Aim: We reviewed the radiographic response of three patients with metastatic castration-resistant prostate cancer treated with CRXL301, a docetaxel nanoparticle. For these three patients, we isolated and analyzed circulating tumor cells (CTCs) to explore microtubule (MT) drug-target engagement (MT-DTE) as a biomarker of response to treatment. MT-DTE was based on a quantitative assessment of the MT cytoskeleton in CTCs from pre- and post-treatment patient samples as a potential read-out of CRXL301 activity.Methods: We isolated CTCs using negative CD45+ depletion and subjected them to multiplex confocal microscopy using our established protocol. CTCs were identified as CD45-/CK+/DAPI+ cells and MT-DTE was determined using our developed imaging algorithm. We quantified MT bundling in CTCs across multiple time points, from baseline to on-treatment to disease progression. Here, we describe the longitudinal analysis of MT-DTE in CTCs from patients treated with CRXL301 and its correlation with response to treatment.Results: We collected CTCs at seven time points from three metastatic castration-resistant prostate cancer patients. Clinical response was evaluated by Response Evaluation Criteria in Solid Tumors (RECIST) v.1.1 criteria in those patients with measurable disease. Of the three patients enrolled, one experienced partial response (-50%) to CRXL301 and two patients were unevaluable given bone only disease. Notably, however, these two patients showed stable disease clinically based on bone scans. MT-DTE across all time points revealed that, early time points within four and 24 h of drug administration exhibited the highest levels of drug engagement (MT-DTE) as compared to baseline. However, these early time points did not correlate with clinical response. We observed that the CTCs collected one week after the first or second dose of CRXL301 treatment in the responding patient had numerically higher levels of MT-DTE as compared to the other two patients.Conclusion: Taxane on-target activity can be detected and analyzed quantitatively in CTCs by tubulin immunofluorescence. Early time points, within 24 h of drug administration, showed high levels of DTE but did not correlate with clinical response. MT-DTE in CTCs collected after one week on treatment correlated best with treatment response. The clinical utility of the 1-week CTC DTE should be tested and validated in future clinical trials involving taxanes. Particularly for prostate cancer, taxanes (docetaxel and cabazitaxel) are the only chemotherapy shown to prolong survival in metastatic castration-resistant prostate cancer (mCRPC) patients, and are now considered standard of treatment-5. Despite their clinical success, unfortunately not all patients benefit from taxane treatment, while the molecular basis for taxane resistance in CRPC remains unknown. Currently, there is no biomarker indicative of taxane clinical activity that can be used to discriminate responding vs. non-responding patients early on during their treatment.Microtubule-targeting drugs represent one of the most clinically active chemotherapy drugs for the treatment of solid tumors-8. Following taxane binding to their primary cellular target, tubulin, MT stabilization and increase in MT polymer mass occur. This is indicative of effective . This MT-DTE is visually evidenced by reorganization of the MT network into distinct MT bundles. Impaired MT-DTE has been associated with taxane resistance in multiple preclinical models,10. However, no clinical studies assessing MT-DTE on tumor samples have been reported yet, and the role of MT-DTE as biomarker of clinical response to taxane chemotherapy is still unclear.At the molecular level, taxanes bind to the β-tubulin subunit of microtubules (MT), which are tubulin polymers involved in many biological functions, such as intracellular trafficking of molecules and organelles, and cell mitosis.Despite their clinical success, the administration of taxanes is a long, and complex procedure, which requires the use of highly allergenic amphiphilic solution  and extensive steroid prophylaxis due to their hydrophobic structure-15.The emergence of nanotechnology, particularly nanoparticle-drug conjugates, has improved docetaxel delivery, because it offers the advantages of unique size, improved drug solubility, passive targeting by enhanced permeability and retention effect, and more controlled drug release to the tumor,17. Molecular analyses of circulating tumor cells (CTCs), isolated from the peripheral blood of cancer patients with a simple blood draw, have emerged as alternative validated sources of tumor cells. Although first described in 1869, only recently CTCs have shown their clinical relevance, after clinical studies utilized CTCs as source of tumor material to analyze biomarkers of response to treatment in cancer patients,20.Currently, the molecular characterization of tumor tissue requires a tumor biopsy. However, biopsies are invasive and risky procedures with significant limitations, including biopsy-site bias due to tumor heterogeneity, and challenges associated with repeat biopsies on the same patientClinicalTrial.gov identifier: NCT02380677), we isolated CTCs from the peripheral blood of CRPC patient receiving the investigational drug CRXL301, a payload docetaxel covalently conjugated to a cyclodextrin-polyethylene glycol co-polymer. This is designed to concentrate in tumors, due to the unique characteristics of tumor’s vasculature, and slowly release docetaxel inside tumor cells. CTCs were used to explore the utility of using immunofluorescence to quantitatively assess the integrity of MT cytoskeleton from on-treatment blood samples compared to baseline, as a potential read-out of CRXL301 clinical activity. CTCs were quantitatively analyzed at the single-cell level for MT-DTE to determine if this measure correlated with treatment response to nanoparticle delivery of docetaxel on an individual basis.In the present study  but were required not to have received any prior taxane-based chemotherapy. Patients were also required to have adequate organ function and an Eastern Cooperative Oncology Group performance score of 0 to 1The study was approved by the institutional review board at MD Anderson Cancer Center where these three patients were treated. Patients provided written informed consent before participation. NewLink Genetics granted the permission to release the clinical data on these three prostate cancer patients whose samples were analyzed.Patients were followed for safety, tolerability and preliminary tumor efficacy. RECIST v.1.1 was used for those patients with measureable disease. Patients continued treatment until they experienced progression of disease or unacceptable toxicity or patient/physician decision to withdraw from study.Ten mL of peripheral blood was collected in EDTA tubes  from three mCRPC patients before initiation of CRXL301 chemotherapy . Additionally, 10 mL of peripheral blood was also collected at the following time points: C1D1 plus four hours, C1D2, C1D8, C2D1, C2D1 plus four hours, and C2D8..Blood samples were shipped at ambient temperature on same day of collection and were processed within 24 h from blood draw. Samples were processed as previously described+ cells  by using the RosetteSepTM Human CD45 Depletion Cocktail ; the enriched CTCs were subsequently cytospun onto coverslips and processed for immunofluorescence staining.Briefly, CTCs were enriched by negative depletion of CD452) for 15 min and blocked overnight in 10% Normal Goat Serum  plus 6% Bovine Serum Albumin (BSA) in PBS .Enriched CTC cell preparations were plated on coverslips and fixed with pre-warmed (37 °C) 2% formaldehyde  in 1× PHEM buffer  catalog # ab3510], and goat anti-rabbit IgG secondary antibody, AlexaFluor 488 ; and (5) DAPI for nuclear counterstaining (Invitrogen). Coverslips were mounted on a glass slide by using Mowiol mounting media .Cells were subsequently immunostained for: (1) the