The primary mode of viral transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is thought to occur through the spread of respiratory droplets. The objective of this study was to investigate droplet and splatter patterns resulting from common endoscopic endonasal procedures.Cadaver simulation series.Dedicated surgical laboratory.After instilling cadaver head specimens (n = 2) with fluorescein solution, endoscopic endonasal procedures were systematically performed to evaluate the quantity, size, and distance of droplets and splatter following each experimental condition.There were no observable fluorescein droplets or splatter noted in the measured surgical field in any direction after nasal endoscopy, septoplasty with microdebrider-assisted turbinoplasty, cold-steel functional endoscopic sinus surgery (FESS), and all experimental conditions using an ultrasonic aspirator. Limited droplet spread was noted with microdebrider FESS , drilling of the sphenoid rostrum with a diamond burr , and drilling of the frontal beak with a cutting burr ; however, the use of concurrent suction while drilling resulted in no droplets or splatter. The control condition of external activation of the drill resulted in gross contamination .Our results indicate that there is very little droplet generation from routine rhinologic procedures. The droplet generation from drilling was mitigated with the use of concurrent suction. Extreme caution should be used to avoid activating powered instrumentation outside of the nasal cavity, which was found to cause droplet contamination. The current evidence suggests that otolaryngologists are particularly at risk for acquiring the infection secondary to the nature of the profession’s close contact with the upper respiratory tract, which harbors a high viral load.4 These have been documented traveling distances of greater than 2 m and contaminating surfaces on which they land.5 In addition, there is a significant concern for airborne transmission of SARS-CoV-2 during aerosol-generating procedures (AGPs).6 This has led to the recommendation from the American Academy of Otolaryngology-Head and Neck Surgery to limit elective procedures involving mucosal disruption or aerosolizing sprays, which may include nasal endoscopy, functional endoscopic sinus surgery (FESS), and endonasal approaches involving the use of powered instrumentation.8The primary mode of viral transmission is thought to occur via the spread of respiratory droplets, which carry virus particles approximately 0.125 microns in size.Despite these recommendations, there is a current lack of evidence quantifying the risk associated with these procedures. The purpose of this study was to investigate droplet and splatter patterns resulting from common endoscopic endonasal procedures in a cadaver-simulated series.This study was exempt from institutional review board because it involved the use of nonliving deidentified human cadaveric tissue specimens (IRB protocol 2004100753). The experiments in the study were all conducted in a dedicated surgical laboratory on 2 fresh-frozen cadaver head specimens prepared in identical fashion and placed in a standard supine surgical position. A direct brow incision was made bilaterally across the midline and the anterior frontal table exposed.External ports into the frontal and maxillary sinuses were created as described below. A 4-mm round cutting burr was used to perform the external trephination opening an anterior window approximately 8 to 10 mm in size into both frontal sinuses. Entry was confirmed with endoscopic visualization of the posterior table. Next, the maxillary sinus was approached with a Caldwell-Luc approach, and a similar bony window was created with a 4-mm round cutter burr with confirmation of entry with endoscopic visualization.9 The nasal cavity was then filled with 1 mg/mL fluorescein solution to the level of the anterior head of the inferior turbinate. After 15 minutes, a tracheal suction was used to suction out the instilled solution. The presence of residual fluorescein with adequate staining was confirmed endoscopically (Fluorescein solution at a concentration of 1 mg/mL was created by mixing 500 mg fluorescein 10% (100 mg/mL) AK-Fluor  with 495 mL sterile saline. KERLIX gauze impregnated in Vaseline was placed transorally into the oropharynx and inferiorly through the cut tracheal edge to completely obstruct the oropharynx, hypopharynx, and larynx. The 1 mg/mL fluorescein solution was instilled using a 14-gauge angiocath through the frontal and maxillary ports. Twice the average volume of the maxillary sinus (30 mL) and frontal sinus (14 mL) was instilled into each sinus.opically Figure 1Figure 2A,B). During the following experiment, a 25-cm × 25-cm piece of nonabsorbent blue paper was also affixed to the surgeon’s gown on the chest. The surgeon also wore a face-shield throughout the procedure. Immediately prior to the dissection, a tracheal suction was used to suction out any pooled fluorescein solution.Each cadaver head was placed in the standard operative supine position with the right side of the head toward the right-handed surgeon. Three pieces of 183-cm (6 feet) × 50-cm (1.64 feet) nonabsorbent blue paper affixed to cardboard were placed 90 degrees from each other in the following directions: (1) superior to the head, (2) left side of the head or across from the surgeon, and (3) inferior to the head (The senior author (E.A.I.) performed all of the experimental conditions. On the first cadaver head, the following surgical procedures were systematically performed: (1) nasal endoscopy, bilaterally; (2) septoplasty with bilateral microdebrider-assisted turbinoplasty; (3) complete left-sided FESS using cold, nonpowered instrumentation; (4) complete right-sided FESS using cold, powered suction microdebrider  at 5000 rpm; (5) powered drilling  of the left sphenoid face and rostrum using a 4-mm diamond burr at 75,000 rpm; (6) powered drilling of the right sphenoid face and rostrum using a 4-mm cutter burr; (7) external activation of the soiled drill; (8) powered drilling of the left frontal recess and beak using a 4-mm cutter burr; and (9) powered drilling of the right frontal recess and beak using a 4-mm diamond burr.On the second cadaver head, the following surgical procedures were performed: (1) complete left-sided FESS using cold, powered suction microdebrider; (2) complete right-sided FESS using cold, nonpowered instrumentation; (3) powered drilling of the left sphenoid face and rostrum with size 10 Frazier suction; (4) use of a ultrasonic aspirator on the left sphenoid sinus ; (5) powered drilling of the right sphenoid face and rostrum using a 4-mm diamond burr with size 10 Frazier suction; (6) powered drilling of the left frontal recess and beak using a 4-mm cutter burr with size 10 Frazier suction; (7) use of the ultrasonic aspirator on the right frontal sinus; and (8) external activation of the ultrasonic aspirator. Following each of the above listed surgical procedures, the size, number, and distance of the droplets and splatter on the nonabsorbent blue paper were evaluated and measured by the following technique, and 25-cm × 25-cm transparent grid graphs were laid side-by-side until the entire length of the paper was covered. The blue paper on the surgeon’s chest was removed and laid flat, and a grid was placed on it as well. The surgeon’s face shield was removed and laid flat, and blue paper with an overlying grid was placed underneath it.10 The evaluators then counted and recorded the number, size, and distance of any illuminated fluorescent spots. All measurements were performed independently by 3 evaluators .Since fluorescein fluoresces yellow under ultraviolet light and blue paper does not, the evaluators illuminated the paper with an ultraviolet light to visualize the droplets and splatter from each experimental condition. An example of this is depicted in No observable fluorescein droplets were noted in the measured surgical field in any direction after any of the following procedures: (1) nasal endoscopy, (2) septoplasty with microdebrider-assisted turbinoplasty, (3) FESS performed with cold instrumentation, (4) drilling of the sphenoid rostrum with a cutting burr, (5) drilling of the frontal beak with a diamond burr, (6) drilling of the sphenoid rostrum with a diamond burr with concurrent suction, (7) drilling of the frontal beak with concurrent suction, (8) ultrasonic aspirator on the left sphenoid sinus, (9) use of the ultrasonic aspirator on the right frontal sinus, and (10) external activation of the ultrasonic aspirator.Limited droplet spread was noted under the following 4 conditions: (1) microdebrider FESS , (2) drilling of the sphenoid rostrum with a diamond burr , (3) drilling of the frontal beak with a cutting burr , and (4) control condition of the drill placed outside the nose . 1114 As of the time of this publication, conflicting information persists among regulatory bodies as to degree of protection required for aerosol-generating procedures.16 While available evidence seems to suggest that spread is primarily through respiratory droplets, there is no consensus on exact transmission and little research available attempting to evaluate risk of specific otolaryngologic procedures. In otolaryngology clinical practice, many procedures involve instrumentation and examination of areas suspected to carry high viral loads: the nose, nasopharynx, and oropharynx.17 As practitioners begin to entertain a reverse surge into practice, knowledge of how these routine procedures generate droplets and aerosols is paramount to keeping health care providers and patients safe.As the SARS-CoV-2 pandemic progresses, knowledge of how to prevent its spread is of utmost concern. With the rapid dissemination globally of the virus, anecdotal evidence from experiences in Wuhan, China, as well as historical literature from similar viral epidemics  has guided otolaryngology protocols.The authors found in this cadaver simulation that common endonasal procedures including nasal endoscopy, septoplasty, turbinate reduction, and FESS with cold instrumentation were not sources of droplet or splatter contamination. There were limited amounts of contamination visualized following FESS with microdebrider and powered drilling of the sphenoid rostrum and frontal beak, and all observed droplet sizes were less than 1 mm. The use of a concurrent suction while drilling resulted in no contamination. The suction was used as per routine in a normal surgical setting to suck away pooled irrigation and accumulated bone dust. The reduced pooling of irrigation may have prevented the drill from splashing and propelling droplets out of the nasal cavity. Interestingly, even in the test conditions that resulted in contamination, there was a very limited spread of droplets. In fact, the farthest contamination distance was found to be 12 cm in the drilling of the sphenoid rostrum without concurrent suction. As expected during the control condition, activating a drill outside of the nose resulted in gross contamination with a maximum contamination distance of 13 cm and larger droplet sizes. These findings suggest that the soft tissue boundaries of the nasal cavity function as a barrier in preventing splatter and droplet contamination.18 reported in a recent study positive droplet contamination after all test conditions involving the drill, which included removing bone at the sphenoid rostrum, nasal beak, and external activation each for 10 seconds. Interestingly, the single cadaver study reported no contamination with either cold or microdebrider FESS, although it is important to note that they tested each condition for 10 seconds and applied the microdebrider to the anterior and posterior septum instead of the standard FESS technique.In contrast to our results, Workman et alRather than a contradiction of their findings, we believe our results add further context to this complex issue due to key methodological differences. All dissections in our study were performed using standard operating technique, minimizing use of powered instrumentation external to the patient or anteriorly in the nose. During the experimental conditions involving a drill, the powered instrument was only activated endonasally, used with the objective of performing key rhinological procedures in standard fashion, and was performed for 3 minutes rather than 10 seconds. Through this approach, we believe our droplet distribution simulates durations seen more frequently in the operating room environment. Running a contaminated drill externally as a control condition replicated the previously reported findings in showing gross contamination. Furthermore, the droplet contamination caused by the drill was completely mitigated by the use of concurrent suction.In an era of heightened alertness for safe technique, we must emphasize judicious utilization of powered instrumentation, which should occur only endonasally. Moreover, we recommend the use of a concurrent suction while performing powered drilling within the sinonasal cavity and anterior skull base to prevent droplet or splatter contamination. Based on these findings, the development of suction capabilities for endoscopic endonasal drills to help mitigate the risks of droplet contamination or automatic off switches when drills or debriders are moved outside of the patient may be important. In reviewing the literature, we also believe it is an important and new finding that the activation of the ultrasonic aspirator both endonasally and externally resulted in no observable droplet or splatter contamination.Several limitations to this cadaveric study merit discussion. First, there was no assessment of forced aerosolization (such as sneezing) in the experimental model. However, it is still vital to understand the quantity, quality, and range of droplet and splatter contamination involved during these common procedures, considering respiratory droplets are considered the primary mode of transmission of SARS-CoV-2. Another limitation is that only droplets and splatter visible to the human eye were measured. Endoscopic visualization and measurement of contamination using a blue light filter was attempted, but unfortunately, the authors found it less sensitive secondary to a glare effect. Moreover, instead of a complete 360-degree assessment, the design model allowed for measurements only in the cardinal directions surrounding the specimen.Due to the high intranasal viral loads found in infections such as SARS-CoV-2, there has been much concern over the potential for transmission associated with endonasal procedures. Our results indicate that there is very little droplet generation from key rhinologic procedures such as functional endoscopic sinus surgery and transsphenoidal pituitary approaches. This droplet generation was completely mitigated with the use of concurrent suction in the anterior nasal cavity. However, extreme caution should be used to avoid activating powered instrumentation outside of the nasal cavity, as this was found to cause droplet contamination. While these findings are encouraging, further study is warranted to determine the safety of these cases in the current environment.


