesis [Na,L in ziprasidone-treated atrial myocytes.Enhanced I+ levels , which mtivities ,37. Incrd CaMKII ,38,39,40c events ,42. Furtogenesis , which m2+ leakage-related overaccumulation of cytosolic Ca2+ may result in a mitochondrial overload of Ca2+, which, in turn, would exacerbate mitochondrial ROS production. Increased ROS production has been shown to activate CaMKII, which enhances INa,L and impairs intracellular Ca2+ regulation, leading to arrhythmias [Oxidative stress has been shown to increase the risk of AF ,45. Our hythmias ,48. Whilhythmias ,50, treahythmias . This imOur data show that ziprasidone-treated atrial myocytes presented higher levels of TNF-α and NLRP3. Upregulated NLRP3-inflammasome signaling has been shown to promote AF ,53. Zipr+ current, it also inhibits the delayed rectifier potassium current (Ikr) and adrenergic receptors in animal models [2+ channel in a CaMKII-independent manner [Na,L and SR Ca2+ leak, which, as a result, producing antiarrhythmic effect in the ziprasidone-treated atriums. ICa,L were decreased in the ziprasidone-treated myocytes. Therefore, the inhibition effect of KN-93 on L-type Ca2+ channel may not be responsible for the primary anti-arrhythmic mechanism in the ziprasidone-treated atriums.Finally, some methodological weaknesses in the present study need to be acknowledged. While ranolazine has been well used as an inhibitor of late Nal models . Howeverl models . The widt manner . Our dat2+ and Na+ homeostasis in atrial myocytes, leading to a higher occurrence of atrial tachyarrhythmias. Oxidative stress and NLRP3 inflammasomes were enhanced in the ziprasidone-treated myocytes. KN-93 and ranolazine inhibited the atrial arrhythmic events. These findings suggest that ziprasidone causes intracellular Ca2+ and Na+ dysregulation, which may result from enhanced oxidative stress and an activated TNF-α/NLRP3 inflammasome pathway in ziprasidone-treated myocytes, providing proarrhythmic substrates in atriums.In conclusion, ziprasidone promoted post-rapid-pacing atrial triggered activities. Ziprasidone exposure affected Ca


This paper reports on Indigenous early career researchers’ experiences of mentoring in Australian higher education, with data drawn from a longitudinal qualitative study. Interviews were conducted with 30 Indigenous participants. A consistent theme in the findings and contemporary critical literature has been a reaction against institutionalised and hierarchical cloning and investment models of mentoring that reinforce the accumulation of White cultural capital, in favour of strength-based relational models tailored to build Indigenous cultural wealth in parallel with career development. We write from an equity-based standpoint addressing mentoring as a complex and raced space where individual Indigenous ECRs articulate a desire and will to develop a successful and meaningful career, rich in cultural wealth and with their identity intact. It is our intent that these findings will also have global significance and support the more sustainable and ethical career development of First Nation early career academics in relationally like colonised contexts. A review of literature about mentoring in higher education reveals an iterative research space describing the theory behind and the efficacy of mentoring paradigms and practices  Yosso, . CCW is CCW nevertheless represents and highlights Indigenous cultural resources for empowerment, collective struggles and the Indigenous principles of respect, relationality and reciprocity, stewardship and paying it forward Brayboy, . CulturaIndigenist research methodologies underpin this study and refer to a research paradigm that respects and honours Indigenous epistemologies, ontology and axiology. Indigenist paradigms are relational and grounded in the principles of an emancipatory imperative, political integrity and the privileging of Indigenous voices Rigney, . Smith  contendsInterviews are being conducted over a 3-year period, with the first round commencing in 2020. This paper reports on this early-stage data of the first round of interviews. Purposeful sampling was conducted (Punch et al., The demographic distribution of the participants is diverse. The 30 participants represent at least 35 Aboriginal and Torres Strait Islander Nations, employed in 21 universities across Australia at the time of writing and working across a range of faculties (Locke et al., All interviews in the first stage of the study were conducted online and voice recorded using Zoom technology due to the limitations imposed by Covid-19 restrictions in Australia at that time. Despite the necessity of conducting interviews online, using an Aboriginal Yarning method (Bessarab et al., first time running a grant, and navigating the system to learn the institution's rules. Though more experienced in the sector, Maree also deemed being mentored by an experienced colleague as a key factor in successfully navigating in the sector:They're the ones that have been in the game for a long time and used to winning these big grants so learning from them the ropes is paramount, as well.Navigating the workplace was a key issue raised by participants and deemed to be an area where mentoring was most helpful; this is despite the varied demographic of the participants. As a newly conferred ECR, Sarah sought knowledge through formal mentoring about institutional mechanisms, such as So it has been my peers that I’d walk in to their office and go, what do I do now? How do I construct this email?Similarly, Patricia identified an ongoing need for support to navigate the system, predominantly through informal mentoring structures:I feel like I get mentored by so many other people in - just by observing and watching, as well. That's got to be included as mentorship, because that's their knowledge and their wisdom, their experiences are benefitting me just by being an observer.For these participants, who came into the ECR role with only some professional knowledge, operational knowledge was a requisite factor for effective mentoring in terms of learning how to navigate the hard metrics of the system. Operational mentoring was not restricted to formal or dyadic mentoring. As an ECR with more experience in the sector, Cate contended:How do I take this and turn it into really good journal articles and then where do I send them - Which publishers are going to work best for me. I'm trying to learn that by doing some co-writing on some action research.Jessie, reflected on co-writing with experienced colleagues as a highly valued experience:Either through formal mentoring, breaking down the fabrication of power in institutions that were not created with Indigenous academics in mind, or creating supportive collegial relationships, ECRs used initiative and navigational skills to position themselves favourably in the sector.I think good mentorship is that capacity building, and not being competitive - actually having a genuine investment in the improvement of our experiences within this white institution.However, in a sector that is not created by or for Indigenous academics, participants also identified systemic and institutional racism, and a hostile culture in the sector, as problematising mentoring experiences. The competitive culture embedded in the institutional meritocracy is echoed in mentoring relationships between Indigenous mentees and non-Indigenous mentors. Eli explained:toxic, in its competitiveness. This toxicity is commonly associated with politics of the institution grounded on meritocracy and individual success; the ways it is set up. Moreover, toxicity is also attributed to racial discrimination. Stopping short of calling it lateral violence, Wiraga’s story tells of the ominous presence of unequal power relations. They identify key challenges as:Negotiating the bureaucracy and the positioning of [tribal affiliation] perspectives against [...] how that can actually manifest. You're always having to watch your back. It's the politics of place.Eli continued, describing the institutional culture as I think mentorship needs to avoid as much as possible […] hierarchy […] So, you go seeking mentorship, but it's not always to your benefit. It's usually to build - because you are, within a white institution, and you're surrounded by non-Indigenous Peoples. You're actually building their profile, rather than building your ownJames’ concerns also centred on hidden agendas, and hierarchical structures, proposing:I would really like an Aboriginal mentor, because there’s just stuff that I have to