leukocyte marker CD45 using an anti-CD45 antibody directly conjugated with QDot-800 ; (2) the epithelial marker cytokeratin, using an anti-pan-cytokeratin mouse monoclonal antibody recognizing human cytokeratins 4, 5, 6, 8, 10, 13, and 18  conjugated to CF594 using Biotium Mix-n- Stain antibody labeling kit ; (3) tubulin , followed by goat anti-rat IgG secondary antibody, AlexaFluor 647 ; (4) androgen receptor AR .The goal of this observational study was to determine the effect of the investigational drug on its target, the microtubule cytoskeleton, in patient CTCs and correlate with response to treatment. We developed a quantitative algorithm to determine the drug-induced MT stabilization, hereafter MT-DTE. To do that we used our established pipeline of CTC enrichment via CD45 negative depletion, followed by a high-throughput imaging algorithm which identifies putative CTCs based on low- resolution scanning using established criteria of DAPIAll patients received single agent CRXL301 for a minimum of two cycles. Patient response to treatment was evaluated according to RECIST. Among the three patients, we observed a differential response to treatment To quantify MT-DTE we analyzed a total of 149 confirmed CTCs from the three patients across all time points. Overall, Patient #1 exhibited lower CTC counts across all the time points ; conversely, the two patients who did not achieve an objective response showed numerically higher CTC counts . CTC counts, mean MT-DTE for all time points and patients’ clinical characteristics are shown in To quantitatively measure MT-DTE, we used tubulin immunofluorescence and high-resolution confocal microscopy to generate three-dimensional reconstructions of MT structures and subjected them to quantitative analysis by ImageJ. MT-DTE was determined on a single cell basis by the mean integrated density of pixels in the top 25% of raw fluorescent intensity values. Representative images of CTCs with different levels of MT-DTE are shown in CTCs with diffuse MT staining pattern and/or lack of discernible filamentous structures had lower MT-DTE. Signs of bundling including thick MT structures, disorganized MT network (distorted pattern throughout cell), and high MT fluorescence staining, had a higher MT-DTE n = 20 and 14, respectively) as well as numerically higher MT-DTE  At baseline (C1D1), the responding Patient #1, had numerically lower MT-DTE (mean: 0.190) compared to the baseline MT-DTE in the two patients with stable disease . In addition, four hours after treatment, Patient #1 had only one CTC detected, while Patients #2 and #3 had higher number of CTCs , compared to baseline in both Patients #1 and #2. For Patient #3, the C1D2 sample was not received. Considering that Patients #2 and #3 had stable disease whereas Patient #1 had a partial response to therapy, these early times points for MT-DTE at 4 and 24 h of treatment do not seem to discriminate the responding patient from the patients with stable . However, it did not discriminate the responding patient from the patients with stable disease. In contrast, the higher MT-DTE in CTCs collected one week after the first or second dose of CRXL301, compared with their respective baseline samples, and was observed only in the responding patient. Taken together, these data suggest that early MT-DTE likely reflects initial drug binding but not sustained target engagement, while persistent MT-DTE after one week of treatment appears to correlate better with treatment response.In our previous studies, we could not implement quantitative analysis of taxane-induced MT bundling in mCRPC patient CTCs, as MT integrity was not preserved in the CTCs isolated via the PSMA-based microfluidic device, in mCRPC patient CTCs one week after treatment initiation with docetaxel or cabazitaxel was significantly correlated with response to taxane chemotherapy. However, MT-DTE was only qualitatively assessed in that study due to loss of cytoskeletal integrity after CTC enrichment. In the current study, we quantified MT-DTE and sought to correlate with AR subcellular localization in the three participating patients. We observed three distinct phenotypes of AR subcellular localization from the on-treatment CTC analysis, ranging from nuclear to cytoplasmic enrichment to downregulated. We did not observe a significant correlation between MT-DTE and cytoplasmic AR enrichment [Supplementary Figure 1], likely due to intra-patient heterogeneity in AR phenotypes together with the small sample size. In addition, we utilized a AR primary antibody which cannot discriminate between full-length AR and AR splice variants, which are constitutively nuclear and associated with taxane resistance; this could in part explain the absence of correlation between MT engagement and AR sub cellular localization.Despite the observational nature of these results and due to the limited sample size, our findings here are consistent with our previous report where we showed that a decrease in nuclear AR, likely as a result of MT stabilization. Better mechanistic understanding and the development of more sophisticated, multi-parametric computational approaches are required to develop a MT-bundling heterogeneity index, which could enable precision medicine strategies for patients receiving treatment with a microtubule stabilizing agent.We also observed intra and inter patient heterogeneity in CTC MT-DTE analysis. We observed CTCs with no bundling from the on-treatment cohort, suggesting MT cytoskeleton response heterogeneity. This mixed response at CTC level could indicate clonal heterogeneity potentially due to inherent mechanisms of taxane resistance present in some but not all cells, such as tubulin mutations or expression of drug efflux pumps that could impair on target-drug engagement. Additional factors not yet studied in the clinical context, such as variability in cell biophysical properties , intracellular viscosity, and MT stiffness, could affect the formation of MT bundles in response to taxane treatment,33. Nevertheless, this observation warrants a bigger cohort to identify whether the presence of MT stabilization prior to taxane treatment may correlate with a lack of taxane drug efficacy.Regarding the inter-patient heterogeneity, we observed a pronounced MT-DTE at C1D1 in Patient #3, in the absence of prior taxane treatment, while there was no additional MT engagement following CRXL301 administration. This high MT bundling at baseline, may reflect aberrant MT stabilization by endogenous or secreted factors, or it may reflect dietary factors, such as the natural flavonoid, Fisetin, found in fruits and vegetables, which was shown to bind tubulin and stabilize microtubules with binding characteristics superior than paclitaxel. Similar to our approach, this study assessed on target drug activity, which was limited to PSMA expression without taking into account MT changes, in response to docetaxel binding. Nevertheless, this approach is commendable and should be extended to all investigational drugs and their targets in patient CTCs. Such analyses can help select patient more likely to respond to treatment and provide mechanistic insight into the molecular basis of clinical response and resistance.Interestingly, a recent study reported on the clinical efficacy of BIND-014, a PSMA-directed docetaxel-containing nanoparticle, in metastatic CRPC patients. The study was performed in 42 patients and identified a significant association between a decline in PSMA-expressing CTCs preferentially and the clinical activity of the investigational agentOverall, these data provide proof of principle that MT bundling induced by taxanes including their nanoparticle formulations can be detected in patient CTCs and that liquid biopsies at early, on treatment time points could be used to inform clinical decision making. Larger prospective clinical studies in solid tumors treated with taxane-based chemotherapy are warranted to validate these observations.


