Whole mount of adult ear skin as a model to study vascular malformations.

BACKGROUND: Genetic analysis in human patients has linked mutations in PIK3CA, the catalytic subunit of PI-3'Kinase, to sporadic incidences of vascular malformations. METHODS: We have developed a mouse model with inducible and endothelial-specific expression of PIK3CAH1047R , resulting in the development of vascular malformations. Systemic induction of this mutation in adult mice results in rapid lethality, limiting our ability to track and study these lesions; therefore, we developed a topical and local induction protocol using the active metabolite of tamoxifen, 4OH-T, on the ear skin of adults. RESULTS: This approach allows us to successfully model the human disease in a mature and established vascular bed and track the development of vascular malformations. To validate the utility of this model, we applied a topical rapamycin ointment, as rapamycin is therapeutically beneficial to patients in clinical trials. We found that the induced ear lesions showed significant attenuation after treatment, which was easily quantified. CONCLUSIONS: These data collectively provide evidence of a new model to study vascular malformations in adult tissues, which should be particularly useful in environments lacking specialized small-animal imaging facilities.

Formulation of cost-effective medium and optimization studies for enhanced production of rapamycin.

BACKGROUND: Enhancing rapamycin production using a cost-effective medium is crucial for wider accessibility, reduced manufacturing costs, sustainable pharmaceutical practices, and advancements in therapeutic applications. It promotes global health, biotechnological innovation, research collaboration, and societal well-being through affordable and effective treatments. This study focuses on the development of a novel cost-effective production medium for the synthesis of rapamycin from Streptomyces hygroscopicus. RESULTS: In the initial screening, more rapamycin production was observed in medium A. Initially, the organism produced 10 µg/mL rapamycin. Based on the OFT results, a novel cost-effective medium composition was designed, incorporating soyabean, sugarcane juice, and dried tomato components. Using RSM, soyabean and tomato was found to be more significant in rapamycin production than sugarcane. In the optimized medium, the production of rapamycin increased significantly to 24 µg/mL. Furthermore, a comparative analysis of the growth kinetics between the production normal medium (referred to as production medium A) and the newly optimized cost-effective production medium revealed that the optimized cost-effective production medium significantly enhanced the production of rapamycin. CONCLUSION: Overall, this study demonstrates the successful development of a cost-effective production medium for rapamycin synthesis from S. hygroscopicus. The findings highlight the potential of using a cost-effective medium to enhance the production of a valuable secondary metabolite, rapamycin, while reducing production costs.

ODF2 Negatively Regulates CP110 Levels at the Centrioles/Basal Bodies to Control the Biogenesis of Primary Cilia.

Primary cilia are essential sensory organelles that develop when an inhibitory cap consisting of CP110 and other proteins is eliminated. The degradation of CP110 by the ubiquitin-dependent proteasome pathway mediated by NEURL4 and HYLS1 removes the inhibitory cap. Here, we investigated the suitability of rapamycin-mediated dimerization for centriolar recruitment and asked whether the induced recruitment of NEURL4 or HYLS1 to the centriole promotes primary cilia development and CP110 degradation. We used rapamycin-mediated dimerization with ODF2 to induce their targeted recruitment to the centriole. We found decreased CP110 levels in the transfected cells, but independent of rapamycin-mediated dimerization. By knocking down ODF2, we showed that ODF2 controls CP110 levels. The overexpression of ODF2 is not sufficient to promote the formation of primary cilia, but the overexpression of NEURL4 or HYLS1 is. The co-expression of ODF2 and HYLS1 resulted in the formation of tube-like structures, indicating an interaction. Thus, ODF2 controls primary cilia formation by negatively regulating the concentration of CP110 levels. Our data suggest that ODF2 most likely acts as a scaffold for the binding of proteins such as NEURL4 or HYLS1 to mediate CP110 degradation.

The Case for the Target of Rapamycin Pathway as a Candidate Circadian Oscillator.