Dendrites undergo extensive growth and remodeling during their lifetime. Specification of neurites into dendrites is followed by their arborization, maturation, and functional integration into synaptic networks. Each of these distinct developmental processes is spatially and temporally controlled in an exquisite fashion. Protein kinases through their highly specific substrate phosphorylation regulate dendritic growth and plasticity. Perturbation of kinase function results in aberrant dendritic growth and synaptic function. Not surprisingly, kinase dysfunction is strongly associated with neurodevelopmental and psychiatric disorders. Herein, we review, (a) key kinase pathways that regulate dendrite structure, function and plasticity, (b) how aberrant kinase signaling contributes to dendritic dysfunction in neurological disorders and (c) emergent technologies that can be applied to dissect the role of protein kinases in dendritic structure and function. Dendrites are specialized neuronal processes that receive and integrate synaptic or sensory input. While dendrites are extremely heterogeneous morphologically, dendrites of a certain neuron type generally exhibit stereotyped morphology , which then undergo autophosphorylation mediated activation. Several CaMKs have been implicated in dendritic development, and these act either via local effects or though transcriptional changes . The four neutotrophins, nerve growth factor (NGF), brain derived growth factor (BDNF) and neurotrophins 3 and 4  signal by binding and activating their receptors, which are members of the tropomyosin receptor kinases (Trk) and the structurally unrelated p75 neurotrophin receptor . In neurons, mTOR senses and integrates nutrient and growth factor availability through extracellular signals such as BDNF, insulin, insulin-like growth factor 1 (IGF1), vascular endothelial growth factor (VEGF), ciliary neurotrophic factor (CNTF) and glutamate, to mediate neuronal proliferation, dendritic growth and synaptic function –Akt–mammalian target of rapamycin (mTOR) signaling pathway promotes dendritic growth and branching through upregulation of protein and lipid synthesis. The lipid kinase PI3K signals through AKT and is inhibited by phosphatase, Drosophila peripheral sensory neurons , or Furry (Fry), a protein required for Trc kinase activity, leads to sensory neurons that produce excessive numbers of dendritic branches, which fail to tile normally  is a neurodevelopmental disorder with a strong genetic basis, and is defined by deficits in social communication, language development as well as repeated behaviors  was identified in a ASD proband  derived neurons differentiated from CDKL5 patients, suggesting that these are physiologically relevant kinase and substrates. Further, this important study also identified the consensus sequence of CDKL5 phosphorylation as RPXpS motif  in the 16p11.2 genomic locus are strongly associated with neurodevelopmental disorders including ASD, schizophrenia, and structural brain changes  is a severe neurodevelopmental disorder caused by presence of an extra copy (or parts) of chromosome 21  is a progressive neurodegenerative disease that affects wide areas of the cerebral cortex and hippocampus. Human neuropathology data from AD patients suggests that dendritic abnormalities in AD are widespread and often are present in the early stages of disease. Dendritic abnormalities associated with AD include dystrophic dendrites, reduction in dendrite complexity, and loss of dendritic spines  is pathologically defined by the neurodegeneration of dopaminergic neurons of the substantia nigra, and is characterized by the presence of cytoplasmic inclusions composed of α-synuclein protein aggregates called Lewy bodies .2+ concentrations found in the ATP binding site of the kinase, paired with a phosphopeptide , more efficiently than ATP, enhancing kinase activity of wild type as well as rescuing effects of low activity PD associated PINK1 mutant  is a metabolic labeling technique that can be used to incorporate amino acids carrying specific heavy and light isotopes of C and N, allowing for simultaneous identification and quantitation of complex protein mixtures  that is associated with a high risk of autism  derived neurons can be a powerful tool to model dendritic development and for studying how kinase signaling contributes to development and dendritic dysfunction. Stem cells can be reprogrammed from affected patient fibroblasts or from health controls, and differentiated into different neuronal and non-neuronal cell types to model neurological diseases Kinase signaling pathways act in a concerted fashion to mediate almost all aspects of dendritic development. These complex signaling pathways are elegantly intertwined, with key kinase signaling elements recurring throughout neuronal development. The human genome encodes a total of 518 kinases (Manning et al., SY and KN wrote the article and approved the submitted version.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.