Vertebrae locations and vertebral segments of lesions were recorded and the impact on CTV delineation was evaluated. (3) Results: Forty-six VM eligible for SBRT following iPET were evaluated in eight patients . Eighty-one vertebral segments were detected using MRI, 26 with FDG or F-DOPA PET/CT, and 70 using iPET. iPET was able to detect more lesions than MRI for vertebral bodies (44 vs. 34). iPET-based delineation modified MRI-based CTV in 70% (32/46) of cases. (4) Conclusion: iPET allows a precise mapping of affected VM segments, and adds complementary information to MRI in the definition of candidate volumes for VM SBRT. iPET may facilitate determining target volumes for treatment with stereotactic body radiotherapy in metastatic vertebral disease.(1) Background: Stereotactic body radiotherapy (SBRT) for vertebral metastases (VM) allows the delivery of high radiation doses to tumors while sparing the spinal cord. We report a new approach to clinical target volume (CTV) delineation based on anti-carcinoembryonic antigen (CEA) positron emission tomography  in patients with metastatic breast cancer (BC) or medullary thyroid cancer (MTC). (2) Methods: All patients underwent iPET, spine magnetic resonance imaging (MRI), and positron emission tomography-computed tomography (PET-CT) using  Stereotactic body radiation therapy (SBRT) has emerged as a treatment option for selected patients with VM, with the objectives of pain-relieving decompression, tumor stabilization, and prevention of fractures ,2. SBRT 18fluorodeoxyglucose (FDG) has been developed with the objective of improving diagnostic precision for the detection of bone metastases, with a good sensitivity and specificity except for lesions measuring less than 10 mm  and 2.79 [range 1.72–57.24] for iPET and F-DOPA respectively. Tumor burden evaluated by functional volumes was equivalent between both iPET (median TTV 294 [IQR: 159–558]) and FDG-PET (median TTV 299 [IQR: 139–410] for FDG-PET) (p = 0.256).In total, 45 metastatic patients expressing CEA were included  were selected as having 46 VM eligible to SBRT , but fewer in pedicles  and a similar number in laminae or transverse processes , and spinous processes   . FDG-PETIn 16/46 VM, a similar number of vertebral segments was determined in iPET and MRI, but in only 14/46 (30%) the CTV was strictly identical between the two modalities, which means that iPET-based delineation modified MRI-based CTV in 70% (32/46) of cases . In the We have evaluated the feasibility of targeting metastatic vertebral lesions using metabolic PET and iPET with a view to treatment with SBRT in patients with metastatic CEA-expressing BC and MTC. We have shown that MRI and iPET provided complementary information, which enables better definition of the CTV and, thanks to iPET, the prospect of a new method for the delineation of VM and SBRT.In the first instance, we have looked for a better characterization of the CTV with the assistance of functional imaging with FDG-PET/CT and F-DOPA-PET/CT. The literature suggests that the sensitivity of FDG-PET in detecting spinal metastases is high , but onlIn the face of this difficulty, iPET, due to its excellent contrast, seems to be an interesting technique to assist with the delineation of target volumes for radiotherapy to secondary bony metastatic lesions in CEA- (or CEACAM5-) expressing tumors. The results seem promising with, in the present case, eleven vertebrae that were positive on iPET that were negative on MRI. This shows pathological vertebrae expressing CEA may not be visible on MRI, especially in oligometastatic disease.Having evaluated the feasibility of iPET in the detection of VM, we have concentrated on each pathological vertebra in order to determine precisely the affected zone and its corresponding CTV with a view to treatment with SBRT. We showed that iPET modified the MRI-defined spinal CTV in seven cases out of 10, by enlarging the CTV in more than one in four cases. However, iPET sensitivity and specificity have yet to be fully determined. There may be secondary lesions shown on MRI which, because they do not express CEA, would be invisible on iPET . Even ifOur studies have certain limitations, since patient selection was on the basis of iPET and not MRI, which is the gold standard for the detection of VM. This choice was made on the basis that the investigation is rarely available and the protocol-based nature of this kind of imaging. Although this was a small study in terms of patient numbers, the number of lesions positive on iPET was significant, and enabled us to have samples of VM sufficient to make comparisons between our results with MRI and PET.iPET, a new technique for phenotype tumor imaging, allows a precise mapping of affected VM segments, and adds complementary information to MRI in the definition of candidate volumes for VM SBRT. iPET may facilitate determining target volumes for treatment with SBRT in metastatic vertebral disease. These results warrant further clinical evaluation.


Coronavirus Disease 19 (COVID-19) caused by Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) it usually manifests with respiratory symptoms , it seems to have a neuroinvasive and neurotropic activity , 2. In tHyposmia has been reported as a possible peripheral nervous system (PNS) symptom caused by COVID-19 infection .In our experience, the smell alteration  seems to be one of the first manifestations of COVID-19 disease, with or without the loss of taste (dysgeusia). Sometimes it remains the only symptom; more often, it comes with fatigue, fever, and cough.We provide a commentary on how COVID-19 could affect the sense of smell and the reason why doxycycline (Dox) could play a role in its recover.The three leading causes of loss of smell reported in the literature are head trauma, chronic sinonasal inflammation and upper respiratory tract viral infections , 4. AnosPost viral temporary chemosensory dysfunction after a common cold is widely reported , 5, 6.The swelling of the mucosa in the olfactory cleft it seems to be cause of the transient olfactory and taste loss typically reported during the common cold. It usually leads to a conductive post-viral loss of smell, and it usually appears 3 months after the upper respiratory tract infection -associated olfactory loss, interferon (IFN)γ- signaling pathways may play a pivotal role in orchestrating immune system function. They are able to modulate inflammatory response and pattern-recognition receptors expressed by the innate immune system during infection . TherefoOn the other side, some post-viral sense of smell impairment may be partly independent of nasal congestion, thus explaining oxymetazoline failure in improving olfaction .Therefore, it has been suggested that HCoV, thanks to their neuroinvasive, neurotropic, and neurovirulent properties may be able to induce neuronal impairment .Entering into the respiratory tract, all HCoV invade and infect intra-luminal macrophages and epithelial cells. HCoV belong to Coronaviridae, enveloped non-segmented, single-stranded, positive-sense RNA viruses (+)ssRNA. Viral spike (S) proteins manage their cell entry program. They act by binding cell-surface receptors and facilitating the fusion of the virus-cell membrane . The spiIFNα/β signaling plays a protective role in reducing the virus spread and modulating T cell non-cytolytic antiviral response in limiting viral load. Moreover, some RNA-viruses have developed mechanisms to counteract innate host defense to establish productive infections in their hosts. This is the case of an RNA virus, the vesicular stomatitis virus (VSV) .in vitro (Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene-5 (Mda-5), seem to have an important role in the recognition of RNA viruses. In particular, it has been shown that immune signaling by RIG-I is involved in the generation of IFN-α/β following VSV infection. Under Dox treatment, cells released high levels of RIG-I proteins eliciting autonomous IFN response, thereby inhibiting viral infection in vitro .In another RNA virus, the Respiratory Syncytial Virus (SRV), viral proteins inhibit IFN-α and IFN-β to establish infection , and it The second-generation tetracycline Dox has an anti-inflammatory and broad spectrum antimicrobial activity , 21.In 1967, Dox was first approved by the FDA . It has In ophthalmology, Dox is usually administered in patients affected by ocular rosacea and posterior blepharitis . The DoxThe rationale in its administration is proteolysis inhibition promoted by matrix metalloproteinases (MMPs) , 25. MMPBesides its well-known use in treating bacterial infections, some studies in the literature report that Dox possesses a broad activity against viral infection too –31.The first who described the Dox antiviral effect was Sturtz in 1998 , and thiin vitro  and significantly affect viral replication in a dose-dependent manner when used against Porcine Reproductive And Respiratory Syndrome virus (PRRSV) infection in cultured cells . Virus mBeing the olfactory neural system able to regenerate throughout life, it can explain why the recovery of olfaction is common .From our observation, anosmia affected mostly young adults rather than elderly patients, confirming existing findings in the literature , 36. It In 2007, Suzuki et al. identified that coronavirus could be associated with anosmia, and he already speculated that nasal inflammation and related obstruction were not the only etiological factors underlying the OD in viral infection . As wellIn our preliminary observation, the administration of Dox 200 mg once daily seems to improve respiratory symptoms and anosmia under Dox treatment in six patients completely recover after only 2 days of treatment. From our experience, it seems reasonable to continue the treatment at least 8 days. The mean patients' age was 35.8 ± 6.8 years, and 4 (66.7%) were females. One patient reported anosmia as the only COVID-19 manifestation; instead of the other five patients who complained about the loss of smell, in which it appeared 5–7 days after mild fever, dry cough, and malaise. The average time of the recovery COVID-19-linked anosmia after the administration of Dox in these patients was 2.5 ± 0.5 days. We noticed a sudden improvement in all symptoms after the administration of Dox, but our most exciting insight is about the rapid recovery of the smell.Unlike olfactory sensory neurons (OSNs), nasal epithelium, which includes the respiratory and olfactory epithelium (OE) expresses high levels of ACE2 . SARS-CoWe are perfectly aware that there is a need for stronger evidence, but our article would intend to underline the importance of considering smell loss as a common symptom of COVID-19, supporting the rationale to treat such patients with Dox based on its interesting antiviral properties.CB and DB: contributed equally to this manuscript, wrote the article, and reviewed the final version. AL and EB: review and editing of the final manuscript. All authors reviewed the manuscript and agreed with its content.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.