encounter that other people don’t encounter. Trying to get around it is really challenging.Participants were forthright in calling for culturally relevant and responsive mentoring that addressed the particularities of an Indigenous ECR in a Eurocentric institution. A paucity of Indigenous academics to fill the role of mentors then is of great concern: Lee explained why this matters:lack on the ground of Indigenous mentors. Additionally, Olive was disappointed about the paucity of Indigenous mentors for research with time to actually help me with anything. Participants also propose cultural awareness was absent from their experiences of mentoring, with Olive arguing: How do I make this argument that Aboriginal people think [it] is important?Olive, who had not received any form of formal mentoring, bemoaned the So, I think if we're mentoring Aboriginal and Torres Strait Islander, we need to have a little bit more understanding of the triggers that might be there for them. I think we also need to know how to advocate on behalf of them and who to go to if it's outside our expertise. I think we do need to have downtime to be able to be just people with each other, you know, and look after each other. I think that's really important because I think our mental health is just as important as well as our spiritual health - to think about how to get things done on time, the timeline stuff.Despite extensive experience in higher education, Julunybarr understood the specific needs of Indigenous academics who are building careers. They explained the significance of an Indigenous mentor who has a shared history and a shared understanding of the place of culturally congruent relationships in mentoring. In particular, Julunybarr spoke about the importance of caring and nurturing, of spiritual health:Fundamentally it’s about a relationship There’s a reciprocal nature to it which implies the whole thing around authority and power as well and I think there’s got to be some – there’s got to be a de-centring of that somewhere in that relationship for me [...] the relationship is the key and especially with our mob there’s shared contexts and understandings, and things like that that differentiate the relationships that I have, mentoring relationships that I have with Aboriginal people than I do with non-Aboriginal people.Skywalker also addressed relationships:, Wirraga placed high value on the place of Indigenous perspectives on the sector and the passing on of this knowledge between mentor and mentee:I think it's helping, it's the tricks along the way of interpretation, interpretation of the bureaucracy that is the university and it's also through a cultural lens, through engaging in Indigenous space.SimilarlyBut I've been blessed in other ways through relationships and community members. There's a number of community members that were basically my support system. Most of my mentoring's probably in my community. There's a couple of aunties and a couple of uncles and brothers that I get to talk to a lot.Shaun and Areau presented a familial understanding of mentoring, one that acknowledges Indigenous ways of intergenerational knowledge transmission and the place of community and Elders in knowledge production. Shaun statedThe academy don't understand community in the same kind of way so they don’t see it as that constant teaching and learning that our community mentors, that our community engages with, that our Elders expect of us that we would pass on our knowledge.Areau’s contribution also acknowledged the significance of community in supporting an academic career, clearly articulating why Indigenous mentoring matters:Sometimes grown from frustration and disappointment, yet always driven by a determination to shape positive change, the 13 participants cited in this paper have, in the spirit of collegiality and trust, shared their perceptions and personal experiences of mentoring. As advocates for Indigenous equity in education, these proud Indigenous ECRs are shining the light on improved and just mentoring practices that are equity-based, collaborative, non-hierarchical and relational.Findings from this study, as voiced by the 30 Indigenous ECR participants, confirm the criticisms made by critical, Indigenous and critical race theory scholars of mainstream mentoring. Calling for culturally responsive equity-based approaches, Indigenous ECRs are challenging the ahistoricism that decontextualised mentoring, advocating for the acknowledgement of Indigenous epistemologies, axiology and ontology within the sector Martin, . ParticiThe following discussion is grounded in acknowledgement of individual and cultural uniqueness. We do not propose a singular approach as we accept the inherent caveat that as the backgrounds, academic trajectories and individual needs of Indigenous mentees vary, so will mentoring needs also vary. This position also acknowledges the limitations of a one-size-fits-all model of institutionalised hierarchical mentoring structures (Alarcón & Bettez, Formal mentoring is one such approach seen as potentially problematic by participants, a position confirmed by scholars critical of investment and cloning models of mentoring (Blackburn et al., In place of hierarchical models that sustain racial injustice and lack of parity in higher education, Indigenous ECRs champion non-hierarchical models that are collaborative, include multiple mentoring relationships that align with Indigenous ways of being, knowing and doing Martin, , and culParticipants confirmed equity-based literature and Indigenous academic leaders by promoting mentoring built on and calling upon community knowledge. Loban  contendsIn sum, Indigenous ECRs are telling of resistance to an imposed view, questioning who is holding the ropes and expressing a desire for Indigenous academics, family and community to be involved in holding reins by using “our own processes to articulate our experiences, realities and understandings” Martin, , p. 211.Findings in this study show unambiguous calls for Indigeneity to sit at the heart of equity-centred mentoring paradigms for Indigenous ECRs. A consistent theme in the findings and contemporary critical literature has been a reaction against institutionalised and hierarchical cloning and investment models of mentoring that reinforce the accumulation of White cultural capital, in favour of strength-based relational models tailored to build Indigenous cultural wealth in parallel with career development. As described by the participants, Indigenous mentors are highly sought after and valued by Indigenous ECRs, who seek equity and acknowledgement that what they bring with them to the institution can be used to benefit and protect the richness of unique cultural identities. With a desire to build on and extend critical mentoring practices in the Australian context to incorporate Indigenous ways of knowing, being and doing, participants in this study are aspirational in the intent to build a robust Indigenous academic community.To conclude and complete the circle, we return to the beginning of our paper with a reminder of the disparity in the number of Indigenous academics in Australian universities, an issue of concern because fewer Indigenous academics in the sector means fewer Indigenous academics available for mentoring Indigenous ECRs. As a closing comment, we propose because the disparity in Indigenous academics impacts negatively on ECR career trajectory, the core business of universities must be to support increased numbers and retention of Indigenous academics with secured funding for Indigenous strategic plans, by embedding Indigenous leadership in the sector (Trudgett et al.,