Breast cancer is a leading cause of cancer-related deaths worldwide, predominantly caused by metastasis. It is generally accepted that the pattern of breast cancer metastasis is largely determined by the interaction between the chemokine receptors on cancer cells and the chemokines expressed at the sites of metastatic disease. Chemokine receptors belong to the G-protein-coupled receptors (GPCRs) family that appear to be implicated in inflammatory diseases, tumor growth and metastasis. One of its members, C-C Chemokine receptor 7 (CCR7), binds chemokines CCL19 and CCL21, which are important for tissue homeostasis, immune surveillance and tumorigenesis. These receptors have been shown to induce the pathobiology of breast cancer due to their ability to induce cellular proliferation and migration upon the binding of the cognate chemokine receptors. The underlying signaling pathways and exact cellular interactions within this biological system are not fully understood and need further insights. Thus, in this review, we summarize the essential roles of CCR7 and its receptors in breast cancer progression. Furthermore, we discuss the mechanisms of regulation that may lead to novel opportunities for therapeutic intervention. Despite the enormous advances in our knowledge of the nature of the chemokines in breast cancer metastasis, research about the involvement of CCR7 in cancer progression is still limited. Therefore, further studies are essential to illustrate the distinct roles of CCR7 in cancer progression and validate its potential as a preventive bio-factor for human breast cancer metastasis by targeting chemokine receptor genes.  Since their discovery over 30 years ago, numerous chemokines have been identified, with new roles emerging past the homing of leukocytes . ChemokiCurrently, 22 chemokine receptors have been identified in humans. These are G-protein-coupled receptors (GPCRs)-integral seven-transmembrane glycoproteins domains (connected by three intracellular and three extracellular loops), a short extracellular N-terminal that will bind to the ligand and an intracellular C-terminal coupled to the group of G-protein receptors. In the literature, four “atypical” receptors have been described capable of binding chemokines with high affinity with no signaling or signaling which is not mediated by G-proteins. This group of receptors has also been named “scavenger” receptors and recently renamed to “ACKR”, the acronym of atypical chemokine receptor . ReceptoChemokines and chemokine receptors are essential in dendritic cell (DC) maturation and (B and T) cells’ development ,7. IndeeA considerable body of evidence highlights the importance of chemokines and their receptors during tumor progression and metastasis. Initially, chemokines selectively regulate the recruitment and trafficking of leukocyte subsets during homeostasis and inflammation ,13. HoweThe discovery that cancer cells overexpress C-C Chemokine receptor 7 (CCR7), which directs them to organs that express their ligands CCL21 and CCL19, led to an increase in reports confirming that chemokine receptors were present in a non-random manner in many other cancers. A positive correlation between chemokine receptor expression and worse prognosis has been found in most but not all cancers. In breast cancer, it has been proved that the chemokine receptors CXCR4, CCR7, CCR6 and CXCR3 and their ligands have been associated with metastasis ,17. DespThe recruitment of immune cells to the tumor site is mediated by the actions of chemokines and their specific ligands, which have also been widely linked to cancer progression and metastasis. Upon the binding of the chemokine ligands, the receptor undergoes a conformational change. This results in the expression of several genes and activates a signaling cascade that, depending on the context, can stimulate cellular growth, migration, pseudopodia formation, adhesion, as well as angiostasis .It was thought that metastasis was regulated through factors such as the size of the vessels or the difference in pressure between the blood and the organs . HoweverC-C Chemokine receptor 7 (CCR7), is a GPCR commonly expressed by T-cell subset central memory cells, thymic T-cells, B cells, mature DCs and other rare cell subsets such as CD4+ CD25+ splenocytes ,23,24,25CCL21 and CCL19 are constitutively expressed at the beginning of the lymphatic vessels and in lymphoid organs. They are present on several stromal cells and on the high endothelial venules (HEVs). Unlike CCL19, CCL21 binds to glycosaminoglycans (GAGs) and immobilizes on the surface of endothelial cells . This meWhile the overexpression of CXCR4/CXCL12 is correlated with the homing of cancer cells to the liver, lung, bone marrow and lymph nodes, the overexpression of the CCR7/CCL21 axis has mainly been related to lymph node metastasis . It alsoCellular migration in vivo and in vitro is dependent on both the physical and biochemical properties of cells and the extracellular matrix (ECM) . For celThe homing and migration of lymphocytes as well as CCR7-positive malignant cells metastasis into secondary lymphoid tissue (SLT) are precisely regulated by complex chemokine–chemokine receptor interaction in the microenvironment. CCR7-mediated T cell migration within SLT is critical for T cell activation and differentiation in adaptive immunity. Investigating the migratory response of CCR7 expressing T cells within a certain chemokine environment will lead to a better understanding of the mechanism of directional migration of T lymphocytes . An inveCCR7 is of particular interest in understanding metastasis because CD4+ T cells and DCs require CCR7 expression to migrate through lymphatic vessels . The lymPre-clinical studies have demonstrated that CCL19/CCL21 producing lymphatic endothelial cells can actively guide the chemotactic migration of CCR7-expressing tumor cells . AdditioCCR7 has also been linked to the creation of new blood and lymphatic vessels in breast cancer patient samples, though the mechanism is still not well understood . This lyThe receptor CXCR4 is the most studied of all chemokine receptors in cancers. Its roles in breast cancer, including cell survival, proliferation, motility, invasion, angiogenesis, recruitment and activation of a number of different cell types, as well as metastasis are well documented . CCR7 isIn breast cancer, hypoxia has been shown to increase CCR7 expression through the hypoxia-inducible factor 1 (HIF-1)-mediated activation of the endothelin receptor A . EpigeneThe interaction between CCR7 and CCL21 has been shown to improve the immunogenicity of CCR7-expressing breast cancer cells . It alsoChemokine signaling in which a cell responds to a self-secreted chemokine is known as autocrine signaling, as opposed to paracrine signaling in which a cell responds to a chemokine secreted by a neighboring cell . Both auThere are a large number of chemokines that regulate the downstream effector molecules which may account for the various effects of these recipients in tumor pathology. To date, the effector molecules and unique mechanisms controlling cell chemotaxis, proliferation, survival and motility have been highly considered in chemokine receptor signaling in cancer ,89.Measuring chemokine ligands’ ability to induce early physiological changes, chemotaxis, actin polymerization and calcium mobilization is routinely performed in vitro to assess chemokine receptor functionality . These fIt is thought that chemokine responses are activated through key survival pathways such as the PI-3K and AKT, JAK/STAT, ERK 1/2, JNK, GSK-3α/β and MAP kinase ,92,93. MChemotactic responses and actin polymerization are not induced following CCR7 ligand-binding activation in non-metastatic breast cancer cells. Chemokine signaling pathways are mediated by the mobilization of the calcium flux through the activation of GPCRs, specifically the Gαi subunit that results from the inhibiting of adenylyl cyclase-mediated cAMP ,101. TheHematopoietic and non-hematopoietic tumors metastatic to lymph nodes are correlated with tumor-dependent CCR7 expression. Physiologically, this indicates mature DCs and naive T lymphocytes recruitment to lymph nodes ,103. HowThe pathways used by CCR7 to facilitate metastasis are not fully understood. It has been reported that CCR7 facilitates cancer progression by manipulating cancer cells’ anoikis and mobility  as well CCR7 expression is regulated by transcription factors, epigenetic and miRNAs mechanisms. Transcription factors such as SP-1, AP-1, NF-κB and NF-AT1 can bind and regulate CCR7’ expression ,109. NevStudies in the field of breast cancer have shown that the correlation between the expression of CCR7 and clinical pathogenicity in human breast cancer is important, including vascular invasion, lymph node metastasis and tumor grade . Howeverthe in vitro and in vivo movement of cancer cells [The up-regulation of specific chemokine/receptor pairs has been reported in many human cancers including lung, breast, colon, prostate, gastric and melanoma ,117, alter cells .The CCR7 expression was evaluated in breast cancer cells in vitro to ascertain whether the rates of expression of a chemokine receptor contribute to the pathogenicity of breast cancer. A group of cell lines was used; each was originally derived from patients with different types of metastatic breast disease displaying various degrees of invasiveness. The examination of the CCR7 expression revealed significant levels of expression in different breast cell lines. Studies reported low CCR7 mRNA levels in MCF-7, MDA-MB-231 and T47D ,118, no Metastasis is one of the problematic tumor processes to study in vitro since the tumor progression relies on sequential events that are dependent on the characteristics of the tumor cells and their interactions with the tissue environment . TherefoTo provide further insight into chemokine-mediated metastatic mechanisms, the expression of CCR7 on mammary epithelial cell lines was modulated using an shRNA system based on the ability to deliver and stably express shRNAs, to study the effect of chemokine modulation on the metastatic propensity of breast cancer cells in vivo . The silCCR7 expression has been reported in particular to increase B16 metastatic capacity via the lymphatic system but not through a blood-borne pathway . At firsHigher levels of CCR7 mRNA was detected in cancer tissue compared to the normal counterpart lung tissues, suggesting that CCR7 expression could be used to predict the metastasis of the lymph node before surgery as a diagnostic tool to improve the overall treatment plan for non-small cell lung cancer (NSCLC) . MoreoveSeveral experimental approaches using specific chemokine antagonists, neutralizing antibodies, knockout mice and targeted gene disruptions have been reported to be useful tools for discerning the role of chemokines and their receptors in vivo ,133. TheFor most chemokine receptors, removing their cytoplasmic tail impairs endocytosis ,135. ForInitial reports suggested that CCL21 and CCL19 were anti-tumorigenic, as they could attract lymphocytes that would attack the tumor . HoweverThe function of CCR7/CCL21 in cancer appears to be controversial; it may play a pro- or anti-tumor function depending on where it is expressed. In tumor cells, it induces development and progression, whereas when expressed by immune cells such as dendritic or T cells, anti-tumor responses are detected . MoreoveThis comprehensive review provided an insight into chemokine-mediated mechanisms performed in breast cancer cells both in vitro and in vivo, specifically CCR7; there are still important questions that remain to be answered. It will be valuable to gain a broader understanding of the functional role of chemokines and their receptors in different cancers other than breast cancer. In addition, given the complex process of metastasis, it is likely that chemokine receptors may function at several steps and therefore, the use of specific in vivo models may assist in elucidating the precise stage at which chemokines and their receptors influence the metastatic pathway. Finally, it would be recommended to examine the effect of chemokine receptor expression on the growth of primary tumors using an orthotopic cancer model to verify the precise role of chemokines during metastasis. The understanding of chemokine-mediated pathways and consequently, their potential disruption may provide useful information for the design of new strategies to treat breast cancer.In recent decades, there have been vast advances in our knowledge regarding the roles of the chemokines in cancer progression. In addition, most chemokines have been reported to act as promoting factors for tumor growth and proliferation, angiogenesis and metastasis. This could be due to the promiscuity of chemokine and chemokine receptor interactions in cancer cells. Participation in cancer cell invasion, metastases and tumor development was reported with the CC Chemokine receptor 7 (CCR7). CCR7 and its receptors have been particularly reported to stimulate the tumorigenesis of breast cancer. It also played a novel role in inducing lymphangiogenesis in the experimental model of breast cancer. It was also accompanied with EMT in human breast carcinoma. Furthermore, CCR7 has been involved in the metastasis of the lymph nodes, invasion, migration and EMT. Findings showed that the EMT progression in breast cancer cells was controlled by the CCL19/CCR7 axis and facilitated the invasion and migration process of tumor cells by triggering several signaling pathways. On the basis of this review, CCR7 has a critical role in breast cancer progression. Thus, it might be elucidating that CCR7 could be a novel target for tumor therapy when used in combination with chemotherapy or immune checkpoint therapy. For verification of CCR7 and its ligands as a therapeutic target for breast cancer progression, further studies are required to illustrate their function and the mechanism of their actions involved in breast cancer progression.