Studies utilizing these techniques employed pregnant female mice for direct introduction of the genome-editing components into the oviduct or were dependent on delivery via tail-vein injection. In mice, embryogenesis occurs within the oviducts and the uterus, which often hampers the genetic manipulation of embryos, especially those at early postimplantation stages (days 6 to 8), owing to a thick surrounding layer of tissue called decidua. In this review, we have surveyed the recent achievements in the production of GE mice and have outlined the advantages and disadvantages of the process. We have also referred to the past achievements in gene delivery to early postimplantation stage embryos and germ cells such as primordial germ cells and spermatogonial stem cells, which will benefit relevant research.The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) systems that occur in nature as microbial adaptive immune systems are considered an important tool in assessing the function of genes of interest in various biological systems. Thus, development of efficient and simple methods to produce genome-edited (GE) animals would accelerate research in this field. The CRISPR/Cas9 system was initially employed in early embryos, utilizing classical gene delivery methods such as microinjection or electroporation, which required ex vivo handling of zygotes before transfer to recipients. Recently, novel in vivo methods such as genome editing via oviductal nucleic acid delivery (GONAD), improved GONAD ( Genome-editing techniques involve the use of sequence-specific nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) that make it possible to induce modifications in a predefined region of the genome . These nThe CRISPR/Cas9 gene editing system requires two components: 1) a guide RNA (gRNA), comprised either of a duplex CRISPR RNA (crRNA)/trans-activating CRISPR RNA (tracrRNA) molecule or of single-guide RNA (sgRNA), a fusion between crRNA and tracrRNA, and 2) a Cas9 endonuclease ,5,6. TheEfficient and safe delivery of components required for genome editing into germ-line-competent cells such as primordial germ cells (PGCs) and spermatogonial stem cells, preimplantation embryos such as zygotes (1-cell embryos) and 2-cell embryos and postimplantation embryos are a prerequisite for a successful genome-editing process.In 2015, a novel in vivo approach called “genome-editing via oviductal nucleic acids delivery (GONAD)” was reported by Takahashi et al. . This teDetailed discussion of each genome-editing approach is discussed in the following sections.In 2013, knock-out (KO) mice were created by the CRISPR/Cas9 system in several laboratories ,26,27,28Although microinjection of genome-editing reagents into zygotes is one of the most useful tools for generating genetically modified mice, it has advantages as well as disadvantages. The advantages of this technology include the delivery of known quantities of nucleic acids into a zygote irrespective of the type of zygote and the introduction of a large size cargo (carrying a gene of interest), which is a significant limiting factor when using viral vectors for gene delivery. The detailed protocols for microinjection-based genome editing have been described in Harms et al.  and JacoEP is a method for delivering exogenous substances  into a cell by forming transient pores into the cell membranes under electrical stimulation in vitro and in vivo.Since Kaneko et al.  first apPGCs are derived from cells included in the epiblast, also called embryonic ectoderm (EEct), adjacent to the extra-embryonic ectoderm (ExEct) at day 6.5 of pregnancy. Such cells are then identified as cell clusters at the base of the allantois at day 7.25 of pregnancy ,47,48. T4)-based gene delivery method to assess which tools can provide efficient transfection of mouse PGCs (isolated from gonads at day 11.5 of pregnancy). They found that EP causes serious damage to PGCs and that liposomal transfection resulted in poor transfection efficiency. In contrast, 18% of PGCs were successfully transfected with plasmid DNA co-precipitated with CaPO4. Unfortunately, these PGCs exhibited growth arrest in few days after being in culture. De Miguel et al. [Murine PGCs can be isolated from genital ridges at days 11.5–12.5 of pregnancy. Successful gene transfer into the isolated PGCs has been reported. For example, Watanabe et al.  evaluatel et al.  first del et al.  providedl et al.  concludeSry) protein, which is important for female-to-male sex-reversal, was injected into isolated genital ridge tissues (from day 11.5 fetuses), which were then subjected to magnetically induced transfection (magnetofection). This was followed by organ culture (culturing on agar blocks) of the transfected genital ridges to enable their further development under in vitro conditions. A fluorescent marker gene, concomitantly introduced into the constructs, at least 5 days after the organ culture was observed to be expressed. To our knowledge, there are no reports of successful genome-editing targeting PGCs in the genital ridges, as it is difficult to maintain the magnetofected genital ridges in vivo through transplantation into a fetal tissue.Svingen et al.  developeNotably, Morohaku et al. ,60 have Spermatogonial stem cells are a subpopulation of spermatogonia that are immature and capable of self-renewal but maintain the ability to differentiate into mature spermatozoa ,62. They−/−)—carrying a single-nucleotide deletion in the crystalline gamma C (Crygc) gene—suffering from cataract. In the GE germline stem cells, the target sequence containing the deletion site was found to be successfully replaced by the ssODNs, as a result of NHEJ or HDR-mediated gene editing. ICSI of spermatogenic cells obtained after male germ-cell transplantation of these GE germline stem cells resulted in the production of cataract-free progeny. This experiment suggests that CRISPR-based gene correction is possible in germline stem cells. Sato et al. [Stra8), which is known to be indispensable to spermatogenesis. However, the Stra8-targeted germline stem cells failed to differentiate into functional sperm cells after male germ-cell transplantation.In 2015, genome-editing experiments with germline stem cells have been reported to be successful in mice ,13,14,15o et al.  performeEGFP) transgene that were examined, all successfully transduced ZP-intact preimplantation embryos (morulae); serotype 6  exhibited a high degree of fluorescence. Notably, infection with rAAV-6 was effective, irrespective of the mouse strain. Yoon et al. [Cas9, which carries Cas9 gene under the control of mouse U1a small nuclear RNA promoter, and rAAV6-gTyr, which contains an EGFP expression unit together with a gRNA expression unit targeted to the tyrosinase (Tyr) gene coding for the protein required for the synthesis of melanin. Incubation of zygotes in the presence of rAAV6-Cas9 and rAAV6-gTyr for 3 days (up to blastocysts) led to the production of blastocysts with 100% indels when treated with the highest concentrations of rAAVs (6 × 109 genome copies). This finding suggests the importance of delivery of AAV particles inside the embryo where genome-editing components are released from AAV capsids, causing genome editing at the target locus.ZP is a layer of glycoproteins with several important functions and considered as a physical barrier for protection from any hazardous substance. As described previously, both lentiviruses and adenoviruses fail to transduce ZP-intact preimplantation embryos; however, they can transduce preimplantation embryos when those are placed in the perivitelline space (the space between the ZP and the membrane of the oocyte) or in a drop of medium containing ZP-free embryos ,71. Yoonn et al.  next assRosa26 homology arms. The KI efficiency in the Rosa26 locus was 15.5% and 6.3%, when assessed at the blastocyst and newborn stages, respectively.Mizuno et al.  observedCas9 and rAAV6-gTyr vectors were injected into the oviduct of pregnant female mice at day 0.5 of pregnancy. Out of the 29 pups that were obtained, 3 were found to have indels. All these mutated founder mice generated albino offspring indicating germline transmission. These results suggest that AAV particles can deliver CRISPR/Cas9 components to ZP-enclosed early embryos in vivo.Yoon et al.  examinedOtx2) or forkhead box protein A2 (Foxa2) and fast green dye into the amniotic cavity of an isolated embryo (Day 7.5 of pregnancy), as shown in As shown in EGFP gene. This was done by direct injection of a solution (1–1.5 μL) containing Cas9 mRNA and gRNA targeted towards EGFP cDNA into the lumen of an expanded area of the oviduct, called ampulla, under a dissecting microscope. After instillation, the entire oviduct was subjected to in vivo EP. Of all the mid-gestational fetuses obtained, 35% were fetuses losing EGFP fluorescence completely and 35% were those with reduced fluorescence. Sequencing analysis revealed that all the fetuses showing complete loss of fluorescence were bi-allelic KO fetuses and that fetuses