Mycobacterium tuberculosis (Mtb) outer membrane remain mostly unknown and, in the absence of classical porins, likely involve specialized transport systems. Calcium ions (Ca2+) are an important nutrient and serve as a second messenger in eukaryotes, but whether bacteria have similar Ca2+ signaling systems is not well understood. To understand the basis for Ca2+ transport and signaling in Mtb, we determined Mtb’s transcriptional response to Ca2+. Overall, only few genes changed expression, suggesting a limited role of Ca2+ as a transcriptional regulator. However, 2 of the most strongly down-regulated genes were the pe15 and ppe20 genes that code for members of a large family of proteins that localize to the outer membrane and comprise many intrinsically disordered proteins. PE15 and PPE20 formed a complex and PPE20 directly bound Ca2+. Ca2+-associated phenotypes such as increased ATP consumption and biofilm formation were reversed in a pe15/ppe20 knockout (KO) strain, suggesting a direct role in Ca2+ homeostasis. To test whether the PE15/PPE20 complex has a role in Ca2+ transport across the outer membrane, we created a fluorescence resonance energy transfer (FRET)-based Ca2+ reporter strain. A pe15/ppe20 KO in the FRET background showed a specific and selective loss of Ca2+ influx that was dependent on the presence of an intact outer cell wall. These data show that PE15/PPE20 form a Ca2+-binding protein complex that selectively imports Ca2+, show a distinct transport function for an intrinsically disordered protein, and support the emerging idea of a general family-wide role of PE/PPE proteins as idiosyncratic transporters across the outer membrane.The mechanisms by which nutrients traverse the  How does Mycobacterium tuberculosis transport nutrients across its outer cell membrane? This study shows that a member of the large PE/PPE protein family imports calcium ions, suggesting a family-wide role as highly atypical bacterial nutrient transporters. Among second messengers, calcium ions  is surrounded by a highly impermeable outer cell wall that is composed primarily of the complex phthiocerol dimycocerosates (PDIMs) that form an outer membrane [Mtb. The impermeable outer membrane and lack of porins raise the question how Mtb transports small molecules such as nutrients, metabolites, but also Ca2+ across the outer membrane. In this way, the very first step in Mtb Ca2+ signaling remains unknown.membrane , creatinmembrane , no equiThe mycobacterial PE/PPE proteins have long been a mystery. They are predominantly found in pathogenic mycobacteria, where they take up a substantial share of the coding capacity , and man2+-mediated processes in Mycobacterium tuberculosis. We identified regulation of ATP levels and biofilm formation by Ca2+. The transcriptional response to Ca2+ was narrow, and the most highly regulated genes were pe15 and ppe20. We show that PE15/PPE20 form a complex, directly bind Ca2+, and facilitate the influx of Ca2+ into Mtb across the outer membrane. These data point to a functional Ca2+ sense-and-response system, identify physiologic processes regulated by Ca2+ and identify a new, specific PE/PPE Ca2+ import system across the outer membrane.Here, we sought to further explore Ca2+ in Mtb physiology is almost entirely unknown. We initially tested for parallels with Ca2+ effects on other bacteria. Ca2+ transport in Escherichia coli depends on ATP, and Ca2+ in turn increases intracellular ATP levels [2+ has a similar effect on ATP levels in Mtb, we exposed Mtb to increasing concentrations of extracellular Ca2+ and measured the intracellular ATP levels. ATP levels increased by >2.5-fold in the presence of 1 mM Ca2+ and by >4-fold in the presence of 10 mM Ca2+  and ppe20 (Rv1387)  . This bepe15 and ppe20 genes are also co-operonic, suggesting that they could be a functional protein pair. According to previously published data, recombinant expression of PE15 and PPE20 individually in E. coli failed to produce soluble protein [Several PE/PPE proteins have been shown to form protein complexes, typically those coding in the same operon . The pe12+, we incubated recombinantly expressed PE15/PPE20 with Ca2+ and tested for protein stability using a thermal shift assay with a gel readout   . Controla2+ Figs  and S2.Mtb cell fractions. PPE20 was only detected in the cell wall fraction  strain by recombineering [pe15/ppe20 KO strain for altered ATP levels. The KO showed reduced Ca2+-dependent increase in ATP production that was reverted to wild-type (WT) levels by complementation with pe15/ppe20 (Comp)  . We nexthenotype . These dpe15/ppe20 in the presence of Ca2+ was consistent with the behavior of a Ca2+ channel. To directly test the idea that PE15/PPE20 has a role in Ca2+ transport, we created an Mtb reporter strain using a fluorescence resonance energy transfer (FRET) system that detects intracellular Ca2+, Twitch [2+-binding domain from troponin C that is optimized for maximal Ca2+ selectivity and ratiometric signal and that has a KD for Ca2+ of 200 nM [Mtb-twitch) and tested for Twitch expression in different cell fractions. Twitch was expressed only in the cytoplasm, indicating that it only reports on cytoplasmic Ca2+ pe15/ppe2mic Ca2+ . We coula2+ Figs . We thus systems  Fig 4C)pe15/ppe2T signal . These d E. coli .2+ transport, we measured Ca2+ uptake in the Mtb-twitch strain and a strain expressing Mtb-twitch in the pe15/ppe20 KO background. The WT produced a robust FRET signal as before upon addition of 5 mM Ca2+. The KO, however, showed reduced influx, consistent with the loss of a selective import channel  with 10% (vol/vol) oleic acid-albumin-dextrose-catalase (OADC) enrichment , 0.5% glycerol (referred to as “7H9+GO”), and 0.05% Tween 80 or 0.05% Tyloxapol (referred to as “7H9+GOT or 7H9+GOTy”) or 7H10 agar supplemented with 10% OADC and 0.5% glycerol or Chelex-treated Sauton’s medium (CTSM) with additional supplements as indicated. CTSM consisted of 0.5 g KH2PO4, 4g L-asparagine, 2 g citric acid, 6% glycerol, adjusted to pH 7, and was treated overnight with Chelex-100 resin (10 g/L) (Sigma) to remove trace metal ion contaminants including Ca2+. After filtration, 0.5 g/L of MgSO4, 0.05 g/L ferric ammonium citrate, 0.1 ml of 1% zinc sulfate, and 0.05% Tween 80 or 0.05% Tyloxapol was added. The pH was adjusted to 6.9 and the medium was sterilized by filtration. Strains bearing antibiotic cassettes were cultured with 50 μg/mL hygromycin or 30 μg/mL kanamycin or 25 μg/ml zeocin as appropriate. Ca2+ concentrations in media were determined using ICP-OES (Perkin Elmer Optima 8300). Media were treated with nitric acid  for 1 h at 65°C and diluted with Chelex-treated water. Ca2+ intensities in each of the digested sample were measured 3 times by ICP-OES at 317.933 nm. Ca2+ standards were measured to generate a standard curve of absorbance versus concentration  was added to each well and incubated for 10 min. Then, 3-fold serial dilutions were read at A600 on a spectrophotometer in a 96-well plate. The represented bar graph is the average readings of 4 biological replicates and Welch’s t test was applied to determine the significance .protocol  in 48-weMtb cells from a 7H9+GOT medium culture (OD600∼1) were washed twice and diluted to an OD600 of 0.01 in CTSM without detergent supplemented with different concentrations of CaCl2 or 1 mM EGTA. Cultures were grown in a 48-well plate at 37°C for 14 days. Cells were treated with 0.1% Tween-80 overnight to create a homogenous suspension. ATP was quantified by incubating 50 μl of the bacteria in triplicate with 50 μl of the BacTiter-Glo reagent for 15 min followed by a luminescence reading. ATP production in the Ca2+-treated cultures were calculated as fold change compared to the calcium-free condition. The represented bar graph is the average fold change of 4 biological replicates and Welch’s t test was applied to determine the significance .Mtb H37Rv was grown to an OD600 of approximately 1 in 7H9+GOT medium. Cells were washed twice with CTSM, subcultured in CTSM starting at an OD600 of 0.05, and grown to an OD600 of approximately 1. The culture was again subcultured in CTSM starting at an OD600 of 0.05 and incubated at 37°C to an OD600 of 0.2. The cultures were supplemented with or without 1 mM CaCl2 in triplicate. At the indicated times, cells were pelleted at 4,000 g for 5 min at 4°C, resuspended in Trizol and lysed by bead beating for 30 s at 6 m/s for 3 cycles with intermittent cooling on ice. Cell debris were pelleted at 20,000 g for 1 min, and the supernatant was transferred to a heavy phase lock gel tube containing 300 μl chloroform. The tubes were inverted for 2 min and centrifuged at 20,000 g for 5 min. RNA in the aqueous phase was precipitated using 300 μl isopropanol and 300 μl high salt solution . RNA was purified using QIAGEN RNeasy kit and ribosomal RNA was depleted using the Ribo-Zero rRNA removal magnetic kit (Illumina). The cDNA library was generated using the NEBNext Ultra II RNA Library Prep Kit, and each replicate was barcoded in the DNA library using the NEBNext Multiplex Oligos for Illumina. Libraries were quantified using the KAPA qPCR quantification kit, pooled, and sequenced at the University of Washington Northwest Genomics Center with the Illumina NextSeq 500 High Output v2 Kit. Read alignment was performed using the Bowtie 2 custom processing pipeline . Gene expression changes were identified using a combination of 5 