Saccharomyces cerevisiae yeast. Commercial wine strains have a number of advantages over natural isolates, and it is their use that guarantees the stability and reproducibility of industrial winemaking technologies. For the highly competitive wine market with new demands for improved wine quality, it has become increasingly critical to develop new wine strains and winemaking technologies. Novel opportunities for precise wine strain engineering based on detailed knowledge of the molecular nature of a particular trait or phenotype have recently emerged due to the rapid progress in genomic and “postgenomic” studies with wine yeast strains. The review summarizes the current achievements of the metabolic engineering of wine yeast, the results of recent studies and the prospects for the application of genomic editing technologies for improving wine S. cerevisiae strains.Modern industrial winemaking is based on the use of starter cultures of specialized wine strains of  For thousands of years, saccharomycetes have been used by humans to produce-wine, bread, beer, and other fermented foods ,2. The bMore than two hundred commercial strains of wine yeast available on the world market are actively used by winemakers to produce different types, varieties and brands of wines. Nevertheless, growing competition in the world market, increasing consumer demands for the quality of wine, its useful properties stimulate winemakers and biotechnologists to search for new cultures and technologies .Starting from the 1990s, classical strain improvement methods (CSI) based on the repeated alternation of successive stages of mutagenesis and selection  have incThe adaptive laboratory evolution (ALE) is a technique of strain optimization that assumes serial or continuous culturing of a particular yeast strain for many generations under selective pressure, such as high ethanol content, high osmolarity etc., thus directing the accumulation of mutants with desired phenotype. As compared to stochastic and laborious CSI techniques, ALE methods are more targeted and convenient . The powFinally, the rapid progress in the field of yeast genomics, systems biology, genetic engineering in recent decades have opened up new possibilities for creating new wine strains based on the knowledge of the molecular nature of the target trait or phenotype ,17,18,19The ML01 strain is capable to perform malolactic fermentation (MLF) simultaneously with alcoholic fermentation due to the presence of integrated copies of a yeast malate permease gene and a bacterial malolactic enzyme gene . During DUR1,2 amidolyase gene [The ECMo01 strain contains and additional copy of the ase gene . Wine prCertain barriers to the widespread use of GM technologies for creating wine strains are associated with regulatory restrictions and negative public perception in many countries of GM technology . Many ofS. cerevisiae strains are discussed.A number of excellent reviews devoted to various aspects of the metabolic engineering of wine and other industrial yeast strains have been published in recent decades ,24,25,26The goals of genetic manipulation of wine strains are aimed at improving such characteristics as the speed and completeness of fermentation, the efficiency of wine processing, preventing the growth of foreign microflora, wine spoilage, refinement of sensory characteristics, nutritional value, including reducing the concentration of harmful and increasing useful compounds, etc.  5,17,2424,25,27.One of the serious problems of winemaking that leads to wine spoilage is “sluggish” or “stuck” fermentation . A commoDepending on grape variety, ripeness, climate, soil etc. YAN content in grape juice may vary from 50–350 mg/L. In general, it is considered that 120–140 mg N/L is sufficient to complete the fermentation of 200 g/L of sugar . HoweverTo fill YAN deficiency, various inorganic ammonium salts, such as diammonium phosphate, are used. However, excessive addition of such salts can also lead to a carbon–nitrogen imbalance and adversely affect the quality of wine , for insMFA2 gene of the a-factor yeast sex pheromone had the greatest effect  sugar catabolism compared to the parent, deletion of MFA1 gene did not have such effect. Authors speculate that the positive effect of MFA2 deletion on sugar consumption under nitrogen limitation may be explained by reduced energy “waste”, but conclude that further work is needed to determine the basis for the link between these genes and fermentation [st cells  by a mulls [STE6 . Many a-ls [STE6 . In the entation .ECM33 gene resulted in the shortest fermentation duration (up to 31% reduction) in either a synthetic medium or grape juice  led to more than two-fold decrease in haziness  that destroy the residual polysaccharides of grape juice had useful properties in terms of facilitating the processing and clarification of wine, stabilizing the color and bouquet of the drink .HSP26 and YHR087W, were introduced into wine strains under the control of their own promoters, the SPI1 gene promoter, induced at ethanol and osmotic stress conditions, or the potent constitutive glycolytic enzyme gene PGK1 promoter, as part of plasmids or as a stable chromosome-integrated copies [A promising way to increase the tolerance of wine strains to ethanol, osmotic stresses is to increase the expression of genes induced under the conditions of “fermentation stress” . Two sucd copies . Stress Brettanomyces/Dekkera [S. cerevisiae are active only against this yeast and cannot be used to control the growth of non-saccharomyces (NS) microflora. Several NS yeast species, such as Kluyveromyces wickerhamii, Pichia anomala, Pichia membranifaciens, and Candida pyralidae do produce killer toxins, but their activity against D. bruxellensis is low [Contamination with extraneous microflora presents a serious threat to winemaking because it can slow down the winemaking process, stop it completely, damage wine . The mos/Dekkera ,66. The /Dekkera . Traditi/Dekkera . A commo/Dekkera . However/Dekkera  can be us is low .S. cerevisiae antimicorbial peptides (AMPs) [Hanseniospora guillermondi cells present in the supernatants of S. cerevisiae CCCMI 885 cell fermentations [S. cerevisiae strains overexpressing corresponing TDH1 and TDH2/3 gene fragments were generated and shown promising to produce this biopreservative, active against both D. bruxellensis and lactic acid bacteria at large scale [Known genetic engineering approaches consist in creation of microbicidal GM yeast strains producing bacteriocins , glucanas (AMPs) . The disntations , the natntations . Recombige scale .One of the most important and difficult areas of metabolic engineering of yeast is the production of “low alcohol” strains, i.e., strains capable of releasing less ethanol during wine fermentation while maintaining all other biochemical and organoleptic parameters of the resulting beverage. Wines with reduced strength are finding increasing demand among consumers due to a number of factors. A decrease in ethanol concentration has a positive effect on the nutritional value and organoleptic parameters of wine, and better corresponds to various regulatory standards .Aspergillus niger glucooxidase (GOX) [Lactobacillus casei lactate dehydrogenase (LDH) [There are various technological and microbiological approaches to reducing the alcohol concentration in wine . A groupse (GOX) , Lactobase (LDH) . Under tS. cerevisiae glucose transporter genes in order to force the obtained GM strains to switch their metabolism from fermentative to respiratory one regardless of glucose concentration in the culture medium [Another approach consisted of the intensive modification of e medium . Howevere medium , fermentAlternative strategies for producing “low alcohol” yeast were aimed at manipulating the endogenous genes of alcohol dehydrogenases , triosepAnother option for reducing the ethanol yield included switching part of the carbon metabolism towards the synthesis of Krebs cycle intermediates. Although both overexpression and deletion of the genes of some enzymes involved in oxidating or reducing TCA branches influenced the content of organic acids, the ethanol yield did not change .HXT2 and the regulator MIG1. However, the decrease in the level of ethanol in this strain was very slight [In order to relieve glucose repression of genes encoding respiratory enzymes, the authors obtained a strain with deletion of genes for the glucose transporter y slight .GPD1 or GPD2 genes of glycerol-3-phosphate dehydrogenase isoenzymes increased glycerol concentration by more than five-fold [ALD6 and butanediol dehydrogenase BDH1,2 genes [Perhaps the most successful and viable strategy for producing “low alcohol” strains is to redirect part of the carbon flux from ethanol to glycerol at the glycolysis stage. Overexpression of the ive-fold . The resive-fold . The dis,2 genes .TPS1 trehalose synthase gene while maintaining complete fermentation and a slight increase in glycerol content.Encouraging results were obtained by parallel analysis of collections of wine and laboratory strains overexpressing or deficient in several central metabolism genes . The greThese studies open up new possibilities for developing approaches for obtaining “low alcohol” strains of yeast, but it is clear that the practical implementation of such strains is still a matter of the distant future. The increased formation of glycerol due to alcohol during fermentation leads to a violation of the redox balance, the formation of wines with high glycerol/ethanol ratio and an unacceptable content of other metabolites that have a negative effect on the sensory wine qualities.Wine taste and aroma are main characteristics that determine the differences between a huge number of brands and varieties of wines produced worldwide. The chemical composition of wine depends on many factors, determined by the enormous variability of the conditions of both grape growth and winemaking technologies. These include the grape variety, geographical and technological features of its growing, microbial ecology of grapes and fermentation processes, winemaking methods, and when using starter cultures, the individual characteristics of the strain used.The contribution of yeast to the formation of the aroma of wine can be due to (a) the production of enzymes that convert “aromatically neutral” grape compounds into aromatically active, (b) biosynthesis of hundreds of aromatically active secondary metabolites, i.e., acids, alcohols, esters, polyolols, aldehydes, ketones, volatile sulfur compounds, and volatile phenols, (c) production of ethanol and other solvents that help to extract aromatic compounds from grape solids, and (d) autolysis of dead yeast cells .Esters make the most significant contribution to the formation of a characteristic bouquet of wine during fermentation. The composition of the wine contains up to 160 of these compounds formed during alcoholic fermentation. The most significant effects on the aroma and bouquet of wine have acetate esters and fatty acid ethyl esters. Among the acetate esters, ethyl acetate (“fruity” and “tart” aromas), isoamyl acetate (“banana” flavor) and 2-phenylethyl acetate  can be distinguished. In the formation of ethyl esters of fatty acids, the alcohol group is ethanol, and the acyl group is derived from medium chain activated fatty acids. This group includes ethyl hexanoate (pear flavor) and ethyl octanoate (apple flavor).ATF1 gene increased the content of ethyl acetate, isoamyl acetate, 2-phenyl acetate, and ethyl caproate during fermentation. Overexpression of the ATF2 gene had a lesser effect. Overexpression of the EHT1 gene enhanced the content of esters responsible for apple, apricot, and banana flavor in wine [To modulate the content of volatile esters responsible for fruit aromas in wine, GM derivatives of strain VIN13 with overexpression of alcohol acetyltransferase genes were obtained. Overexpression of the  in wine .An interesting approach to increase the content of