showing reduced fluorescence were the ones that had mosaic mutations for the integrated EGFP gene. Gene delivery to the blastomere of an embryo is a possibility when GONAD is performed on day 1.4 of pregnancy (which corresponds to 2-cell stage), and this could be one of the causes for frequent generation of mosaic mutations. To circumvent these mosaic mutations, Ohtsuka et al. [Cas9 mRNA and gRNA, as the former is known to induce genome editing faster than the latter, and GONAD was performed on day 0.7 of pregnancy, a stage corresponding to late 1-cell embryos. By this stage, the cumulus cells—which surround the zygotes tightly at early 1-cell stage and are thought to hinder EP-mediated transfer of exogenous substances to an embryo—begin to detach. As a result, the efficiency of obtaining mice with indels was very high (~98%). Furthermore, this modified method created large deletions at a target gene as well as KI alleles. The resulting mutated traits were transmitted to the next generation. Based on these results, Ohtsuka et al. [i-GONAD). The i-GONAD was also proven to be effective in rats [i-GONAD have been reported by Gurumurthy et al. [i-GONAD has been recently published by Ohtsuka and Sato [i-GONAD is schematically shown together with the analysis of the embryos (morulae) isolated 2 days after i-GONAD (unpublished).As previously mentioned, Takahashi et al.  performea et al.  modifieda et al. . RNP wasa et al.  renamed  in rats ,83. The y et al. ,85. A reand Sato . In FiguInfection of ZP-intact preimplantation embryos floating in the oviductal lumen has been proved to be possible by a simple intraoviductal instillation of rAAV-6-containing solution, similar to GONAD . This apIn 1995, Tsukamoto et al.  first deEGFP cDNA simultaneously; EGFP transgenes in a homozygous (Tg/Tg) state on day 12.5 of pregnancy  and possible indels revealed the presence of the Cas9 gene and indels at the target loci of all the three samples. Notably, the indels were composed of normal and mutated cells  or somite (day 8.5) stage embryos have focused on ex vivo manipulation of the isolated embryos. However, a study by Sheehy et al.  is an exSftpcI73T)—a mutated version of the pulmonary-associated protein C gene—that codes for a protein that helps prevent the lung from collapsing when emptied. Embryonic expression of SftpcI73T is known to cause severely diffused parenchymal lung damage leading to the early death of affected individuals. Alapati et al. [SftpcI73T gene. Gene editing was successful in the target cells of the lungs of 20% of the mice born. In addition, the report states that the genetic alteration was not observed in the germ cells, indicating that genetic changes cannot be transmitted to the offspring. This study has the potential to aid the development of novel fetal gene-editing methods that target human monogenic disorders.Experiments using in utero gene delivery methods are usually performed on mid- to late-gestational fetuses (from days 9–18 of pregnancy), probably because they are relatively easier to be identified by visual observation  promises to overcome this limitation. Wild-type SaCas9 has been shown to reduce the off-target activities by about 90% for gene sequences that are highly similar and are therefore susceptible to mutations [In this review, we have described the applicability of CRISPR/Cas9 system for producing GE animals via ex vivo manipulation of embryos and germ cells as well as direct in vivo gene delivery to pregnant female mice. While the CRISPR/Cas9 technique is considered to be one of the promising tools for inducing genome modification at the desired target loci, the possibility of off-target mutations is a limitation of the technique. This results in concerns regarding the safety of this technique when used for gene therapeutic studies in humans. The use of newly engineered nuclease variant called “SaCas9-HF” which is originally derived from Cas9 nuclease of the utations . This moi-GONAD, TPGD-GEF, germ cell-based genome editing using germline stem cells, and in utero gene delivery. As summarized in Though the CRISPR/Cas9-based genome-editing technique is highly advanced, various other technologies for producing GE experimental animals have also been developed. These include zygote microinjection, in vitro EP of zygotes, GONAD,


This study investigated optometrists’ attitudes and self-reported practice behaviors towards omega-3 fatty acids for eye health, and knowledge and understanding of their potential risks and benefits. An anonymous online survey was distributed to optometrists in Australia and New Zealand. Questions included practitioner demographics and practice modality; self-reported practices and recommendations relating to diet, nutritional supplements, and omega-3 fatty acids for age-related macular degeneration (AMD) and dry eye disease (DED); and practitioner knowledge about omega-3 fatty acids. Of 206 included surveys, most respondents (79%) indicated recommending for their patients to consume omega-3 fatty acids to improve their eye health. Sixty-eight percent of respondents indicated recommending omega-3-rich foods for AMD management, while 62% indicated recommending omega-3 supplements. Most respondents (78%) indicated recommending omega-3-rich foods or supplements for DED. For DED, recommended omega-3 supplement dosages were  2000 mg [1000–2750 mg] per day. The main sources of information reported by respondents to guide their clinical decision making were continuing education articles and conferences. In conclusion, optometrists routinely make clinical recommendations about diet and omega-3 fatty acids. Future education could target improving optometrists’ knowledge of differences in the evidence for whole-food versus supplement sources of omega-3 fatty acids in AMD. Further research is needed to address uncertainties in the evidence regarding optimal omega-3 dosage and formulation composition in DED.  Diet is a major lifestyle factor that can influence eye health . There iOmega-3 fatty acids can be obtained from several sources. Short-chain omega-3, alpha-linoleic acid (ALA), is found mostly in plant-based foods . Long-chain omega-3 fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are predominantly obtained from marine-based foods . As the biological conversion of ALA to EPA and DHA is incomplete in vivo (estimated in the range of 5 to 20% conversion) ,5, direcDED is one of the most common reasons for patients to seek ophthalmic care . DED canIn addition to its anti-inflammatory effects, DHA is implicated in maintaining the structural and functional properties of the retina . Diets rEye care clinicians have an important role in informing their patients and the public about modifiable risk factors for ocular disease, including diet. Optometrists, as major providers of primary eye care in Australia and New Zealand, are ideally positioned to counsel patients about the benefits and risks of nutritional supplements and their potential impact on eye health. Two studies have investigated the self-reported recommendations made by optometrists relating to nutrition. In the UK, Lawrenson and Evans (2013) reported active engagement by optometrists in providing nutritional advice relating to AMD, but highlighted a need for increased awareness of the relevant research evidence . In an AIn both Australia and New Zealand, the professional scope of optometry includes the diagnosis, assessment, and management of ocular disorders, including the prescription of topical and, in New Zealand, oral medications. In both countries, optometrists are assessed to equivalent competency standards . ResearcThe aim of this study was to investigate the current attitudes and self-reported practice behaviors of optometrists towards omega-3 fatty acid recommendations for eye health, and to assess their opinions and understanding of the potential benefits and risks associated with oral omega-3 fatty acid intake. An anonymous, web-based survey was distributed to optometrists in Australia and New Zealand from June to October 2019. The survey was circulated electronically through professional organizations  and at a major national professional education conference, held in Melbourne Australia (July 2019). The project was approved by both the University of Melbourne Human Research Ethics Committee (HREC #1853287.1) and University of Auckland Human Participants Ethics Committee on 12 March 2019 for three years (ref. 022730).This survey was administered using Qualtrics  and comprised 39 questions, divided into four sections. Questions within each section sought responses that included: Select yes/no, select all that apply, select one option only, and optional free-text boxes. Reviewing or altering prior responses was not permitted. The survey was piloted to optimize the clarity of questions and the flow. Respondents were informed at the beginning of (1)I make recommendations for my patients to consume omega-3 fatty acids  to improve their ocular health;(2)I do not make recommendations for my patients to