differential expression (DE) tools within DuffyTools. The 5 DE tools included round robin, RankProduct, significance of microarrays (SAMs), EdgeR, and DeSeq2. Each DE tool measurement was combined using the weighted average of fold change and significance . Genes with averaged absolute fold change more than 1.5-fold and p-value <0.01 were considered differentially expressed. RNA-seq data is available at NCBI-GEO (accession no. GSE214266).Mtb Rv1386 (PE15) and Rv1387 (PPE20) genes were amplified from Mtb H37Rv genomic DNA using the primers Duet 1–5 that included Gibson overlap sequence (Table B in E. coli BL21(DE3) and a single colony was picked and was grown at 37°C in Terrific Broth medium containing ampicillin. Protein expression was induced by addition of 0.5 mM isopropyl-β-d-thiogalactoside (IPTG) at OD600 of approximately 0.4; culture was then maintained at 16°C for 16 h. Cells were harvested, and pellets were processed for purification. For complex purification through His-PE15, pellets were resuspended in buffer A (50 mM Tris (pH 8.0), 150 NaCl, and 10% glycerol) containing 20 mM imidazole and 1 mM AEBSF and lysed by sonication. Lysate was centrifuged at 35,000 g for 30 min at 4°C and the supernatant was loaded on Ni-NTA column. The column was thoroughly washed with buffer A containing 20 mM imidazole followed by elution with buffer A containing 250 mM imidazole. For PPE20-strep II tag, pellets were resuspended in buffer A containing 1 mM AEBSF and lysed by sonication. Lysate was centrifuged at 35,000 g for 30 min at 4°C and the supernatant was loaded on Strep-tactin column. The column was thoroughly washed with buffer A followed by elution with buffer A containing 2.5 mM desthiobiotin. Both the purified proteins were immediately dialyzed against buffer A and stored at −80°C. Interaction of PE15/PPE20 was confirmed by western blot. The wash and elution fraction from the Ni-NTA and strep tag purification were loaded on either SDS-PAGE or Native-PAGE, and the proteins were transferred onto a nitrocellulose membrane. Blots were probed with mouse α-His antibody followed by IRDye 680RD Goat α-Mouse IgG Secondary Antibody (LI-COR) to detect the His tagged PE15 and were also probed with IRDye 800CW Streptavidin to detect the PPE20-strep tag. The blots were scanned on LI-COR Odyssey platform using the 700 nm and 800 nm channel.pETDuet dual expression plasmid was used to co-express PE15 and PPE20. PE15 was cloned in the MCS-1 region with N-terminal His tag and PPE20 was cloned in the MCS-2 region with a C-terminal Strep II tag. 2 in a thermocycler for 5 min at 25°C followed by incubation at varying temperature (55 to 70°C) for 3 min. The reaction was cooled to 4°C and centrifuged at 20,000 g for 30 min at 4°C to separate the native (supernatant) and the denatured (pellet) fractions. Pellet fraction was solubilized by boiling with 2xSDS loading buffer. PPE20 in each of the supernatant and pellet fraction was detected by western blot using rabbit α-strep II tag antibody followed by IRDye 800CW Goat α-Rabbit IgG Secondary Antibody. To determine the KD value, PPE20 was incubated with varying concentration of CaCl2 (12.5 μm to 3.2 mM) for 5 min at 25°C, 3 min at 65°C followed by incubation at 4°C. PPE20 in the pellet fraction was detected by western blot as described above. The band intensities were calculated using the Image studio software and converted to fraction denatured relative to the total protein. The graph of fraction denatured v/s log Ca2+ concentration was plotted using GraphPad Prism 9.3, and the KD value was determined by fitting a dose-response curve using the least square regression method. For equilibrium dialysis, Ca2+-free PE15/PPE20 was prepared by EGTA treatment and dialysis against Chelex-treated buffer A. Protein (10 μm) was placed in the upper chamber and dialyzed against 50 mM Tris (pH 7.4), 150 mM NaCl buffer containing 200 μm of Ca2+ in the lower chamber for 24 h and the Ca2+ concentration in both chambers was determined by ICP-OES. Samples (100 μl) were digested with 100 μl of nitric acid  for 1 h at 65°C and diluted with 2.8 ml of Chelex-treated water. Ca2+ intensities were measured 3 times by ICP-OES and converted to Ca2+ concentration using the standard curve described above. Percentage ΔC was calculated as per the following formula:Purified PE15/PPE20 protein was incubated with or without 1 mM CaClEncephalitozoon cuniculi as a positive control (obtained from SSGCID # EncuA.01276.a.A1.PS00484), and Rv0831c as a negative control. The bar graph in t test was applied to determine the p-value.The thermal shift and equilibrium dialysis experiments were repeated with buffer control (no protein), calcium-binding protein (CBP) from pe15 (primers KO1 and KO2), 500 bp downstream of ppe20 (primers KO3 and KO4) and hygromycin cassette (primers KO5 and KO6) were amplified separately (Table B in Mtb H37Rv strain carrying recombineering plasmid pNIT [pe15 (His-tag) and ppe20 (FLAG-tag) genes were separately PCR-amplified using primers Comp 1–3 and Comp 4, 5, respectively  under the anhydrotetracycline (ATc)-inducible promoter. The plasmid was electroporated into wild type Mtb H37Rv (WT), pe15/ppe20 KO and pe15/ppe20 complemented strain (Comp), and the expression of Twitch was induced using 100 nM ATc. The expression of Twitch was confirmed by measuring the fluorescence of uninduced and induced cells using a plate reader at 3 different wavelengths . The localization of Twitch and PPE20 was confirmed by western blot. Briefly, 100 ml culture of uninduced and induced Mtb-twitch complemented with the pe15/ppe20 genes was grown in 7H9+GO medium. Cells were washed twice in PBS and resuspended in lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl) containing protease inhibitor cocktail. Cells were lysed by bead beating and the following fractions were prepared according to published protocol [The Ca Addgene  using thCultures were grown in 50 ml 7H9+GO for 7 days and washed 3× with PBS. Cell pellets were resuspended in 9 ml chloroform-methanol (2:1) and incubated overnight at room temperature with shaking. The extract was filtered through 0.2 μm PTFE filters and dried under a stream of nitrogen. The total lipid was weighed and resuspend at a concentration of 20 mg/ml in chloroform-methanol (2:1). Lipids (500 μg) were spotted onto aluminum-backed TLC plate and run in petroleum ether-ethyl acetate (98:2) as the solvent system. Control PDIM was obtained from BEI resources, solubilized in chloroform-methanol (2:1), and 100 μg was run on TLC. The TLC plate was dried, and the lipids were stained by spraying 5% sulfuric acid in methanol followed by heating.Mtb-twitch was grown in either 7H9+GOT or CTSM to an OD600 of 1. Cells (50 μl) were aliquoted in duplicate wells and spiked with Ca2+ (5 mM) and EGTA (5 mM) at indicated time points and fluorescence was continuously measured at wavelengths 432/475 nm (blue) and 432/532 nm (green) at 37°C. Fluorescence readings were corrected by subtracting the readings from wells containing uninduced culture. The FRET ratio was calculated as the green:blue fluorescence ratio and plotted against time. An increase in the FRET ratio indicates an increased intracellular Ca2+ concentration. To determine the dose-dependent effect of Ca2+ on the FRET signal, Mtb-twitch was incubated with increasing concentration of Ca2+ for 30 min at 37°C and an emission scan (from Em450-550 nm) was recorded using excitation at 432 nm.Mtb H37Rv (WT), pe15/ppe20 KO and complemented strains (Comp) expressing twitch were grown to OD600 of approximately 0.5 in CTSM/tyloxapol with or without 100 nM ATc. Cells were washed twice and resuspended in CTSM/tyloxapol at a dilution of 1 OD/ml. Cells were spiked with Ca2+ (5 mM) at indicated time point and blue and green fluorescence were measured continuously at 37°C. FRET ratios were calculated and plotted against time. The assay was repeated by treating cells with permeabilizing reagent [ reagent   ICP-OES standard curve for the determination of Ca2+ concentrations. (D) Ca2+ concentrations in standard 7H9 and Sauton’s media. The data underlying all the plots in this figure are included in 2+, calcium ion; CTSM, Chelex-treated Sauton’s medium; ICP-OES, inductively coupled plasma optical emission spectrometry.(A) CTSM and (B) 7H9 medium containing glycerol, OADC, and Tween-80 (7H9+GOT) supplemented with different concentrations of CaCl(TIF)Click here for additional data file.S2 FigEncephalitozoon cuniculi shows a Ca2+-dependent increase in the melting temperature and concomitant precipitation. (B) A protein not known to bind Ca2+ shows no difference in melting temperature and resulting precipitation. Uncropped images in this figure are included in 2+, calcium ion; CBP, calcium-binding protein.