monoterpenoids in wine is to create GM strains of yeast capable of de novo synthesis of these aromatizing agents. Expression of the sweet basil geraniol synthase gene in the wine strain increased the geraniol content to 750 μg/L, which is more than an order of magnitude higher than the sensory perception threshold of the compound. The total content of other terpenoids increased by more than 200 times in comparison with the control .Aspergillus awamori arabinofuranosidase along with Aspergillus kawachii β-glucosidase. Wine obtained using a strain expressing both enzymes contained higher concentrations of monoterpenes  than wine obtained by processing with a commercial enzyme preparation and possessed improved sensory characteristics [Important aromatic components of Muscat and Riesling grape varieties are monoterpenes. For the full manifestation of their aromatic properties, these compounds must be released from complexes with various wine wort polysaccharides. Researchers from the Institute of Wines Biotechnology, Stellenbosch University (South Africa), obtained derivatives of the VIN13 wine strain producing secreted eristics .BDH1,2 genes in the S. uvarum strain, the authors were able to almost halve the concentration of this unpleasant impurity component [High concentrations of diacetyl give an unpleasant, oily, aftertaste to the wine. Diacetyl is a side prolapse of the valine metabolism and can be converted to acetoin and butanediol by the action of the Bdh1p and Bdh2p Butandiol Dehydrogenases. Due to the coexpression of omponent .FBA1 gene promoter were integrated into the H0 locus of the AWRI strain  in chardonnay grape juice while retaining the ability to completely ferment wine wort .Resveratrol (RV) is a potent plant antioxidant with multiple beneficial effects on human health and is therefore used in medical, food, and cosmetic areas . In grapS. cerevsiae strain, engineered to express 4-coumaroyl-CoA ligase gene from poplar and resveratrol synthase gene from grapevine [S. cerevisiae strain through overexpression of bacterial TAL genes, A. thaliana 4CL gene and VST gene from Vitis vinifera [S. cerevisiae was engineered by introducing A. thaliana AtPAL2, AtC4H At4CL2 genes and V. vinifera VST1 gene in the same CEN.PK102-5B strain under the control of strong constitutive pTEF1 and pPGK1 promoters [Recombinant RV production was first shown in laboratory rapevine . The obtrapevine ,89. In tromoters . Cultureromoters .S. cerevisiae strains used in a number of traditional technologies for production of biologically aged wines [A special group of winemaking microflora is represented by flor ed wines ,91, suched wines .In the course of prolonged biological exposure under the flor yeast vellum, wine acquires specific taste and aroma characteristics caused by changes in the yeast metabolism from enzymatic to oxidative one ,93,94. GKey flor yeast strain characteristics, such as the ability for efficient vellum formation, resistance to high alcohol and acetaldehyde concentrations, to low pH etc are largely associated with the properties of cell surface proteins.FLO11 adhesin gene for flor yeast biofilm formation was proven in experiments on its genetic inactivation [Ccw14p and Ygp1p [BNT2 gene encoding one of the vesicular transport proteins [The key role tivation  or overetivation . Overexpnd Ygp1p , as wellproteins , can alsHSP12 [HSP150 [SOD1 and SOD2 superoxide dismutase genes, genes for enzymes of the gluthathione biosynthesis pathway [Promising targets for further genetic and genetic engineering manipulations with flor strains aiming at improving their biofilm formation ability and resistance to various types of stress are HSP12  and HSP1 [HSP150 , heat sh pathway  , which can slow down or stop this important technological process. Therefore, a strain of wine yeast capable of simultaneously performing NMB would be of great interest both to winemakers and to consumers.Strain ML01  is capabSchizosaccharomyces pombe mae1 gene encoding malate permease and the O. oeni malolactic enzyme gene mleA. Both genes are placed under the control of the strong constitutive promoter of the S. cerevisiae PGK gene. The strain is able to completely ferment malic acid at 5g/L concentration in the wort within 5 days, without negative effect on the sensory wine properties. Further detailed phenotypic, transcriptome, proteomic analysis showed that strain ML01 is equivalent to the original parental wine strain [Strain ML01 obtained on the basis of strain S92 contains two chromosomally integrated genes- the e strain .Another GM wine yeast strain approved for use in USA and Canada was obtained in order to reduce ethyl carbamate (EC) content in wines . EC is aDUR1,2 under the control of the regulatory sequences of the PGK1 gene [DUR1,2 gene is 17 times increased, which leads to a decrease in the urea concentration, and the ammonia produced is utilized as a nitrogen source. The concentration of EC in wine obtained using the ECMoO1 strain was reduced by 90%, while the phenotypic characteristics of the strain are equivalent to the original strain 522.Strain ECMo01 contains an additional copy of the amidolyase gene GK1 gene . Dur1,2pS. cerevisiae, an extensive and diverse set of tools for genetic engineering and directed modification of the genome has been developed quite a long time ago and are widely used for research in the fields of functional genomics, synthetic biology, biotechnology, and metabolic engineering [For laboratory strains of ineering . At the ineering .S. cerevisiae was published back in 2013 [The use of CRISPR-Cas genome editing systems can successfully overcome these limitations. The first work on the application of the CRISPR-Cas system for  in 2013  and the  in 2013 .Examples of the successful application of CRISPR-Cas systems for industrial yeast strains relate to such aspects as the production of bioethanol from lignocellulosic raw materials, metabolic engineering for the production of vitamins and antibiotics, the improvement of aromatic and taste properties of beer, and a number of others ,110,111.CAN1 gene [CAN1 gene encodes arginine permease, which along with GAP1 amino acids permease is responsible for the transport of arginine to yeast cells from the culture medium. During the subsequent stages of catabolism, arginine is cleaved by Car1p arginase to ornithine and urea, which is either excreted by Dur4p permease or converted to carbon dioxide and ammonia by Dur1p/Dur2p urea amidolyase. The resulting recombinant strains were characterized by reduced urea production  under experimental micro-winemaking with the ability to ferment a synthetic substrate, although at a slightly reduced growth rate. The authors believe that further verification of the strains is necessary under the conditions of industrial winemaking. The advantage of introducing a mutation into the CAN1 gene compared to other methods of modifying arginine utilization pathways is that this technique is less sensitive to fluctuations in the content of nitrogen sources in the wort and less affects the growth parameters of yeast strains [One recent study describes the use of the CRISPR-Cas system for producing wine strains with reduced urea production. A group of scientists from Canada and Italy constructed derivatives of wine strains EC1118 and AWRI1796 defective in both alleles of the AN1 gene . The CANFAS2 genes (encodes the α subunit of fatty acid synthase) and TOR1 (a growth regulator in response to the availability of a nitrogen source), linked to the trait of increased PEA production. Then, using CRISPR-Cas in commercial wine strains, wild alleles were replaced with mutant ones. As a result, the production of PEA increased by 70% [A promising area of application of genome editing methods is the directed change in the pathways of biosynthesis of aromatic compounds. Thus, in a recent work, yeast strains with increased production of phenylethyl acetate (PEA) were obtained using the CRISPR-Cas system . PEA is d by 70% .S. pastorianus/bayanus beer yeast strain [FDC1 gene. Using the CRISPR-Cas system, the authors introduced a mutation characteristic of ale strains into all four copies of the FDC1 gene in the lager strain. The result was a strain containing a cis-gene mutation that lacks the ability to produce 4VG and has significant potential for use in the beer industry.In another work, the CRISPR-Cas system was used to reduce the production of 4-vinyl guaiacol (4VG) in a hybrid t strain . It is kThe CRISPR-Cas system is an extremely convenient tool for research in the field of functional genomics of wine strains. Until recently, the vast majority of experiments in the field of functional genomics of yeast were performed using laboratory strains. Nevertheless, according to the latest information from the SGD database (27 June 2020), when classified in terms of gene ontology, a significant number of yeast genes remain “unknown” . Such uncertainty is partly determined by the lack of specific conditions in which these genes are important. At the same time, these unknown genes experience regular changes in expression during many technological processes, including at different stages of wine fermentation .Characteristic changes in the expression pattern of a number of “unknown” genes were revealed in our recent work during the transcriptome analysis of the sherry strain at different stages of film formation . CRISPR-S. cerevisiae can be successfully used in oenology for the metabolic engineering of wine strains. GM strains of wine yeast with targeted changes in various stages of the central or secondary metabolism have proven effective both in optimizing the winemaking process itself and in improving the quality of the resulting wines. Selected examples discussed in the review are shown in The extensive arsenal of genetic manipulation methods developed for laboratory strains of


The epigenetic regulation of microRNA (miRNA) expression related to the FK506-binding protein 5 (FKBP5) gene may contribute to the risk of stress-related disorders such as posttraumatic stress disorder (PTSD). Here, we identified candidate miRNAs derived from FKBP5 knockout mice as a potential diagnostic biomarker of PTSD. Using a translational approach, candidate miRNAs found to alter in expression within the medial prefrontal cortex of FKBP5 knockout mice were selected. Each candidate miRNA was examined in the serum of 48 recently traumatized individuals with PTSD and 47 healthy individuals. Multimodal imaging was also conducted to identify the neural correlates for the expression of candidate exosomal miRNAs in response to trauma exposure. Differential miRNA expression was found according to PTSD diagnosis in two composite marker groups. The differential miRNA expression between the composite marker groups contributed to PTSD symptom severity, which may be explained by differential recruitment of prefrontolimbic activity in brain imaging. The present study reveals that a set of circulating exosomal miRNAs showing altered expression in FKBP5 knockout mice play a potential role as epigenetic markers of PTSD. The corroborative evidence from multiple levels including molecular, brain, and behavioral indicates that these epigenetic biomarkers may serve as complementary measures for the diagnosis and prognosis prediction of PTSD in recently traumatized individuals. Emerging evidence suggests the contribution of epigenetic regulation of microRNA (miRNA) expression in the risk and protective factors associated with PTSD3, including recent reports of altered miRNA expression in response to an array of environmental stressors5. This suggests that changes in miRNA expression following trauma may serve as a promising diagnostic biomarker as well as a molecular target for mechanism-based treatments3. Given the multilevel regulatory role of miRNAs in the brain genomic response to environmental stress5, understanding the concerted actions of various miRNAs in relation to target gene pathways may provide insight with regards