consume omega-3 fatty acids  to improve their ocular health;(3)I recommend for my patients not to consume omega-3 fatty acids to improve their ocular health.After completing Participants who selected option (1) were directed to complete Section 3a . Participants who selected options (2) or (3) were directed to complete Section 3b .Data contributed by optometrists practicing outside of Australia or New Zealand, or from participants who did not complete the survey up to the end of https://www.ibm.com/analytics/spss-statistics-software). Graphs were plotted in GraphPad PRISM . Descriptive statistics were used to analyze participant demographics, clinical practices relating to diet and nutrition, clinical practice relating to omega-3 fatty acids, and practitioner knowledge of omega-3 fatty acids. Fisher’s exact test was used to compare data relating to the proportions of respondents across different groups. Specific recommendations relating to omega-3 supplementation, derived from responses in the free-text boxes, were represented using a ‘word cloud’ . The size of the lettering in the ‘word cloud’ reflects the frequency with which respondents reported the individual words.Statistical analysis was performed using IBM SPSS statistics software version 21.0 . The five knowledge questions surveyed and the answers that were considered as ‘correct’ are provided in Of 234 total responses received, 206 responses were included in the analysis. Twenty-eight surveys were excluded because they were either not completed up to the end of Most respondents completed their optometry training in Australia or New Zealand (94%); the remainder completed their degree in the UK, South Africa, or US . Australian respondents practiced in all 6 states; most were practicing in Victoria (48%), New South Wales (25%), and Queensland (12%). From New Zealand, responses were received from optometrists in all 16 regions except Gisborne, Tasman, Marlborough, West Coast, and Southland. Most New Zealand optometrists practiced in Auckland (35%), Waikato (14%), Wellington (13%), and Hawkes Bay (13%). Approximately half of the respondents worked in independent optometric practice (49%), 30% worked principally in corporate practice, 11% in a hospital or public health clinic, and 8% within academia. The vast majority of respondents (92%) selected one or more areas of clinical interest, with 50% self-declaring interest in DED or ocular surface disease, 26% in retinal disease, and 8% in gerontology or aged care.As responses from Australian and New Zealand practitioners were similar, pooled data across both locations are presented. n = 2) or an online dietary intake tool (n = 1). Optometrists reported more frequently  asking their patients about their nutritional supplement intake than about their diet : 2.83, 95% confidence interval, CI: 1.83 to 4.37; p < 0.001). Approximately one in three optometrists reported often or always (32%), and one in two, sometimes (52%) providing patients with general or specific dietary advice. Of the 91% of respondents who asked their patients about their diet, 98% did not use any quantitative tools to survey dietary habits. Three respondents indicated that they used either a food frequency questionnaire (Most respondents (79%) indicated that they recommend the consumption of omega-3 fatty acids to patients, in either supplement form or from food sources, to improve eye health. The remaining (21%) respondents indicated that they did not make any recommendations to their patients regarding omega-3 fatty acids. None of the respondents recommended that their patients avoid consuming omega-3 fatty acids.For the 44 respondents who reported not making any recommendations relating to omega-3 fatty acids, almost half (47%) indicated that they felt like they did not know enough about omega-3 fatty acids to justify making recommendations. Thirteen respondents felt there was insufficient published research evidence to support omega-3 fatty acid recommendation for eye health, and three indicated that the published evidence they were aware of showed that omega-3 fatty acids were not beneficial for ocular health. Seven respondents either themselves considered or perceived that their patients considered nutritional advice to be outside the scope of optometric practice. n = 1), recent evidence had changed their opinions (n = 1), or they would recommend other forms of therapy  over dietary interventions (n = 2). Of the 44 respondents who did not make any recommendations relating to omega-3 fatty acids, 20% (n = 9) indicated recommending supplement formulations based on the results of the Age-Related Eye Disease Studies (AREDS and AREDS II) for their patients with AMD.Four respondents indicated that they previously recommended omega-3 fatty acids to their patients, but were no longer doing so. Reason(s) provided included that: they did not see any clinical benefits in their own patients (p = 0.019), had a self-declared clinical interest in DED or ocular surface disease , or if they recognized that omega-3 fatty acid supplements had potential side effects . In multivariate analysis, both a clinical interest in DED or ocular surface disease  and recognition that omega-3 fatty acids have side effects  were significant factors in predicting whether omega-3 fatty acids were recommended to improve ocular health. n = 17). About one-third of respondents (29%) provided specific dosages in their recommendations (n = 23), and these ranged from 250 mg to 5000 mg of omega-3 fatty acids per day . Twelve percent of respondents (n = 10) indicated that they do not make specific recommendations relating to the brand or dosage of omega-3 supplement, and 11% percent indicated that they would advise their patients to consult either a pharmacist or a general practitioner for advice relating to specific supplementation products and the dosage.Participants who indicated recommending omega-3 supplements were invited to detail the specific recommendation(s) given to their patients . p < 0.001).Self-reported clinical recommendations relating to dietary sources of omega-3-rich foods and frequency of intake in DED were similar to those for AMD D. Of theOral omega-3 supplements were recommended by 78% of optometrists for managing DED F. The mon = 10) of respondents advised their patients to consult either the instructions on the supplement bottle or the advice of a pharmacist. Half of the respondents (50%) were not aware of a difference between the biological effects of plant-based (short-chain) and marine-based (long-chain) omega-3 fatty acids. About one-third (31%) correctly considered plant-based omega-3 fatty acids to have less, and 12% considered plant-based omega-3 fatty acids to have greater, biological effect than marine-based omega-3 fatty acids when consumed at the same dose. Just over half (51%) of respondents considered omega-3 fatty acids to be mostly anti-inflammatory, 15% of optometrists recognized that omega-3 fatty acids are always anti-inflammatory, and 42% of optometrists correctly considered omega-6 fatty acids to be mostly pro-inflammatory. The perceived ideal ratio of omega-6 to omega-3 in the human diet was most frequently selected as 1 to 4 (27% of optometrists), 16% of respondents correctly selected the ideal ratio to be 4 to 1, and 39% of optometrists were unsure of the ideal ratio. From a forced-choice list of five options ranging from 150 mg/day to 1500 mg/day, 65% of respondents nominated a daily recommended dietary target of long-chain omega-3 fatty acids (the remaining respondents selected ‘don’t know’). The most common daily targets selected were 1000 mg per day (24% of optometrists) and 1500 mg per day (17% of optometrists). One in 11 respondents correctly selected the ideal approximate target of 500 mg/day for adults. About half of respondents (52%) considered there to be a safety limit for daily omega-3 fatty acid consumption; the perceived limit selected by respondents encompassed a 20-fold dose range. From the four multiple choice options offered, 500 mg per day was considered the upper limit by 4% of all optometrists, 1500 mg per day by 15% of optometrists, 3000 mg per day by 18% of optometrists, and 5000 mg per day by 8% of optometrists. Over half (53%) of respondents believed that omega-3 fatty acids have potential side effects. Based on multiple-choice options see , these wp = 0.034) relative to those without such interests. Compared with respondents working in academia, those who were based principally in a corporate practice setting had a lower omega-3 knowledge score .The level of respondents’ knowledge of omega-3 fatty acids was assessed using an ‘omega-3 knowledge score’, derived from five knowledge-specific questions in the survey see . A ‘passThis study describes the self-reported clinical practices and opinions of Australian and New Zealand optometrists, as related to omega-3 fatty acids and eye health. To our knowledge, this is the first study to consider clinicians’ awareness and routine practices relating to omega-3 fatty acids