The molecular mechanisms that drive circadian (24 h) rhythmicity have been investigated for many decades, but we still do not have a complete picture of eukaryotic circadian systems. Although the transcription/translation feedback loop (TTFL) model has been the primary focus of research, there are many examples of circadian rhythms that persist when TTFLs are not functioning, and we lack any good candidates for the non-TTFL oscillators driving these rhythms. In this hypothesis-driven review, the author brings together several lines of evidence pointing towards the Target of Rapamycin (TOR) signalling pathway as a good candidate for a non-TTFL oscillator. TOR is a ubiquitous regulator of metabolism in eukaryotes and recent focus in circadian research on connections between metabolism and rhythms makes TOR an attractive candidate oscillator. In this paper, the evidence for a role for TOR in regulating rhythmicity is reviewed, and the advantages of TOR as a potential oscillator are discussed. Evidence for extensive feedback regulation of TOR provides potential mechanisms for a TOR-driven oscillator. Comparison with ultradian yeast metabolic cycles provides an example of a potential TOR-driven self-sustained oscillation. Unanswered questions and problems to be addressed by future research are discussed.

Use of everolimus following kidney transplantation during pregnancy: A case report and systematic review.

OBJECTIVE: There is limited safety data on the use of everolimus during pregnancy. In this study, we present the maternal and neonatal outcomes of everolimus used throughout the course of pregnancy and conducted a systematic review of reports of everolimus use after organ transplantation during pregnancy. CASE REPORT: A woman with type 1 diabetes who underwent kidney transplantation was treated with tacrolimus, everolimus, and prednisolone. Two years later, she became pregnant. At 27 weeks of gestation, an emergent cesarean delivery was performed owing to severe preeclampsia and fetal distress. No congenital malformation was noted in the baby at a corrected age of 4 months, and the maternal renal function remained stable. CONCLUSION: Our systematic review did not identify evidence of teratogenicity in babies exposed to everolimus as an immunosuppressant after transplantation. To better assess the risk of exposure to everolimus during pregnancy, all cases of new pregnancies occurring in transplant recipients receiving treatment with mammalian target of rapamycin inhibitor inhibitors should be reported.

Toxic effects of sirolimus and everolimus on the development and behavior of zebrafish embryos.

Sirolimus and everolimus have been widely used in children. These mammalian target of rapamycin (mTOR) inhibitors have shown excellent efficacy not only in organ transplant patients as immunosuppressive agents but also in patients with some other diseases. However, whether mTOR inhibitors can affect the growth and development of children is of great concern. In this study, using zebrafish models, we discovered that sirolimus and everolimus could slow the development of zebrafish, affecting indicators such as survival, hatching, deformities, body length, and movement. In addition to these basic indicators, sirolimus and everolimus had certain slowing effects on the growth and development of the nervous system, blood vessels, and the immune system. These effects were dose dependent. When the drug concentration reached or exceeded 0.5 µM, the impacts of sirolimus and everolimus were very significant. More interestingly, the impact was transient. Over time, the various manifestations of experimental embryos gradually approached those of control embryos. We also compared the effects of sirolimus and everolimus on zebrafish, and we revealed that there was no significant difference between these drugs in terms of their effects. In summary, the dose of sirolimus and everolimus in children should be strictly controlled, and the drug concentration should be monitored over time. Otherwise, drug overdosing may have a certain impact on the growth and development of children.

The mTOR Inhibitor Rapamycin Counteracts Follicle Activation Induced by Ovarian Cryopreservation in Murine Transplantation Models.