to the phenotypic heterogeneity of PTSD10. However, discrepancies exist among previous studies regarding the identification of miRNAs that are related to the pathophysiology of PTSD11. Furthermore, despite the immediate neuroplastic effects as modulated by miRNA following traumatic exposure13, little is known about the dynamic changes that occur in miRNA expression profiles during the acute stages of PTSD. Known for its fundamental regulatory role of the hypothalamic-pituitary-adrenal (HPA) axis and glucocorticoid receptor (GR) function in the presence of stress15, FK506-binding protein 5 (FKBP5) is a key modulator of stress-related disorders including PTSD17. Since the landmark study on the genetic association of FKBP5 with PTSD18, several preclinical and clinical studies examined the genetic modulation as well as peripheral and central expression of FKBP5 in stress-related disorders including PTSD and major depression20. Studies also revealed that having the risk genotype of FKBP5 may affect the prefrontolimbic circuit of the brain, an area known to regulate stress response25. Taken together, FKBP5 overexpression may be precipitated by both genetic predisposition and additional epigenetic modulation, and may therefore increase the risk for stress-related disorders with various phenotypes28.Posttraumatic stress disorder (PTSD) is a psychiatric condition that results from trauma, and its pathology influences various biological processes within the body29, then examined in the human serum samples. We hypothesized that these candidate miRNA levels alter in the case of recently traumatized individuals with PTSD as compared to trauma-unexposed healthy individuals, and that these alterations in miRNA levels are associated with the structure and functional activity of the prefrontolimbic regions of the brain.The present study identified clusters of circulating miRNA according to their alteration in expression level in recently traumatized individuals diagnosed with PTSD, and examined the mediating role of this stress-induced epigenetic modification on the prefrontolimbic circuit of the brain. Using a translational approach, candidate miRNAs were derived from an FKBP5 knockout (KO) mouse model). These miRNAs were then selected as the final candidate miRNAs for further analyses in the human PTSD cohort.The medial prefrontal expression profiles of miRNAs were compared between FKBP5 KO and FKBP5 wild-type (WT) mice. In total, 23 downregulated and 18 upregulated miRNAs were revealed in FKBP5 KO mice compared with FKBP5 WT mice Table . ConservS2The exploratory analyses to examine the relationships between the expression profiles for the subset of identified miRNAs in the two compartments of serum exosomes and medial prefrontal cortex (mPFC) were performed and are presented in Fig. Among the aforementioned 18 candidate miRNAs selected from the FKBP5 KO mouse model, 3 of the circulating exosomal miRNAs displayed levels below the detection limit in more than 10% of the human serum samples , and were excluded from the final analysis.30, the principal component analysis based on a correlation matrix between the 15 candidate miRNA expression values identified three distinct clusters of ‘composite marker 1’, ‘composite marker 2’, and ‘composite marker 3’, each of which comprised of 8 miRNAs , 4 miRNAs , and 3 miRNAs , respectively  and composite marker 2  as compared with the control group. However, there were no between-group differences in composite marker 3 . Fold changes of the PTSD group in each individual miRNA are described in Fig. The means of the standardized miRNA expression values were compared between the two groups. The PTSD group displayed higher expression of composite marker 1  and cortisol  between the PTSD and control groups  and composite marker 2  in the PTSD group, but not in the control group   and control groups    , while there was no between-group difference in GM volume ratio   ratio and the gray matter (GM) volume ratio between prefrontal versus limbic regions were measured to approximate the functional and structural prefrontal control over the limbic regionsIs) Fig. . The PTS51) Fig. .Figure 3r = 0.32, permutation-adjusted P = 0.03) as well as a higher GM volume ratio  in the PTSD group  nor GM volume ratio  in the PTSD group  or the prefrontal/limbic CBF ratio . Moreover, there were no significant interaction effects between the expression levels of composite 2 and trauma type on both the prefrontal/limbic GM volume ratio  or the prefrontal/limbic CBF ratio .In addition, robust regression analyses were performed to examine whether the correlations between the expression profiles of candidate miRNAs belonging to composite 1 or 2 and the prefrontal/limbic GM volume or CBF ratio differ according to trauma types. These exploratory analyses showed no interaction effects between the expression levels of composite 1 and trauma type on both the prefrontal/limbic GM volume ratio , indicating a significant relationship between the predominant expression of composite marker 2 in relation to higher severity of PTSD symptoms oms Fig. .To our knowledge, this is the first study to identify the concerted actions of a set of miRNAs showing the differential expression in FKBP5 KO mice on the pathophysiology of PTSD at the molecular, brain, and behavioral levels using a human cohort, as illustrated in Fig. One of the strengths of our translational approach of using an FKBP5 KO mouse model is the allowance to identify candidate miRNAs in a more targeted manner as compared with whole-genome miRNA expression profiling. Our findings may also have merit through having collected a comprehensive dataset from a relatively large sample size of recently traumatized individuals, where each participant underwent a battery of assessments including miRNA quantification, multimodal neuroimaging, and clinical/behavioral assessments on PTSD symptoms.It is important to note that the majority of the currently identified miRNAs associated with PTSD have been previously implicated to take part in the underlying neural mechanisms of stress regulation and stress-related disorders Table . Our fin19, our proof-of-concept study found that altered expression levels of a series of candidate miRNAs derived from FKBP5 KO mice may relate to the compensatory or pathological adaptations of the prefrontolimbic circuit, a major neural correlate of PTSD. Specifically, the enhanced miRNA expression levels of composite marker 1 were associated with the increased prefrontal control over the limbic activity in the PTSD group. As this relationship was unique and exclusively applicable to miRNAs of composite marker 1 and not of composite marker 2, this may suggest a coping or compensatory role of miRNAs clustered into composite marker 1 in response to trauma exposure. While considering this potential role of miRNAs of composite marker 1, it is also noteworthy that the elapsed time since trauma exposure was relatively short in the current PTSD sample (median 4.4 months). As such, the miRNA expression profiles of the current sample may be more dynamic or differ in the case of chronic PTSD. Future studies that investigate the miRNA expression profiles of chronic PTSD may provide further insight with regards to the potential roles of each composite marker groups in the pathophysiology of PTSD.Consistent with a growing number of studies that have reported the involvement of FKBP5-associated genetic or epigenetic regulation in PTSDThe diverging functions between miRNAs categorized into composite markers 1 and 2 may be corroborated by our findings from the correlation analyses, which demonstrated that the imbalance in composite marker expression levels may influence PTSD symptom severity. While the evidence suggesting the direct effects of the current candidate miRNAs on FKBP5 protein expression levels within the prefrontolimbic regions is scarce, we cautiously suggest that miRNAs clustered into composite markers 1 and 2 may play a distinct role in PTSD patients through their response to altered FKBP5 protein expression in PTSD condition. However, it is also noteworthy that alteration in miRNA levels upon the ablation of FKBP5 gene may not necessarily indicate alterations in FKBP5 protein expression or activity. As such, future mechanism studies that examine the relationship between alterations in the current miRNA candidate levels and the resultant changes in FKBP5 protein expression would be necessary to support this speculation.17. It should also be noted that the significant correlation between serum levels of hsCRP and the expression profile of the candidate miRNAs as shown in the PTSD group was not observed in the control group. Although there was no between-group difference in serum hsCRP level, this lack of correlation may be cautiously explained by previous literature, which suggested HPA axis activity is altered in individuals with PTSD34. Considering that HPA axis activity is partly reflected by serum hsCRP levels, the association between serum hsCRP and candidate miRNA expression profiles in the PTSD group may differ from those in the control group. Further studies to examine whether HPA axis activity may differentially influence miRNA expression profiles between being under a stressful versus normal condition would be warranted.Among the serum markers evaluated in the current study that reflect HPA axis activity, the peripheral levels of hsCRP were significantly correlated with FKBP5-associated miRNA expression profiles in the PTSD group. This finding may imply a potential feedback link between alteration in HPA axis function and the resultant modulation of FKBP5 expression or activity, as suggested in previous research35.There were also no significant relationships between serum cortisol levels, the other marker for HPA axis activity, and candidate miRNA expression profiles in the PTSD group. While this finding may be contrary to our expectation, we suggest that this may be partly due to the less efficient feedback actions of cortisol as a result of increased glucocorticoid resistance, a key pathological feature of HPA axis activity related to PTSD36. Specifically, we anticipate that the expression profiles of the circulating exosomal miRNAs may reflect disease-specific alterations in miRNA expression within the brain in response to trauma. Based on these assumptions, the currently selected exosomal miRNAs may be valid neurobiological markers of PTSD in the case of those who have been recently exposed to trauma. In addition, the stability of expression patterns is another advantage for the application of exosomal miRNAs as potential biomarkers, given the fact that exosomes may serve as a RNase-protective vesicle that protects miRNAs from RNase-rich environments in the peripheral blood38. However, although a significant relationship between altered expression levels of exosomal miRNAs and mPFC miRNAs was suggested in our FKBP5 KO mouse model  for mapping. Future studies and re-analyses that implement up-to-date versions of the bioinformatics tools, including genome annotation, are warranted to ensure the accuracy of the findings.The following limitations should be taken into consideration when interpreting the results. First of all, the current study implemented a less conservative approach using the uncorrected Moreover, the current study selected candidate miRNAs based on the expression profile at the basal level from the FKBP5 KO mouse model. As such, potential alterations in miRNA expression that may occur as a reactive response to stress or trauma were not considered. Namely, the current set of miRNA candidates was not selected in the FKBP5 KO mouse model under a stress-induced condition.As for the validity of the current findings, although internal validation using bootstrapping was performed, the current findings should be externally validated in an independent sample with a larger sample size. While taking a targeted approach by designating a predefined subset of