in primary health care. The chosen focus on omega-3 fatty acids for this survey was based on their reported potential to improve outcomes in certain chronic ocular conditions, such as DED and AMD. The current findings provide new insights into optometrists’ understanding of current research evidence relating to omega-3 fatty acids in the context of eye health, and their self-reported application of this knowledge in practice. Furthermore, this survey considered the sources of information and evidence used to guide optometrists’ clinical decision-making as a foundation for informing the optimal mode(s) of delivering future education programs relating to nutrition in eye care practice. This survey, which was made available to an estimated 6500 practicing optometrists via professional optometry networks in Australia and New Zealand, received responses that were considered representative of the major regions in both countries. About half of respondents described their principal practice setting as independent (49%), which indicates a possible over-representation of responses received from independent practices compared to corporate or public health practices . GlobalOptometrists, as major providers of primary eye care, are ideally placed to ask patients about their diet and to offer evidence-based advice about nutrition-based strategies for reducing the risk and/or progression of ocular disease. In a previous survey-based study, it was reported that two in five patients attending for optometric care in Australia expected their optometrists to ask them about their diet and/or nutritional supplement intake, although only one in five recalled being asked about these factors when attending an optometric consultation . In the Over 50% of respondents indicated that they would frequently recommend for their AMD patients to increase their intake of omega-3 rich foods. This is comparable to a previous survey, conducted in the UK, which found that about half of eye care professionals  reported frequently recommending eating oily fish at least twice per week to individuals with AMD and/or individuals considered to be at risk of AMD . Several prospective cohort studies have associated higher intakes of foods containing long-chain omega-3 fatty acids with a reduced risk of AMD progression ,46. The With respect to omega-3 fatty acid supplementation, 62% of respondents reported recommending either plant- or marine-based supplements for AMD management. Although it might be argued that a person’s diet can be augmented through supplementation if adequate levels of certain nutrients are not being obtained from food sources, the current best-available evidence does not support omega-3 oral supplements for preventing or slowing the progression of AMD . In contThe absorption of EPA and DHA from nutritional supplementation is influenced by the formulation, chemical preparation, and a person’s background dietary fats ,53. PhosAbout 80% of respondents reported recommending omega-3 fatty acids, in either diet or supplement form, for DED management. This proportion is comparable to previously reported values of 70% of Australian optometrists  and 80% Although the pathogenesis of DED is unclear, tear instability, tear hyperosmolarity, and neuro-sensory abnormalities are understood to contribute to a vicious cycle of inflammation that perpetuates on the ocular surface . The priOur study established that practitioners are less likely to report recommending omega-3 fatty acids for milder forms of DED. This trend has been reported in previous evaluations of self-reported practices relating to recommending omega-3 fatty acids for DED, by eye care practitioners in New Zealand, Australia, and the UK ,56. It iRespondents indicated recommending a variety of daily omega-3 fatty acid dosages for managing DED, ranging from 250 mg to 6000 mg per day (median [IQR]: 2000 [1000–2750] mg/day). Published guidelines exist with regard to the optimal dose of omega-3 fatty acids for several other health conditions. For example, the American Heart Association recommends 4000 mg EPA, or combined EPA and DHA, per day for reducing systemic triglycerides in cases of hypercholesterolemia . There aRandomized controlled trials (RCTs), comparing the efficacy of long-chain omega-3 fatty acids relative to placebo or to no intervention for treating DED, have adopted a wide range of dosages, varying across a 20-fold range, from 135 mg to 3000 mg of combined EPA and DHA per day . This laThe two commercially available formulations reported to be most frequently recommended by survey respondents were Thera Tears™  and Lacritec™ , 192 mg linolenic acid, 434 mg Borago officinalis seed oil fixed, and 93.5 mg gamma-linolenic (GLA) acid). In addition to omega-3 fatty acids, Lacritec contains short-chain omega-6 fatty acids. Approximately one in five optometrists reported recommending combined omega-3 and omega-6 supplements for managing DED. While eicosanoids derived from the metabolism of the long-chain omega-6 fatty acid, AA, are pro-inflammatory, dietary intake of the short-chain omega-6, GLA, and its precursor, LA, can yield anti-inflammatory effects by acting as precursors to the eicosanoid prostaglandin-E1 (PGE1) . The potThe US Women’s Health Study found an association between a higher ratio of dietary omega-6 to omega-3 and an elevated risk of DED . A recenBaseline dietary omega-3 fatty acid intake is another important consideration for identifying patient populations likely to benefit from enhancing their omega-3 fatty acid intake. Supplementation in individuals already achieving sufficient essential fatty acid levels through their diet would not be expected to demonstrate the same biological response as in those who are deficient . While 9It has been estimated that in Australia, 80% of adults may not be meeting the suggested dietary intake of long-chain omega-3 fatty acids , which, High-dose omega-3 fatty acids, >2000 mg per day, tend to be safe and well tolerated but are not without potential adverse effects. The most common side effects are gastrointestinal , dermatological , and hematological . Several studies examining the safety profile of omega-3 fatty acids, dosed at up to 4000 mg per day, have concluded that supplementation is generally well tolerated and that associated adverse events are unlikely to be of clinical significance ,73,74,75Four in five respondents selected continuing education conferences and/or articles as the main source of information used to guide their clinical decision making about omega-3 fatty acids. Two in five respondents indicated that they source primary research articles and/or systematic reviews for advice. The importance of evidence-based practice is well recognized in both optometric training and practice . A previA number of recent initiatives have sought to support optometrists in making evidence-based dietary recommendations that are relevant to eye health. Optometry Australia’s 2019 AMD Clinical Practice Guide recommends that optometrists inform patients about how AMD risk can be modified by diet and other lifestyle factors, such as smoking . With reThe current study identified current clinical practice patterns in primary eye care, as related to oral omega-3 fatty acids, and highlighted knowledge gaps and perceived barriers to clinical implementation. There were some limitations to the present study. While the cohort represented a broad range of optometric practice locations and modalities, there was the potential for self-selection bias, and this was reflected in the proportion of participants declaring a clinical interest in DED or ocular surface disease. Also noted was a relative over-representation of younger optometrists, and optometrists working in independent (private) practice. These limitations may affect the generalizability of our findings, but are not uncommon considerations in survey-based research.Our results suggest that most Australian and New Zealand optometrists routinely provide clinical recommendations about diet and nutritional strategies, as relevant to eye health. While many optometrists indicated recommending diet-based strategies for managing AMD, the potential benefits of whole-food omega-3 sources relative to the limited benefits of omega-3 supplements were not consistently appreciated. Most respondents recommended omega-3 fatty acids for managing DED, despite conflicting data from recent randomized trials. Respondents were more likely to recommend omega-3 fatty acids to patients with moderate or severe (rather than mild) DED, and to use omega-3 supplementation to treat predominantly evaporative DED. Further research is needed to address uncertainties in the evidence relating to the optimal prescribing regimen  for treating DED. This information will be of value for informing future education programs about eye health and nutrition, particularly at events such as clinical education conferences that were reported to form the main source of information used by optometrists to guide clinical decision-making.