(A) A known CBP from (TIF)Click here for additional data file.S3 Fig2+ over the congener Mg2+. The His-tagged Twitch protein was purified from the cytosolic fraction using Ni-NTA column, treated with EDTA and desalted. An emission scan of the purified protein was recorded upon incubation with either Ca2+ or Mg2+. (C) Thin-layer chromatography of the cell wall component PDIM shows no difference in PDIM content between WT and pe15/ppe20 KO strains. (D) Permeabilization of the cell wall diminishes PE15/PPE20’s effect on Ca2+ import. WT, pe15/ppe20 KO, and complemented strain were transformed with Twitch, permeabilized (P) with lysozyme and Triton-X100, and the Ca2+-FRET signal measured. The data underlying all the plots and uncropped images in this figure are included in 2+, calcium ion; FRET, fluorescence resonance energy transfer; KO, knockout; PDIM, phthiocerol dimycocerosate; WT, wild type.(A) Western blot showing localization of Twitch-2B (His tagged) in the cytosolic fraction. The same cell fractions as in (TIF)Click here for additional data file.S1 Table2+ in Mtb and (B) primers used in this study.(A) Differentially expressed genes in response to Ca(PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file. 5 Oct 2022AttachmentResponse to reviewers.docxSubmitted filename: Click here for additional data file. 24 Oct 2022Dear Dr. Grundner, Thank you for submitting your manuscript entitled "Calcium transport by the Mycobacterium tuberculosis PE15/PPE20 proteins" for consideration as a Research Article by PLOS Biology.Your manuscript has now been evaluated by the PLOS Biology editorial staff, as well as by an academic editor with relevant expertise, and I am writing to let you know that we would like to proceed towards publication of your manuscript as a *Discovery Report*. 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Lupinus spp.). The effect of the total substitution of SBM by white lupine (WL) and yellow lupine (YL) seeds in the diets of growing rabbits and of gender on meat quality and the fatty acids (FA) profile were evaluated. Sixty hybrid weaned rabbits  (20 rabbits per diet), were fed diets that contained 150 g/kg of SBM (SBMD) and WL (WLD) or YL (YLD) for 35 to 69 days. At the end of this period, 30 rabbits (10 rabbits per diet) were slaughtered to evaluate the carcass and meat characteristics and the FA profile of the longissimus dorsi (LD) muscle. In general, the carcass and meat characteristics (pH and colour) were not affected (p > 0.05) by diet or gender. Further, there was no observed effect (p > 0.05) of gender on meat FA and on the calculated indexes related to human health. However, diet had an effect (p < 0.05) on the FA profile, FA categories, and calculated indexes related to human health. The meat from rabbits fed SBMD presented higher (p < 0.05) saturated FA  and lower (p < 0.05) polyunsaturated FA . Our results showed that SBM may be completely replaced by WL or YL, improving the quality of LD muscle FA in terms of nutritional quality for humans.In Europe, the most appropriate strategy to replace soybean meal (SBM) in animal feed has been the development of diets containing locally produced protein sources. One of these sources is lupine ( Lupinus spp.), faba beans (Vicia faba), and peas (Pisum sativum), are well adjusted to the Mediterranean soil and climate and have been studied as alternative protein sources for animal feed. Even though these Mediterranean legumes present lower protein contents than soybean meal (SBM), several studies have showen that they could totally or partially replace SBM in lamb [For the past ten years, the European Union has been trying to change its dependence on imports of soybean for animal feed. Different legume species, such as lupines  ,15,16,17The results found on the carcass traits and meat quality of rabbits fed WL are also relevant, indicating that it is an adequate substitute for SBM. Volek and Marounek  found a Regarding meat quality, Volek and Marounek  observedThe chemical composition of meat from rabbits, especially fat content and the FA profile, is influenced by sex. The meat of female rabbits presents higher fat content than males slaughtered at 77 and 90 days  or at 93Lupinus albus, cv. Nacional) or YL  seeds and of gender on the carcass characteristics and meat quality, namely the FA profile of the LD muscle, of growing hybrid rabbits  of both sexes .The objective of this study was to evaluate the effects of the total replacement of SBM with sweet varieties of WL  at Vila Real , and the animals were handled according to Portuguese law on animal welfare in experimental research  and the Sixty weaned hybrid rabbits  of both sexes  were reared in a building with closed air-conditioning and maintained at 18–23 °C. The day/night cycle was 12/12 h (light from 07:00 to 19:00).Rabbits weaned at 35 days of age and with similar live weights  were lodged in 30 metabolic cages  and were randomly distributed to three different dietary treatments. The sex distribution was random for all groups.Three diets were formulated to be nearly isonitrogenous and isoenergetic, according to the recommendations for growing rabbits by De Blas and Mateos , and theEach experimental diet was randomly assigned to 10 cages. In order to avoid digestive disturbances, a feed restriction (80%) was performed during the first 14 days of the experiment. This restriction was based on recommendations for rabbits during the post-weaning period, and the amount of feed supplied to the animals was planned daily. After this period, the animals were fed ad libitum until the end of the trial. Diets were offered in pelleted form throughout the experimental period. The rabbits had free access to drinking water through nipple drinkers, and during this study, no antibiotics were used in the feed or drinking water. More detailed information on animal handling and diets can be found in Garcia-Santos et al. .At 69 days of age, 10 rabbits per treatment (of both sexes (average LW 2692 ± 137.9 g)), were slaughtered by sudden cervical dislocation, without fasting, which is the method accepted by Portuguese law on animal welfare in experimental research  and as pThe carcasses traits were measured and calculated according to the recommendations of the World Rabbit Science Association . The ski2 + b*2) ½.At 24 h post-mortem, the carcass and meat colour were assessed on the surface over the HL (biceps femoris muscle) and on the transversal section of LD muscle, respectively, in accordance with Ouhayoun and Dalle Zotte . The colThe ultimate pH (pHu) was measured 24 h post-mortem by directly inserting the electrode into the LD muscle using a pH meter and allowing the temperature setting .The LD muscle was dissected between the first and seventh lumbar vertebrae from the two sides of refrigerated carcasses (24 h at 3 °C). Samples of the LD muscle were excised and stored (−20 °C) for determination of the FA and cholesterol contents.Dried samples of the experimental diets were ground to pass a 1 mm screen and were analysed, according to the AOAC  procedurv/v) methanol/sulfuric acid solution, with heating for 30 min at 80 °C, centrifugation at 3000 rpm for 5 min, and collection of the final supernatant. The analysis of the FA methyl esters was performed in duplicate by gas chromatography with a 30 m × 0.25 mm capillary column and a flame ionization detector . The applied carrier gas was helium at a flow rate of two mL/min. The oven temperature was programmed to increase at 35 °C per minute between 150 °C and 180 °C and at 5 °C per minute up to 220 °C. A temperature of 250 °C was considered for the injector and detector. The relative percentage of each individual FA in relation to the total FA was considered. The FA were identified by comparison of the retention times with a known standard Supelco®37 Component FAME Mix . Thirty-three FA were detected and quantified. The proportion of SFA , PUFA , and MUFA  was calculated. Short and medium SFA were considered when C < 16, according Panth et al. [The fatty acids were determined by the procedures described by Argemi-Armengol et al. . The inth et al. .g for 3 min in order to accelerate phase separation. An aliquot of the upper layer (n-hexane) was transferred into a small screw Teflon-lined cap tube, and a spatle tip of anhydrous sodium sulphate was added. Finally, the