candidates may be appropriate for proof-of-concept studies such as our own, our results should be replicated with a genome-wide expression profiling of miRNAs for the future application of exosomal miRNAs as biomarkers in the diagnosis of PTSD.It should also be noted that the current study lacks information regarding the polymorphism of the FKBP5 gene in the human cohort of PTSD patients and trauma-unexposed individuals, despite the fact that such data may have allowed better characterization of the results. Future studies that include this important information of genetic data may provide stronger evidence on the specific vulnerability to clinical symptoms according to FKBP5 allele as well as the differential link between candidate miRNA levels and the prefrontolimbic activity in PTSD patients who carry the risk allele. Furthermore, the results from the current control group without any exposure to major trauma could not elucidate the exact effects of candidate miRNA expression on the conditional risk for PTSD among trauma-exposed individuals. The inclusion of an additional study group of trauma-exposed individuals who did not develop PTSD is warranted in order to examine whether the current miRNA candidates may differentially influence the development of PTSD in recently traumatized individuals. As such, the lack of a study group of trauma-exposed who have not developed PTSD is a notable limitation in the current study.Finally, since alterations in miRNA expression levels may not necessarily reflect their effects on the target mRNA of the canonical pathway, further studies are necessary to elucidate the detailed mechanisms underlying the associations between the current miRNA candidates, their respective target mRNAs, and the overall biological processes that may be involved. The current results should be interpreted with this important limitation in mind.In conclusion, the current proof-of-concept study suggests the possibility of utilizing exosomal miRNA as biomarkers that are complementary to predicting the prognosis of PTSD in recently traumatized individuals. Further applications using FKBP5-associated miRNAs as biomarkers may be an important step towards the development of mechanism-based, disease-modifying or preventive treatment of PTSD.Strains of mice (Fkbp5tm1Dvds/J) used for this study included male and female of FKBP5 knockout  and littermate wild type . FKBP5 KO mice were obtained from the Jackson Laboratory . Three to four-month-old KO mice (JAX stock #017989) and WT mice were used for the study. RNA extraction was performed from the dissected mPFC using a miRNeasy mini kit . Total RNA extracted from the brain was used for small RNA enrichment. Column-purified small RNA was used for library preparation, using the NEXTflex Small RNA Sequencing Kit V3 . In addition to examining the miRNA expression within the mPFC, circulating miRNA expression levels were also examined in the blood of FKBP5 KO and WT mice.Detailed procedures of the animal study are described in Supplementary Methods. All animal use and related protocols were in accordance with the Institutional Animal Care and Use Committee of Chung-Ang University and Konkuk University.The study recruited 50 recently traumatized individuals diagnosed with PTSD at the time of assessment (PTSD group) and 50 trauma-unexposed healthy individuals (control group), all of whom were between the ages of 20 and 50. Among the 100 participants that were enrolled, 2 individuals from the PTSD group and 3 individuals from the control group were excluded due to insufficient exosomal miRNA yield and quality. The final analysis included 48 individuals from the PTSD group and 47 individuals from the control group. The demographic and clinical characteristics of the study participants are presented in Table Exosomal RNA was isolated from human serum using ExoQuick solution . Isolated exosome RNAs were reverse-transcribed using the TaqMan MicroRNA Reverse Transcription kit  and the miRNA-specific reverse transcriptase primers. All miRNA expression values were normalized to that of miR-16 and relative expression values of miRNAs were used for subsequent statistical analyses. Further details regarding the examination of peripheral miRNA levels in a human cohort are provided in Supplementary Methods.Serum levels of cortisol and hsCRP were measured to reflect HPA axis activity. For each serum sample, levels of cortisol and hsCRP were examined in duplicate using commercialized kits containing reagents and automated devices, as further described in Supplementary Methods.Structural and perfusion magnetic resonance (MR) imaging scans were obtained using a 3.0 Tesla Philips Achieva MR scanner  equipped with a 32-channel head coil. High-resolution T1-weighted images and arterial spin labeling (ASL) images were acquired using a three-dimensional T1-weighted magnetization-prepared rapid gradient echo imaging sequence and a pseudocontinuous ASL single-shot echo-planar imaging sequence, respectively.Absolute CBF (ml/100 g/min) and GM volumes were measured as neural correlates of FKBP5-associated miRNA expression in relation to PTSD in predefined ROIs, mainly including the prefrontolimbic regions. The prefrontal regions included the medial prefrontal cortex, paracingulate cortex, and subcallosal cortex, and the limbic regions included the amygdala, hippocampus, and nucleus accumbens.a priori defined ROIs are described in Supplementary Methods.Details regarding the image acquisition, preprocessing, selection of the prefrontolimbic ROIs, and measurements of CBF and GM volumes of the A principal component analysis using a correlation matrix between miRNA expression levels followed by a varimax rotation was performed to extract the initial set of components as relevant composite markers for miRNAs.Group comparisons of FKBP5-associated exosomal miRNA levels between the PTSD and control group were performed using logistic regression analysis. The AUCs for the miRNA candidates was calculated with an internal validation of 1,000 bootstrap resampling.P values were calculated in order to correct for multiple comparisons40.Pearson correlation analyses were performed between the relative expression profiles of miRNA candidates and serum cortisol and hsCRP levels, respectively. In addition, the relationships between standardized expression profiles of the miRNA composite markers and CBF as well as GM ratio of the prefrontal versus limbic regions were examined. Permutation-adjusted Further details regarding the statistical analyses performed are described in Supplementary Methods.This study was approved by the Institutional Review Board of Ewha W. University and conforms to the Declaration of Helsinki. All participants gave written informed consent. All animal experiments and related protocols were in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of Chung-Ang University and Konkuk University.Supplementary information.


Transcriptional riboswitches involve RNA aptamers that are typically found in the 5′ untranslated regions (UTRs) of bacterial mRNAs and form alternative secondary structures upon binding to cognate ligands. Alteration of the riboswitch's secondary structure results in perturbations of an adjacent expression platform that controls transcription elongation and termination, thus turning downstream gene expression “on” or “off.” Riboswitch ligands are typically small metabolites, divalent cations, anions, signaling molecules, or other RNAs, and can be part of larger signaling cascades. The interconnectedness of ligand binding, RNA folding, RNA transcription, and gene expression empowers riboswitches to integrate cellular processes and environmental conditions across multiple timescales. For a successful response to an environmental cue that may determine a bacterium's chance of survival, a coordinated coupling of timescales from microseconds to minutes must be achieved. This review focuses on recent advances in our understanding of how riboswitches affect such critical gene expression control across time. Over the past several decades, RNA has emerged as a key player beyond a “message” between DNA and protein. Non-coding RNAs (ncRNAs) are vital for countless cellular mechanisms, such as ribozyme mediated catalysis of RNA processing reactions, RNA mediated gene silencing, and stabilization of phase separated particles, to name a few Eddy, . The fun1), to cations (such as Mn2+), anions (such as F−), and even other RNAs (such as tRNAs)  ligand influx, which can be as fast as the rate of diffusion, (2) ligand binding to the RNA, establishing specific hydrogen bonding, stacking and ionic interactions (nanoseconds to single seconds), (3) alterations in RNA secondary structure (milliseconds to multiple seconds), and (4) regulation of transcription elongation/termination and their downstream biological consequences (seconds to minutes)  Al-Hash. Until rEscherichia coli (E. coli) bacteria have even been shown to directly couple transcription and translation, with ribosomes binding to mRNA during active transcription elongation by RNAP  . When Salmonella is deficient in Mn2+, transcription elongation of the mntH gene becomes permissive, subsequently allowing for expression of the transporter and resulting in an increase in Mn2+ uptake  to relatively slow global gene regulation is a critical modulator for sustaining the life of prokaryotes. For example, the MnE. coli contains an efflux transporter protein, called mntP (2+ transport protein)  is one approach by which the coupling of ligand influx to ligand-induced folding of a translational riboswitch and the subsequent impact on the expression of a reporter gene can be monitored directly  was probed in E. coli, confirming and complementing expression studies of a GFP-coupled reporter. Through intracellular icSHAPE, this study revealed stronger preQ1 mediated occlusion of the ribosome binding site than was observed in the previously described structure, which was solved in purified form via X-ray deffraction  to allow for the ligand binding kinetics to outpace and thus drive the kinetics of RNA folding. A commonly observed mechanism called the induced fit (IF) mechanism describes riboswitch ligand binding events where ligand binding occurs faster than the conformational changes of the riboswitch, whereas ligand dissociation from the unfolded RNA is slower , which unlocks a linchpin-gated structure that promotes transcription termination. The presence of F− stabilizes the gated conformation toward a functional response within a narrow range of ligand concentrations over a wide range of transcription rates  structural rearrangement occurs faster than ligand binding, hence gene regulation occurs via CS  and optical tweezers, have quickly become essential tools for monitoring ligand-dependent structural changes in the aptamer region  Savinov. smFRET 1-sensing riboswitch follows the two ligand binding mechanisms of IF and CS dependent on the ligand and metabolite conditions as well as specific sequence adaptations found in various bacteria  has been observed to favor a CS pathway where the ligand primarily binds to a pre-selected conformation of the aptamer -seq revealed that the riboswitch is controlled by the kinetics of co-transcriptional folding, which drives the RNA into a short-lived folded state, even in the absence of F−. Binding of ligand favors a kinetically trapped, stably folded state, which delays the nucleation of the terminator hairpin until RNAP has escaped the terminator poly(U) sequence to continue transcription  footprinting and SHAPE have allowed for monitoring of structural changes upon addition of a ligand  and zero-mode waveguide (ZWM) microscopy at increasingly higher throughput of individual molecules while maintaining high sensitivity (Duss et al., CS, SD, RR, and NW wrote the manuscript. CS and SD produced the figures. All authors contributed to the article and approved the submitted version.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.