Background and Objectives: Ovarian tissue cryopreservation followed by autotransplantation (OTCTP) is currently the only fertility preservation option for prepubertal patients. Once in remission, the autotransplantation of frozen/thawed tissue is performed when patients want to conceive. A major issue of the procedure is follicular loss directly after grafting mainly due to follicle activation. To improve follicular survival during the OTCTP procedure, we inhibited the mTOR pathway involved in follicle activation using rapamycin, an mTOR inhibitor. Next, we compared two different in vivo models of transplantation: the recently described non-invasive heterotopic transplantation model between the skin layers of the ears, and the more conventional and invasive transplantation under the kidney capsule. Materials and Methods: To study the effects of adding rapamycin during cryopreservation, 4-week-old C57BL/6 mouse ovaries, either fresh, slow-frozen, or slow-frozen with rapamycin, were autotransplanted under the kidney capsule of mice and recovered three weeks later for immunohistochemical (IHC) analysis. To compare the ear with the kidney capsule transplantation model, fresh 4-week-old C57BL/6 mouse ovaries were autotransplanted to either site, followed by an injection of either LY294002, a PI3K inhibitor, vehicle control, or neither, and these were recovered three weeks later for IHC analysis. Results: Rapamycin counteracts cryopreservation-induced follicle proliferation, as well as AKT and mTOR pathway activation, in ovaries autotransplanted for three weeks under the kidney capsule of mice. Analyses of follicle proliferation, mTOR activation, and the effects of LY294002 treatment were similar in transplanted ovaries using either the ear or kidney capsule transplantation model. Conclusions: By adding rapamycin during the OTCTP procedure, we were able to transiently maintain primordial follicles in a quiescent state. This is a promising method for improving the longevity of the ovarian graft. Furthermore, both the ear and kidney capsule transplantation models were suitable for investigating follicle activation and proliferation and pharmacological strategies.

Reduced Prevalence of Dementia in Patients Prescribed Tacrolimus, Sirolimus, or Cyclosporine.

BACKGROUND: Evidence suggests patients prescribed calcineurin inhibitors (CNIs) have a reduced prevalence of dementia, including Alzheimer's disease (AD); however, this result has never been replicated in a large cohort and the involved mechanism(s) and site of action (central versus periphery) remain unclear. OBJECTIVE: We aim to determine if prescription of CNIs is associated with reduced prevalence of dementia, including AD, in a large, diverse patient population. Furthermore, we aim to gain insight into the mechanism(s) and site of action for CNIs to reduce dementia prevalence. METHODS: Electronic health records (EHRs) from patients prescribed tacrolimus, cyclosporine, or sirolimus were analyzed to compare prevalence, odds, and hazard ratios related to dementia diagnoses among cohorts. EHRs from a random, heterogeneous population from the same network were obtained to generate a general population-like control. RESULTS: All drugs examined reduced dementia prevalence compared to the general population-like control. There were no differences in dementia diagnoses upon comparing tacrolimus and sirolimus; however, patients prescribed tacrolimus had a reduced dementia prevalence relative to cyclosporine. CONCLUSION: Converging mechanisms of action between tacrolimus and sirolimus likely explain the similar dementia prevalence between the cohorts. Calcineurin inhibition within the brain has a greater probability of reducing dementia relative to peripherally-restricted calcineurin inhibition. Overall, immunosuppressants provide a promising therapeutic avenue for dementia, with emphasis on the brain-penetrant CNI tacrolimus.

Inhibitors of the Mechanistic Target of Rapamycin Can Ameliorate Bortezomib-Induced Peripheral Neuropathy.

Bortezomib, an anticancer drug for multiple myeloma and mantle cell lymphoma, causes severe adverse events and leads to peripheral neuropathy. The associated neuropathy limits the use of bortezomib and could lead to discontinuation of the treatment; therefore, effective intervention is crucial. In the present study, we statistically searched for a drug that could alleviate bortezomib-induced peripheral neuropathy using adverse event self-reports. We observed that specific inhibitors of the mechanistic target of rapamycin (mTOR) lowered the incidence of bortezomib-induced peripheral neuropathy. These findings were experimentally validated in mice, which exhibited long-lasting mechanical hypersensitivity after repeated bortezomib treatment. This effect was inhibited for hours after a systemic injection with rapamycin or everolimus in a dose-dependent manner. Bortezomib-induced allodynia was accompanied by the activation of spinal astrocytes, and intrathecal injection of mTOR inhibitors or an inhibitor of ribosomal protein S6 kinase 1, a downstream target of mTOR, exhibited considerable analgesic effects in a dose-dependent manner. These results suggest that mTOR inhibitors, which are readily available to patients prescribed bortezomib, are one of the most effective therapeutics for bortezomib-induced peripheral neuropathy.

Efficacy of Percutaneous Coronary Intervention With Synergy Stents in Patients Aged ≥75 Years: 1-Year Clinical Outcomes from the Synergy Elderly Registry.