tube was briefly shaken, and an aliquot of the n-hexane layer was filtered through a 0.45 µm nylon syringe filter  into an amber screw-cap vial with a Teflon septum. The HPLC systems used were an Alliance 2695 model  and a 996 Photodiode Array Detector . Empower 3TM advanced software  was employed in order to control system operation and results management. The analysis of cholesterol was carried out using a normal-phase silica column , with photodiode array detection for cholesterol (208 nm). The solvent (2% v/v isopropanol in n-hexane) flow rate was 1 mL/min, the run lasted for 17 min, and the temperature of the column oven was adjusted to 30 °C. The content of total cholesterol in foal meat was quantified based on the external standard technique from a standard curve of peak area vs. concentration. The results were expressed as mg of cholesterol/100 g of meat [To determine the total cholesterol, saponification, extraction, and identification were carried out according to the procedures described by Dominguez et al. , with mo of meat ,36,37.The atherogenic (AI), thrombogenic (TI), and saturation (S/U) indexes were calculated according to Ulbricht and Southgate . The hypYij = dependant variable;µ = general mean;Di = fixed effect of diet ;Gj = fixed effect of gender ;DiGj = interaction effect between diet and gender; andeijk = residual random error.To perform the statistical analysis, the JMP version 14 program was used  and the p value of < 0.05, and a p value of between 0.05 and 0.1 was interpreted as a trend. If the diet effects were significant, the Tukey’s multiple comparison test was applied to the compare the means.Statistical significance was accepted at a The effects of diet and gender on the carcass characteristics of 69-day-old rabbits are presented in p > 0.05), and only HCW and CCW showed a tendency to be higher  in males. Colour  was not affected by gender (p > 0.05). Carcass parts were also not affected (p > 0.05) by gender, with the exception of the hind part, which was lower (p = 0.047) in males (29.2 vs. 30 g/100 CCW).Regarding the effect of gender on carcass characteristics, no significant differences were observed (p > 0.05) were observed in the carcass characteristics of 69-day-old rabbits between the diets, although there was a tendency (p = 0.054) for rabbits fed the WLD to have a lower slaughter weight . The colour characteristics and carcass parts were not affected by diet (p > 0.05).No differences (p > 0.05)The interaction effect on the carcass characteristics was non-significant  were found between sexes for pHu. However, for colour, a significant difference (p < 0.05) was found for yellowness , redness , and hue , with higher values for males. For a*, the value found for males was 57% higher than that observed for females (3.31 vs. 5.26). The diet and the interaction diet ×gender had no significant effect (p > 0.05) on the physical characteristics (pHu and colour) of the LD muscle of the rabbits.No significant differences (The effects of diet and gender on FA (g/100 g) and cholesterol (g/100 g) composition of the LD muscle of rabbits at 69 days of age are presented in p > 0.05) effect on the LD muscle FA profile of the rabbits. However, a tendency for a high stearic (C18:0) acid content (p = 0.001) in females was observed .Gender had no significant (p < 0.05) on almost all of the FA presented in p < 0.05), though no significant differences (p > 0.05) between WLD and YLD (average 28.38 g/100 g FA) were found. Meat from the rabbits fed the WLD presented the highest (p < 0.05) content of oleic  and α-linolenic (3.87 for WLD and 2.6 g/100 g FA for SBMD and YLD) acids. The cholesterol content of the LD muscle was affected by diet (p = 0.02), with the lowest (p < 0.05) value of YLD (25.5 g/100 g).Diet had an effect (p > 0.05).The interaction effect on the LD muscle FA profile of the rabbits was not significant (p > 0.05) were observed in any FA categories or in the calculated indexes related to human health presented in The FA categories and the nutritional indexes related to human health of the LD muscle of rabbits at 69 days of age are presented in p < 0.05) on FA categories and indexes related to human health of the LD muscle of rabbits at 69 days of age (p < 0.05) on lupine diets . The same was observed for all categories of SFA . The opposite was observed in total PUFA content, which was higher (p < 0.05) on lupine diets . The n − 6/n − 3 ratio for LD meat presented the highest (p < 0.05) value for rabbits fed the YLD (8.8), followed by the SBMD (7.1) and then the WLD (5.4), which had the lowest value.In general, diet had an effect (s of age . The totp < 0.05) DFA in the meat from the rabbits fed these diets . The h/H index was significantly higher (p < 0.05) in rabbits fed the lupine diets, about 30% higher than that observed in those fed the SBMD. The LD meat of the rabbits fed the WLD presented the lowest (p < 0.05) TL value (0.886), followed by YLD (1.033) and then by SBMD, which had the highest value (1.190). The TI was 58% lower (p < 0.05) in rabbits fed the WLD than that observed in rabbits fed the SBMD.The lower levels of SFA and higher levels of PUFA in meat from the rabbits fed the lupine diets promoted a higher  measured in the LD muscle were not affected by gender or diet, and no interaction between diet and gender was observed. Most studies have described no significant difference between females and males in the ultimate pH (pH 24 h after slaughter) in the LD muscle of animals slaughtered between 70 and 120 days of age ,47,48,49p < 0.01 for a* and p < 0.05 for H*) in the LD muscle at 24 h post-mortem. Dalle Zotte et al. [p < 0.05) in the LD muscle of females than in males at 24 h post-mortem. However, most studies showed no effect of sex on meat (LD muscle) colour at 24 h after slaughter, with males presenting high values for a* and b* [The meat colour parameters which were assessed 24 h post-mortem were not affected by gender. Nonetheless, a trend toward lower a*, b*, and hue (H*) values in females compared to males was observed. The a* value in males was 57% higher than the values observed in females, indicating redder meat in males than in females. Cavani et al.  also obse et al.  found th* and b* ,42,49,51p = 0.02 [p = 0.01 [p < 0.05) in males and C18:3n-3 higher (p < 0.05) in females. The FA categories  were not affected by sex, which agrees with the results obtained by Gasperlin et al. [No differences due to gender were found in the FA composition of LD muscle, with the exception of the content of stearic acid (C18:0), which was higher in the female rabbits compared to the male rabbits. The effect of gender in the FA profile of rabbit meat varies greatly among studies. The effect of gender on the FA content of meat (LD muscle) was noted only for capric acid  or oleip = 0.01 ), which p = 0.01  observedn et al.  and Polan et al.  on rabbin et al.  and Daszn et al.  on LD mun et al. ,22,52,53The substitution of SBM by WL or YL promoted an effect on almost all FA content. The meat of the rabbits fed lupine diets was characterised by a more desirable FA composition and a higher concentration of MUFA and PUFA. We were not able to find any work comparing an SBM diet with a WLD or YLD. However, Kowalska et al.  also fouThe PUFA n − 6/n − 3 ratio and the nutritional indexes related to human health were improved in rabbits fed the WLD and the YLD, indicating that lupine diets yielded LD meat that may provide a nutritional benefit to humans. Our results confirm the previous findings of Volek and Marounek  and VoleRabbits fed the two lupine diets studied, the WLD and YLD, presented non-significant differences for almost all of the FA and the FA categories. The same was observed for all the indexes related to human health analysed, except for the TI, which was lower in the LD muscle of the rabbits fed the WLD.From the present study, it follows that SBM may be completely replaced with WL or YL (150 g/diet) in the balanced diets of growing rabbits, supplemented with amino acids, and having no negative effect on the carcass characteristics and meat physical properties (pH24 and colour). However, the total substitution of SBM by WL or YL improved the quality of the LD muscle FA in terms of its nutritional quality for humans. Thus, as a general conclusion, the use of lupine as an ingredient in the diet of rabbits has a positive effect on its nutritional quality without affecting the productivity of the animals, and it is an important strategy for reducing dependence on soybean meal for animal feed.