Idiopathic Pulmonary fibrosis (IPF) is a catastrophic disease with poor outcomes and limited pharmacological approaches. Heat shock protein 90 (HSP90) has been recently involved in the wound-healing pathological response that leads to collagen deposition in patients with IPF and its inhibition represents an exciting drug target against the development of pulmonary fibrosis. Under physiological conditions, HSP90 guarantees proteostasis through the refolding of damaged proteins and the degradation of irreversibly damaged ones. Additionally, its inhibition, by specific HSP90 inhibitors  has proven beneficial in different preclinical models of human disease. HSP90 inhibition modulates a complex subset of kinases and interferes with intracellular signaling pathways and proteome regulation. In this review, we evaluated the current evidence and rationale for the use of HSP90 inhibitors in the treatment of pulmonary fibrosis, discussed the intracellular pathways involved, described the limitations of the current understanding and provided insights for future research. Idiopathic Pulmonary Fibrosis (IPF) is a devastating disease, characterized by the progressive substitution of the lung parenchyma with a fibrotic scar. It is associated with poor prognosis and an estimated mean survival of 2–5 years from the time of diagnosis . The patHeat shock proteins (HSPs) constitute a large family of co-chaperones classified by molecular weight and involved in the correct folding of a large number of “client proteins”. HSPs react to heat, hypoxia and oxidative stress, by assisting the assembly, stabilization, and translocation of oligomeric proteins . FurtherHSP90, the most abundant of HSPs, is increased in patients with IPF  and has It is important to understand, however, that HSP90 interacts with a great number of proteins, kinases, and transcription factors and that its inhibition is followed by a strong modulation of the proteome and phosphoproteome, a large part of which is not completely understood. From this perspective, proteomic analysis of IPF represents one of the most promising methodologies in defining unknown pathways of IPF pathogenesis and in revealing how HSP90 inhibition may affect these pathways.The aim of this review is to discuss the current understanding of HSP90′s role in the development of pulmonary fibrosis, the current proteomic understanding of IPF, evaluate available preclinical data and describe how inhibition of HSP90 could be a therapeutical target for patients with IPF.bacteria and eukaryotes [HSP90 is one of the most expressed heat shock proteins, found in both karyotes . It playkaryotes .The NTD is conserved among HSPs and shares homology with the ATPase/kinase GHKL  superfamily . It presHSP90 chaperones form homodimers through the binding of the N-domain acquiring a V shape-dimer with ATP-depending conformational shifts . This coHSPs represent a sophisticated protein quality-control network, which assist protein folding during assembly, and selectively degrade irreversibly damaged proteins. HSP90, the most studied and abundant of HSPs, is crucial for maturation of signaling proteins involved in cell division and development, such as steroid hormone receptors, kinases and key oncogenic proteins like the tumor suppressor p53 ,37. HSP9www.hsp90.org [Professor Didier Picard greatly contributed to the overall understanding of the HSP90 interactome, collecting the results of different studies and creating an online platform to help researcher in the general understanding of this complex network (sp90.org ). HoweveDuring stress, the Heat Shock Response (HSR) regulates the cytoplasmic proteostasis response, through the transcription of stress genes and the de novo synthesis of heat shock proteins in order to guarantee cell survival, adaptation to circulating hormones and protection of proteins from environmental insults ,43. BesiThe “HSPome”, defined as the part of the proteome affected by HSPs activity and modulation, as we previously mentioned, is constituted by an enormous number of functional proteins, transcriptional factors and ubiquitinases, whose expression could be regulated by the use of inhibitors resulting in profound changes in cell morphology and functionality, especially in response to stress.In cancer, HSP90 regulates a high number of proteins involved in cellular signaling and tumor promotion, making HSP90 inhibitors a therapeutic approach with great potential . SimilarCurrent proteomic understanding of IPF is derived from peripheral blood, bronchoalveolar lavage fluid (BALF), and lung tissue analysis. Patients with IPF exhibit variable rates of disease progression and for this reason many studies have focused on finding new biomarkers or indicators of therapeutic response to monitor these patients . ProteomEven though the profile of circulating proteins differs from lung proteins, it can still provide insight into biological pathways linked to a persistent overexpression of inflammatory markers, adhesion molecules, complement proteins and HSPs.BALF proteomic analysis of patients with IPF, compared to matched healthy controls, displayed an increased number of eosinophil and neutrophil proteins ), profibrotic cytokines , and various collagens . Carleo Furthermore, isoforms of Receptor for Advanced Glycation End-products (RAGE)—whose activity is related to activation of kinases (MAPK) and transcriptional factors (NF-κB) —were anaC-myc, as well as inhibition of protein kinases [Isobaric tag for relative and absolute quantitation (iTRAQ) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze proteomic changes in lung tissue of IPF patients and controls . From mo kinases .In a murine model of bleomycin-induced PF, the proteome and secretome were studied at different time points in order to define fibrotic and regenerative stages . Two uneTaken together, these data strongly suggest that IPF could be classified as a disease with “unbalanced” proteostasis, whose main affected proteins could function as new biomarkers for disease severity. This data also indicates that HSP90 should be considered as a potential target for new therapeutic approaches.There is a rising amount of literature suggesting that HSP90 plays an important role in fibrogenesis  and thatHSP90α is able to promote the phosphorylation of Protein kinase B (AKT) in Thr308, P38, and extracellular signal–regulated kinase (ERK) signaling pathways, which are known downstream non-Smad-pathways of TGFβ1 signaling and participate in the development of pulmonary fibrosis . Indeed,Strong expression of both HSP90α and HSP90β was observed in abnormal bronchiolar structures overlying fibroblast foci, as well as in hyperplastic bronchioles of IPF lungs. Hyperplastic type-II alveolar epithelial cells (AECII) near areas of dense fibrosis displayed robust immunostaining of HSP90β in IPF lungs compared to healthy donors, whereas HSP90α was absent in AECII. Smooth muscle cells located in the wall of bronchioles showed expression of HSP90α and α-SMA, but not of HSP90β . CollageHSP90 has also been found increased in animal models of pulmonary fibrosis. In the bleomycin-induced mouse model, BALF and serum displayed high levels of HSP90α and this increase was related to fibrosis . ActivatHSP90 inhibitors have shown already efficacious results in cancer  and infeHSP90 inhibitors strongly decrease HSP90 interactions with kinases . Sharma By modulating the HSPome, HSP90 inhibitors affect complex intracellular networks involved in the development of pulmonary fibrosis and offer strong rationale as useful therapeutical interventions directing cells through protein degradation.IPF is complex disease with unclear mechanisms of pathogenesis. Proteomic analysis together with genomic studies represent a promising approach towards defining unknown pathways of genetic predisposition and proteome unbalance in IPF. In recent years, several new molecules have been implicated in its pathogenesis, creating new potential biomarkers and therapeutical targets. However, it is important to note that every cell type presents its own proteome and some of the present data arise from mixed cell populations. In order to characterize cell-to-cell differences, it would be ideal to isolate either from BALF, lung tissue or peripheral blood each distinct cell type and carry its proteomic evaluation. This would greatly contribute to a further description of the role that each cell, either pneumocytes, endothelial or immune cells, play during the development of the disease.Limitations also arise from the analysis of HSP90ome. HSP90 is involved in the stress response and consequently expression and activity levels are affected by experimental and environmental conditions that need to be considered when comparing different studies.Further, HSP90 exists in multiple isoforms  that have a distinct role in cellular metabolism, as some function in the cytosol, others in the nucleus, and others in the mitochondria, thus establishing distinct interactive networks whose understanding is far from clear . NotablyOther disadvantages are related to the techniques used to study the relationship between HSP90 function and the proteome. Current technology is unable to distinguish between direct or indirect interactions between the chaperone and other proteins. In addition, defining the client proteins (HSPome) of HSP90 and how HSP90 inhibitors change their expression remains challenging. Large proteomic analyses describe changes in proteins expressed in relatively high concentration while HSP90 modulates many signaling and transcription factors which, are expressed at low levels in cells, but are characterized by a strong metabolic activity.Further issues arise when comparing the main proteomic studies on HSP90 inhibitors. Thus, results from studies by Song , MaloneyEven though more studies are required, proteomic studies from IPF patients and laboratory analysis of HSP90 inhibitors provide a strong mechanistic rationale for their investigation in IPF, justifying further experimentation in preclinical studies to better identify their anti-fibrotic effects, optimal dose strategy, and potential side effects.IPF is a disease characterized by unbalance of the proteome. HSP90 plays an important role in the pathogenesis of the fibrotic process in the lung. Controlled interventions, as with HSP90 inhibitors, represent an exciting approach for the modulation of the proteome and phosphoproteome that has been shown to result in reduction of pro-fibrotic markers, ECM proteins and improved lung mechanics in several pre-clinical studies. Furthermore, HSP90 inhibitors critically interfere with Smad and non-Smad intracellular signaling of TGF-β. As the modulation of HSP90 results in changes in multiple pathways, further studies are necessary to define in detail how HSP90 inhibitors exert their beneficial effect in IPF, the optimal dose strategy, and potential side effects.