Data regarding the clinical outcomes of older patients after Synergy everolimus-eluting stent (S-EES) implantation are limited. This study investigated the 12-month clinical outcomes of older patients who underwent percutaneous coronary intervention with new-generation drug-eluting stents according to ischemic risks. This prospective multicenter study targeted patients aged ≥75 years who underwent S-EES implantation. The primary and secondary end points included 12-month device-oriented composite end point (DOCE) (cardiovascular death, target vessel myocardial infarction, or target lesion revascularization) and major adverse cardiac and cerebrovascular events (MACCEs; all-cause death, myocardial infarction, target vessel revascularization, stent thrombosis, or stroke), respectively. A stratified analysis was conducted according to high-ischemic risk (HIR), defined as complex coronary intervention (number of stents implanted ≥3, total stented length >60 mm, chronic total occlusion, left main, or bifurcation), diabetes, or chronic kidney disease. In total, 650 enrolled patients aged ≥75 years were categorized into HIR (n = 425) and non-HIR groups (n = 225). In the total population, the 1-year incidence of DOCEs was 2.5%. The rates of DOCEs were not significantly different between the HIR and the non-HIR groups, whereas the MACCE rate was higher in the HIR (9.4%) than the non-HIR group (4.9%, p = 0.035), and the DOCE and MACCE components did not differ significantly in the occurrence between the groups. The independent predictors for the DOCEs or MACCEs included age, anemia, or left ventricular ejection fraction <40%. In conclusion, in older patients, S-EES implantation demonstrated favorable device-related outcomes, regardless of procedural complexity or co-morbidities. However, it requires careful attention because older patients with HIR are associated with worse clinical outcomes.

Paradigm shift in the treatment of tuberous sclerosis: Effectiveness of everolimus.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterised by abnormal cell proliferation and differentiation that affects multiple organs and can lead to the growth of hamartomas. Tuberous sclerosis complex is caused by the disinhibition of the protein mTOR (mammalian target of rapamycin). In the past, various therapeutic approaches, even if only symptomatic, have been attempted to improve the clinical effects of this disease. While all of these therapeutic strategies are useful and are still used and indicated, they are symptomatic therapies based on the individual symptoms of the disease and therefore not fully effective in modifying long-term outcomes. A new therapeutic approach is the introduction of allosteric inhibitors of mTORC1, which allow restoration of metabolic homeostasis in mutant cells, potentially eliminating most of the clinical manifestations associated with Tuberous sclerosis complex. Everolimus, a mammalian target of the rapamycin inhibitor, is able to reduce hamartomas, correcting the specific molecular defect that causes Tuberous sclerosis complex. In this review, we report the findings from the literature on the use of everolimus as an effective and safe drug in the treatment of TSC manifestations affecting various organs, from the central nervous system to the heart.

Influence of genetic variants on the pharmacokinetics and pharmacodynamics of sirolimus: a systematic review.

Sirolimus is an antiproliferative and immunosuppressive compound inhibiting the mTOR pathway, which is often activated in congenital low-flow vascular malformations. Studies have demonstrated the efficacy of sirolimus for this disease. Studies in kidney transplant patients suggest that genetic variants can influence these pharmacokinetic parameters. Therefore, a systematic literature search was performed to gain insight into pharmacogenetic studies with sirolimus. Most studies investigated CYP3A4 and CYP3A5, with inconsistent results. No pharmacogenetic studies focusing on sirolimus have been performed for low-flow vascular malformations. We analyzed two common variants of CYP3A4 and CYP3A5 (CYP3A4*22 and CYP3A5*3, respectively) in patients (n = 59) with congenital low-flow vascular malformations treated with sirolimus. No association with treatment outcome was identified in this small cohort of patients.

Rapamycin insensitive regulation of engineered ligament structure and function by IGF-1.