The inter-netted limbs throughout the central, sympathetic, and parasympathetic systems perform these functions. They generate their common second messenger, cyclic GMP to affect the physiology. This review describes an historical account of their sequential evolutionary development, their structural components and their mechanisms of interaction. The foundational principles were laid down by the discovery of its first limb, the ACTH modulated signaling pathway (the companion monograph). It challenged two general existing dogmas at the time. First, there was the question of the existence of a membrane guanylate cyclase independent from a soluble form that was heme-regulated. Second, the sole known cyclic AMP three-component-transduction system was modulated by GTP-binding proteins, so there was the question of whether a one-component transduction system could exclusively modulate cyclic GMP in response to the polypeptide hormone, ACTH. The present review moves past the first question and narrates the evolution and complexity of the cyclic GMP signaling pathway. Besides ACTH, there are at least five additional limbs. Each embodies a unique modular design to perform a specific physiological function; exemplified by ATP binding and phosphorylation, Ca2+-sensor proteins that either increase or decrease cyclic GMP synthesis, co-expression of antithetical Ca2+ sensors, GCAP1 and S100B, and modulation by atmospheric carbon dioxide and temperature. The complexity provided by these various manners of operation enables membrane guanylate cyclase to conduct diverse functions, exemplified by the control over cardiovasculature, sensory neurons and, endocrine systems.One monumental discovery in the field of cell biology is the establishment of the membrane guanylate cyclase signal transduction system. Decoding its fundamental, molecular, biochemical, and genetic features revolutionized the processes of developing therapies for diseases of endocrinology, cardio-vasculature, and sensory neurons; lastly, it has started to leave its imprints with the atmospheric carbon dioxide. The membrane guanylate cyclase does so  Existing initially in the shadows of cyclic AMP signaling, cyclic GMP has slowly rose to prominence as a critical signaling molecule controlled by the activities of soluble and membrane guanylate cyclases (MGCs) and phosphodiesterases. After a brief outline of the chronological development of the MGC field, we focus on the uniqueness of the modular design which adds layers of complexity and enables the enzyme to perform various physiological functions.Some of the advancements made by the authors and their collaborators are featured. The narration is partially borrowed, with appropriate citations, from the authors’ earlier reviews: , 2014a,bBeginning with the discovery of cyclic AMP in 1958, understanding of the molecular principles of hormonal signaling began to undergo a radical change. The new paradigm was that the hormone did not directly target its signaling site. Instead it did so through its second messenger, cyclic AMP .For constructing the building blocks of this “cyclic AMP second messenger” concept Sutherland in 1971 and Rodbell and Gilamn in 1994 were awarded Nobel prizes in physiology and medicine.In Sutherland’s original “second messenger” concept, hormone was the first messenger and its interacting cell surface receptor product, cyclic AMP, the second. Glycogen, catecholamines, and other polypeptide hormones manifested their biological activities within the cell in this manner. The principal features of this concept were envisioned to be applicable to all hormonal systems . The recIn the 1970’s the term “transduction” was introduced by the Rodbell’s group . It means, and inhibitory, Gi; the former stimulates and the latter inhibits the transduction process. The molecular nature of the receptor specified the effect of the hormone. The cyclic AMP signaling system was thus a three-component system, composed of three distinct proteins – the receptor, the G protein, and the adenylate cyclase , a lipid signaling molecule into two separate second messengers, diacylglycerol and inositol triphosphate (IP3). IP3 signal mobilized the intracellular Ca2+ pool that could open TRP channels, activate protein kinase C or act on other Ca2+ sensors. Diacylglycerol also activated protein kinase C and certain TRP channels.A similar G protein modulated pathway was discovered, subsequently. It was the phosphatidylinositol-signaling  “The ACTH-modulated biosynthetic step is the cleavage of the (20S)-20-hydroxycholesterol to corticosterone. This step is dependent on the generation of cyclic GMP, yet, not on the cyclic AMP. Accordingly, the step requires the labile synthesis of protein and is rate limiting” .(2) In the model of the carcinoma cells, steroidogenesis gets out of ACTH control, yet not of cyclic GMP.(3) The physiological concentrations of ACTH, 2.5 to 10 μunits, do not increase the synthesis of cyclic AMP but stimulate the peak synthesis of cyclic GMP with a simultaneous rise in corticosterone synthesis.(4) Thus, “the hormone, ACTH, modulates the steroidogenic production was supported by purification from the bovine adrenal cortex of the cyclic GMP-dependent protein kinase, the enzyme directly locked in with cyclic GMP” .(5) The Mr. of the cyclic GMP-dependent protein kinase is 145,000. Two identical subunits  form the functional enzyme. The enzyme binds two molecules of cyclic GMP per holoenzyme and self phosphorylates. Calmodulin and troponin C, markedly stimulate the enzyme. The ability of cyclic GMP and its analogs to stimulate steroidogenesis matches their ability to activate cyclic GMP-dependent protein kinase. Thus, this protein kinase is totally different from the cyclic AMP-dependent protein kinase which dissociates into its regulatory and catalytic subunits upon binding cyclic AMP.These facts, and purification of the MGC from the rat adrenal cortex , allowedvia nitric oxide (NO) gas  gas . (2) The(NO) gas .They reinforced their concept by reporting the presence of NO-dependent MGC in almost all mammalian tissues, ranging from peripheral to the central nervous system. The response to NO varied, however; ranging from 3-fold stimulation in the liver to 14.7-fold in the rat cerebellum .This passionate pursuit on the physiological irrelevance of the hormone-dependent MGC was extended to the cyclic GMP-dependent protein kinase. The belief was that it did not phosphorylate any protein with higher specificity than cyclic AMP-dependent protein kinase did. The implication was that in the instances where a stimulus  generated cyclic GMP in the intact cell, it acted through a cyclic AMP transduction component instead of the cyclic GMP. Again, reinforcing the concept that the cyclic GMP signaling pathway was a subservient to the cyclic AMP transduction system .Remarkably, for the discovery of the NO, and NO-modulated cyclic GMP signaling system, Robert Furchgott, Louis Ignarro and Ferid Murad were awarded the 1998 Nobel Prize in Physiology or Medicine. The underlying mechanism for the NO action was cited as: the NO gas generated in the endothelial cell layer, signaled guanylyl cyclase activation, and the generated cyclic GMP caused relaxation of the blood vessels.Also, notably, in this concept, the soluble guanylate cyclase was the sole cellular signaling pathway.The details for these awards, none-the-less, are misleading for two reasons. (1) It totally ignored the historical evolution of the MGC signal transduction. Factually, in the beginning these laboratories not only denied its cellular existence, they aggressively negated the ongoing results of the other laboratories that had established the physiological, biochemical and structural integrity of the membrane guanylate cyclase signal transduction. Before 1998, the date of the award, other