Following rupture, the anterior cruciate ligament (ACL) will not heal and therefore more than 400,000 surgical repairs are performed annually. Ligament engineering is one way to meet the increasing need for donor tissue to replace the native ligament; however, currently these tissues are too weak for this purpose. Treating engineered human ligaments with insulin-like growth factor-1 (IGF-1) improves the structure and function of these grafts. Since the anabolic effects of IGF-1 are largely mediated by rapamycin complex I (mTORC1), we used rapamycin to determine whether mTORC1 was necessary for the improvement in collagen content and mechanics of engineered ligaments. The effect of IGF-1 and rapamycin was determined independently and interactions between the two treatments were tested. Grafts were treated for 6 days before mechanical testing and analysis of collagen content. Following 8 days of treatment, mechanical properties increased 34% with IGF-1 and decreased 24.5% with rapamycin. Similarly, collagen content increased 63% with IGF-1 and decreased 36% with rapamycin. Interestingly, there was no interaction between IGF-1 and rapamycin, suggesting that IGF-1 was working in a largely mTORC1-independent manner. Acute treatment with IGF-1 did not alter procollagen synthesis in growth media, even though rapamycin decreased procollagen 55%. IGF-1 decreased collagen degradation 15%, whereas rapamycin increased collagen degradation 10%. Once again, there was no interaction between IGF-1 and rapamycin on collagen degradation. Together, these data suggest that growth factor-dependent increases in collagen synthesis are dependent on mTORC1 activity; however, IGF-1 improves human-engineered ligament mechanics and collagen content by decreasing collagen degradation in a rapamycin-independent manner. How the anticatabolic effects of IGF-1 are regulated have yet to be determined.NEW & NOTEWORTHY IGF-1 increases and rapamycin decreases mechanical and material properties of engineered human ligaments by regulating collagen content and concentration. There was no interaction between IGF-1 and rapamycin, suggesting that IGF-1 and rapamycin work independently. We found that IGF-1 improves collagen content by decreasing collagen degradation in a rapamycin-independent manner, whereas growth factor-dependent increases in collagen synthesis are blocked by rapamycin. These data may explain why interventions to increase IGF-1 have not helped rehabilitation.

Biodegradable-Polymer or Durable-Polymer Stents in Patients at High Bleeding Risk: A Randomized, Open-Label Clinical Trial.

BACKGROUND: Limited information is available on the comparative efficacy and safety of different stent platforms in patients at high bleeding risk undergoing an abbreviated dual antiplatelet therapy duration after percutaneous coronary intervention (PCI). The aim of this study was to compare the safety and effectiveness of the biodegradable-polymer sirolimus-eluting stent with the durable-polymer zotarolimus-eluting stent in patients at high bleeding risk receiving 1 month of dual antiplatelet therapy after PCI. METHODS: The Bioflow-DAPT Study is an international, randomized, open-label trial conducted at 52 interventional cardiology hospitals in 18 countries from February 24, 2020, through September 20, 2021. Patients with a clinical indication to PCI because of acute or chronic coronary syndrome who fulfilled 1 or more criteria for high bleeding risk were eligible for enrollment. Patients were randomized to receive either biodegradable-polymer sirolimus-eluting stents or durable-polymer, slow-release zotarolimus-eluting stents after successful lesion preparation, followed by 1 month of dual antiplatelet therapy and thereafter single antiplatelet therapy. The primary outcome was the composite of death from cardiac causes, myocardial infarction, or stent thrombosis at 1 year, and was powered for noninferiority, with an absolute margin of 4.1% at 1-sided 5% alpha. RESULTS: A total of 1948 patients at high bleeding risk were randomly assigned (1:1) to receive biodegradable-polymer sirolimus-eluting stents (969 patients) or durable-polymer zotarolimus-eluting stents (979 patients). At 1 year, the primary outcome was observed in 33 of 969 patients (3.6%) in the biodegradable-polymer sirolimus-eluting stent group and in 32 of 979 patients (3.4%) in the durable-polymer zotarolimus-eluting stent group (risk difference, 0.2 percentage points; upper boundary of the 1-sided 95% CI, 1.8; upper boundary of the 1-sided 97.5% CI, 2.1; P<0.0001 for noninferiority for both tests). CONCLUSIONS: Among patients at high risk for bleeding who received 1 month of dual antiplatelet therapy after PCI, the use of biodegradable-polymer sirolimus-eluting stents was noninferior to the use of durable-polymer zotarolimus-eluting stents with regard to the composite of death from cardiac causes, myocardial infarction, or stent thrombosis. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT04137510.

Intrathecal rapamycin attenuates the mechanical hyperalgesia of paclitaxel-induced peripheral neuropathy in mice.

Paclitaxel is an extensively used chemotherapy antitumor drug and paclitaxel-induced peripheral neuropathy (PIPN) is one of the most common side effect. Rapamycin, originally used as an adjuvant drug for chemotherapy, has recently been found to possess potential neuroprotective activities. Our purposes of this study are to verify the effect of rapamycin on PIPN, which contributes to a new target for PIPN treatment. Mice were given paclitaxel or rapamycin with different injection methods. Paw withdrawal threshold was tested at different time points for mechanical sensitivity assessment. Administration of paclitaxel, both 2 mg/kg and 5 mg/kg, could induce mechanical hypersensitivity. 0.01 mg intrathecal injection of rapamycin showed the best effect on attenuate the mechanical hyperalgesia of PIPN. Intrathecal injection of only rapamycin would not induce the mechanical hyperalgesia while when rapamycin and paclitaxel were used together the mechanical hyperalgesia induced by paclitaxel could be attenuated. Paclitaxel could induce mechanical hyperalgesia in mice and rapamycin could attenuate such mechanical hyperalgesia of PIPN.

Multi-targeting liposomal codelivery of cisplatin and rapamycin inhibits pancreatic cancer growth and metastasis through stromal modulation.

Pancreatic cancer treatment faces challenges due to drug resistance as well as liver metastasis. As a new strategy for treating pancreatic cancer, combination therapy is now available, but the dense mesenchymal barrier in the tumor tissue blocks drug delivery and impairs its therapeutic efficacy. To address this issue, we prepared an ATF peptide-decorated liposomal co-loaded with cisplatin and rapamycin (ATF@Pt/Rapa Lps), which targets both tumor cells and cancer-associated fibroblasts that express uPAR receptors. In tumor sphere penetration experiments, ATF peptide modified liposomes significantly enhanced deep penetration. More importantly, the ATF@Pt/Rapa Lps disrupted the stroma, as demonstrated by the downregulation of ɑ-SMA, I collagen, and fibronectin protein in vivo and in vitro. In this way, highly effective drug delivery to tumor cells can be achieved. As expected, there was a stronger inhibition of cell proliferation and migration by ATF@Pt/Rapa Lps in vitro compared to free Pt/Rapa and Pt/Rapa Lps. Furthermore, ATF@Pt/Rapa Lps showed greater therapeutic effects in PANC02 transplanted tumor mice and liver metastasis mice models. Ultimately, multi-targeting nanomedicines co-loaded with Rapa and cisplatin may provide a new approach to treating metastatic pancreatic cancer.

Effective Attenuation of Arteriosclerosis Following Lymphatic-Targeted Delivery of Hyaluronic Acid-Decorated Rapamycin Liposomes.

Background: The activation of lymphatic vessel function is the crux to resolving atherosclerosis (AS), a chronic inflammatory disease. Rapamycin (RAPA) recently has attracted considerable attention as a potent drug to induce atherosclerotic plaque attenuation. The objective of this work was to develop a ligand-decorated, RAPA-loaded liposome for lymphatic-targeted delivery of drugs to improve abnormal lymphatic structure and function, resulting in highly effective regression of atherosclerotic plaques. Methods: Hyaluronic acid-decorated, RAPA-loaded liposomes (HA-RL) were fabricated by emulsion-solvent evaporation. The average size, zeta potential, entrapment efficiency were characterized, and the stability and drug release in vitro were investigated. Furthermore, the in vitro and in vivo lymphatic targeting ability were evaluated on lymphatic endothelial cells and LDLR-/- mice, and the efficiency of this nano-system in inducing the attenuation of atherosclerotic plaques was confirmed. Results: HA-RL had a size of 100 nm, over 90% drug encapsulation efficiency, the storage stability was distinguished, demonstrating a slow release from the lipid nano-carriers. The mean retention time (MRT) and elimination half-life (t1/2ß) achieved from HA-RL were 100.27±73.08 h and 70.74±50.80 h, respectively. HA-RL acquired the most prominent efficacy of lymphatic-targeted delivery and atherosclerotic plaques attenuation, implying the successful implementation of this novel drug delivery system in vivo. Conclusion: HA-RL exhibited the most appreciable lymphatic targeting ability and best atherosclerotic plaques attenuation efficiency, opening a new paradigm and promising perspective for the treatment of arteriosclerosis.