groups, including ours in 1982, had already demonstrated that the guanylate cyclase exists in two forms, soluble and the MGC . Only, tThe earliest documented facts, however, were that the membrane form is not NO-modulated and it is structurally and functionally different from the soluble form as summarized in The present understanding is that the soluble guanylate cyclase is modulated by NO, it is encoded by two genes , is heteThe proposed structural model of the MGC purified from rat adrenocortical carcinoma  and late2+ was needed for stimulation of steroidogenesis  the source of ANF is the heart atria; (2) the heart is the endocrine gland; (3) ANF is the most potent hypotensive agent.The studies initiated by the De Bold’s group and followed by others’ revolutionized the field of cardiovasculature and provided an opening for advancing the ANF research toward the development of drugs for hypertension . It was via its surface receptor domain, activates the catalytic domain of ANF-RGC and generates cyclic GMP?With the knowledge that ANF-RGC is present in the adrenal gland, we asked two critical questions: (1) Does the heart-originated ANF signal modulate the steroidogenic machinery of the adrenal gland? And (2) how does ANF, In reference to (1), ANF-RGC presence in the glandular tissues of the adrenal gland and testis supported the idea that the heart signals the metabolic processes of steroidogenesis in these endocrine tissues. Accordingly, studies from various groups showed that ANF raises the levels of cyclic GMP and down-regulates the aldosterone formation in the adrenal glomerulosa cells . Noting It was now possible to elucidate the molecular principles of this SWITCH and conquer the diseases of hyper-and hypo-tension. Indeed, as the future holds, this was one of the game changer contribution in the field of cardiology.In hindsight, additional investigations started to flood in. They ranged from the purification of ANF-RGC from other glandular tissues to its cloning and its’ varied modes of steroidogenesis , was cloned first in a joint effort  and thenNow, the identities of three forms of the MGCs were known. They, constituted a SURFACE RECEPTOR family. Two members, ANF-RGC and CNP-RGC were locked in with the physiology of cardiovasculature; third, STa-RGC, with the intestine. In a common theme, the family relaxed the vasculatures of their respective target organs and ANF-RGC also toned down the hypertensive activity.via the Northern-blot analysis presence of the CNP-RGC was detected in the central nervous system and in the cells of the neural crest  of ANF-RGC indeed houses the hormone’s (ANF) binding site. A GCα mutant of ANF-RGC, cloned from the adrenal cDNA library differed from ANF-RGC in two amino acid residues within the ExtD: position 338 was occupied by His instead of Gln and in position 364 Pro substituted for Leu . GCα exh338 and/or Leu364 are obligatory for ANF stimulation. Ensuing point mutation analyses singled out Leu364 as the key residue for both activities.Thus, Gln364 of this domain is critically necessary for ANF binding, (3) in the intracellular domain the catalytic domain follows the kinase-like domain, and (4) the catalytic domain possesses both the basal and the hormone-dependent activities. Binding of ANF to the ExtD controls the catalytic activity in the intracellular domain  ExtD, (2) the Leur domain .From the historic perspective, GCα, that was decisive in determining the ANF binding site and was the first mutant of any MGC linked with hormonally-dependent catalytic activity .358 corresponds to Gln338 and Glu332 was found to be vital for CNP binding and CNP-dependent cyclase activity; similar to Leu364 of ANF-RGC it does not have any effect on the basal cyclase activity of CNP-RGC  and larger of 185 residues C-terminal 587–771; . ATP bint system .In a genetically modified mouse model in which the WTAPELL motif was deleted from the ANF-RGC gene, hypertension and cardiac hypertrophy were observed  that gets deleted post-translationally. The calculated molecular mass of ROS-GC with the 56-amino acid LS is 120,361 Da; without it, it is 114,360 Da . The 114Proper identification of ROS-GC had an important historical impact. It corrected an earlier structural flaw.via in situ hybridization analysis was detected in the inner segments and outer nuclear layers of the monkey’s retina and the cyclase was different from ANF-RGC and CNP-RGC, the authors suggested that it may be a part of the phototransduction machinery , yet not published, to show its complete identity with the structure of ROS-GC, published in the early part of 1994 . Thus, RThe discovery of mutations in ROS-GC1 linked with retinal disorders made it possible to establish the critical role of ROS-GC1 in phototransduction and define its abnormalities in molecular terms .2+-modulated ROS-GC1 was known, Koch and Stryer provided the evidence that a calcium binding protein stimulates MGC activity in ROS in a [Ca2+]-dependent fashion  Significantly, cyclic GMP is the second messenger of phototransduction; it increases the conductance of ion channels  that is deleted post-translationally; the calculated molecular mass of the protein with the LS is 120,361 Da; without it, 114,360 Da. (5) No extracellular modulator was found for ROS-GC. (6) Ca2+-free GCAP1 activates ROS-GC1 but Ca2+-bound (IC50 ~ 100 nM), becomes an inhibitor  In recombinant system, GCAP2 is a more powerful stimulator of ROS-GC1 activity at low Ca2+ but with significantly higher than for GCAP1 EC50  Vision begins with PHOTOTRANSDUCTION, i.e., the conversion of light (photon) into an electrical signal in the photoreceptors outer segments. (2) Cachannels . (4) ROSnhibitor . Therebyto do so ; thus, G503-I522 ; GCAP2 a965-N981 .In contrast, one group proposes that both GCAPs bind to the same site on ROS-GC1 ,b.657-TAPELL663 motif for stimulation of the cyclase catalytic activity at low Ca2+ ((10) Even though GCAPs bind to opposite sides of the ROS-GC1’s CCD, they both use the Wlow Ca2+ . Finally2+-dependent manner similar to that of GCAP1. Its role in phototransduction is unclear, however  Do the vision linked retinal neurons embody an additional ROS-GC linked pathway where [Ca2+]i stimulates it? (2) If, “yes,” how does it operate?With the discovery of GCAPs the dogma developed that they are the sole modulators of ROS-GC activity. The curious aspect was, however, that the GCAPs/I and rises once again as [Ca2+] reaches its peak level. The reason for these different modes trace to the nature of the Ca2+ sensor involved – GCAPs, S100B, neurocalcin δ, hippocalcin, and frequenin. (6) In an amazing arrangement, the function of GCAP1 is its MGC partner-dependent; at low Ca2+, it stimulates ROS-GCs and inhibits them at high Ca2+. Yet, it stimulates ONE-GC at high Ca2+. (7) ROS-GC1 has the ability to link with varied forms of Ca2+ sensors  With conservation, the CCD structure of every MGC responds to the bicarbonate signals. The response of ROS-GC to bicarbonate and Ca2+-modulated GCAPs and S100B in combination exceeds the sum of the individual modulations. Thus, bicarbonate provides a means to amplify the transductions. (11) Probably, the bicarbonate signaling can amplify transduction of orthosteric ligand binding. (12) The modulation and regulation by phosphorylation plays a critical part in hormone receptor MGCs.What Are Its coded molecular features that make It achieve such complex tasks? (1) A modular structure. It makes the MGC enormously elastic and multimodal. Unlike its predecessor three component second messenger systems, adenylate cyclase and IP sensors , in partBoth authors have made a substantial, direct, intellectual and equal contribution to the work and approved it for publication.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. 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