Implication of mTOR Signaling in NSCLC: Mechanisms and Therapeutic Perspectives.

Mechanistic target of the rapamycin (mTOR) signaling pathway represents a central cellular kinase that controls cell survival and metabolism. Increased mTOR activation, along with upregulation of respective upstream and downstream signaling components, have been established as oncogenic features in cancer cells in various tumor types. Nevertheless, mTOR pathway therapeutic targeting has been proven to be quite challenging in various clinical settings. Non-small cell lung cancer (NSCLC) is a frequent type of solid tumor in both genders, where aberrant regulation of the mTOR pathway contributes to the development of oncogenesis, apoptosis resistance, angiogenesis, cancer progression, and metastasis. In this context, the outcome of mTOR pathway targeting in clinical trials still demonstrates unsatisfactory results. Herewith, we discuss recent findings regarding the mechanisms and therapeutic targeting of mTOR signaling networks in NSCLC, as well as future perspectives for the efficient application of treatments against mTOR and related protein molecules.

Structural Insights into the Giardia lamblia Target of Rapamycin Homolog: A Bioinformatics Approach.

TOR proteins, also known as targets of rapamycin, are serine/threonine kinases involved in various signaling pathways that regulate cell growth. The protozoan parasite Giardia lamblia is the causative agent of giardiasis, a neglected infectious disease in humans. In this study, we used a bioinformatics approach to examine the structural features of GTOR, a G. lamblia TOR-like protein, and predict functional associations. Our findings confirmed that it shares significant similarities with functional TOR kinases, including a binding domain for the FKBP-rapamycin complex and a kinase domain resembling that of phosphatidylinositol 3-kinase-related kinases. In addition, it can form multiprotein complexes such as TORC1 and TORC2. These results provide valuable insights into the structure-function relationship of GTOR, highlighting its potential as a molecular target for controlling G. lamblia cell proliferation. Furthermore, our study represents a step toward rational drug design for specific anti-giardiasis therapeutic agents.

TRPML3/BK complex promotes autophagy and bacterial clearance by providing a positive feedback regulation of mTOR via PI3P.

TRPML3 is a Ca2+/Na+ release channel residing in both phagophores and endolysosomal membranes. It is activated by PI3P and PI3,5P2. Its activity can be enhanced by high luminal pH and by replacing luminal Na+ with K+. Here, we report that big-conductance Ca2+-activated potassium (BK) channels form a positive feedback loop with TRPML3. Ca2+ release via TRPML3 activates BK, which in turn facilitates TRPML3-mediated Ca2+ release, potentially through removing luminal Na+ inhibition. We further show that TRPML3/BK and mammalian target of rapamycin (mTOR) form another positive feedback loop to facilitate autophagy induction in response to nutrient starvation, i.e., mTOR inhibition upon nutrient starvation activates TRPML3/BK, and this further reduces mTOR activity, thereby increasing autophagy induction. Mechanistically, the feedback regulation between TRPML3/BK and mTOR is mediated by PI3P, an endogenous TRPML3 activator that is enriched in phagophores and is up-regulated by mTOR reduction. Importantly, bacterial infection activates TRPML3 in a BK-dependent manner, and both TRPML3 and BK are required for mTOR suppression and autophagy induction responding to bacterial infection. Suppressing either TRPML3 or BK helps bacteria survival whereas increasing either TRPML3 or BK favors bacterial clearance. Considering that TRPML3/BK is inhibited by low luminal pH but activated by high luminal pH and PI3P in phagophores, we suggest that TRPML3/BK and mTOR form a positive feedback loop via PI3P to ensure efficient autophagy induction in response to nutrient deprivation and bacterial infection. Our study reveals a role of TRPML3-BK coupling in controlling cellular homeostasis and intracellular bacterial clearance via regulating mTOR signaling.