Irritable bowel syndrome (IBS) is a common clinical label for medically unexplained gastrointestinal (GI) symptoms, recently described as a disturbance of the brain-gut-microbiota (BGM) axis. To gain a better understanding of the mechanisms underlying the poorly understood etiology of IBS, we have designed a multifaceted study that aim to stratify the complex interaction and dysfunction between the brain, the gut, and the microbiota in patients with IBS.Deep phenotyping data from patients with IBS (n = 100) and healthy age- (between 18 and 65) and gender-matched controls (n = 40) will be collected between May 2019 and December 2021. Psychometric tests, questionnaires, human biological tissue/samples , assessment of gastric accommodation and emptying using transabdominal ultrasound, vagal activity, and functional and structural magnetic resonance imaging (MRI) of the brain, are included in the investigation of each participant. A subgroup of 60 patients with IBS-D will be further included in a 12-week low FODMAP dietary intervention-study to determine short and long-term effects of diet on GI symptoms, microbiota composition and functions, molecular GI signatures, cognitive, emotional and social functions, and structural and functional brain signatures. Deep machine learning, prediction tools, and big data analyses will be used for multivariate analyses allowing disease stratification and diagnostic biomarker detection.To our knowledge, this is the first study to employ unsupervised machine learning techniques and incorporate systems-based interactions between the central and the peripheral components of the brain-gut-microbiota axis at the levels of the multiomics, microbiota profiles, and brain connectome of a cohort of 100 patients with IBS and matched controls; study long-term safety and efficacy of the low-FODMAP diet on changes in nutritional status, gut microbiota composition, and metabolites; and to investigate changes in the brain and gut connectome after 12 weeks strict low-FODMAP-diet in patients with IBS. However, there are also limitations to the study. As a restrictive diet, the low-FODMAP diet carries risks of nutritional inadequacy and may foster disordered eating patterns. Strict FODMAP restriction induces a potentially unfavourable gut microbiota, although the health effects are unknown.NCT04296552 Protocol version 1 May 2019 In the absence of a structural or organic cause, growing evidence suggests that there is a plethora of factors that may elicit abnormal responses in patients with IBS. Collectively, these factors are described as dysregulation of the brain-gut-microbiota (BGM) interaction. As a bidirectional link between the brain and the gut, the axis reciprocally interacts with key sensorimotor functions, sympathetic and parasympathetic branches of the autonomic nervous system, the endocrine and immune system, the hypothalamic-pituitary-adrenal-axis, the enteric nervous system (ENS), and gut microbiota and metabolites. Alterations in gut microbiota composition or functions is known to impact human behaviour and brain physiology, and dysbiosis is commonly reported in patients with IBS.,5 However, whether IBS-symptoms are caused by changes in microbiota still remains elusive and multiple pathways and mechanisms have been suggested to be involved. These include immune, endocrine, and neural signalling pathways, which the gut microbiota and metabolites may modulate. For instance, structural components of intestinal bacteria and bacterially produced metabolites  may regulate immune function and cytokine production, with upstream effects on the blood–brain barrier or brain function through modulation of neuroinflammation. The gut microbiota, directly or through interactions with the host or diet, produce neuroactive compounds  that may affect neuroendocrine functions locally or through humoral or neural routes such as signaling along the vagal nerve.Irritable bowel syndrome (IBS) is a symptom-based diagnosis characterized by chronic abdominal pain associated with altered bowel habits, in the form of diarrhea, constipation, or a mix between the two. In this study, we will employ cutting-edge omics and advanced computational methods to unravel gastrointestinal (GI) pathophysiology of IBS, link this information to results from multimodal brain imaging examination, gastric accommodation test, and information from psychometric tests and questionnaires, and translate these into clinical biomarkers. By this, we aim to stratify the diagnosis and improve treatment for patients with IBS.Indeed, there is a substantial knowledge gap in regards to understanding pathophysiology in both the brain and the gut. The current view on disorders of the BGM axis has replaced the conventional focus on individual brain regions and cell types of the gut, integrating brain networks (brain connectome) and networks of gut cells and microbiota (gut connectome).22.1The primary objective of this study is to increase knowledge and understanding of BGM axis dysfunction and identify covariance across multiple phenotypes leading to identification of biomarkers.The secondary objectives are addressing the multifaceted manifestations of BGM axis dysfunction, specificallyTo identify and quantify structural and functional brain connectivity signatures, including visceral pain provocation and psychophysiological measures, using advanced neuroimaging techniques and statistical approaches.To investigate clinical biochemistry in blood .To evaluate upper GI motility patterns using transabdominal ultrasound.To investigate GI biopsies for multi-omics analysis and complex molecular subclassification.To identify microbiota signatures and metabolites associated with gut-brain equilibrium or dysfunction in IBS patients and healthy controls.To investigate clinical effects of short and long-term strict low-FODMAP diet.To examine and compare the compositional and functional response of gut microbiota at baseline and after the low-FODMAP-dietary intervention, and the relationship with clinical and psychophysiological outcomes.To evaluate if the GA-map technology can be used to predict the response to treatment (to distinguish responder and non-responders prior to low-FODMAP diet).To investigate cognitive and emotional function associated with brain structures and functions known to be affected in adults with IBS, and personality traits and behaviour expected to be of importance to the patient's everyday functioning and effects of a strict low-FODMAP diet.To define phenotypes and biomarkers from all our abovementioned data using high dimensional data-driven and machine learning techniques.2.2www.braingut.no.) and are individually informed by telephone by a study nurse, before inclusion. If a patient decides to participate in the study, (s)he signs an informed consent. Patient recruitment will take place between May 2019 and December 2021. Inclusion criteria for the studyincluded age between 18 and 65 years; fulfillment of the Rome-IV criteria for IBS: Reports of recurrent abdominal pain on average at least 1 day per week during the previous 3 months that is associated with alterations in bowel habits; duration at least 6 months and an IBS-symptom severity score (SSS) >175. Exclusion criteria for the study included pharmacological treatment affecting the GI-tract, including treatment for depression; presence of an organic disease such as coeliac disease, inflammatory bowel disease, diabetes, active Helicobacter pylori infection, polycystic ovary syndrome, and neurological diseases such as multiple sclerosis, Parkinson's disease, and amyotrophic lateral sclerosis; treated with systemic antibiotics within the last 3 months; probiotics or low-FODMAP-diet within the last 3 weeks or being vegan or vegetarian; regular use of analgesics; pregnancy; previous intestinal surgery except appendectomy; claustrophobia or having metallic implants that are not MRI compatible; been travelling outside Europe within the last 3 weeks (or plan to travel in the nearest future); participation in any other simultaneous clinical study; inability to comprehend and respond to questionnaires or follow dietary guidance.Eligible patients from the Bergen area are recruited from the IBS outpatient clinic at Haukeland University Hospital and through social media. Patients are informed about the study online  at baseline. Sixty candidates with IBS-D will enroll in the 12-week strict low-FODMAP dietary intervention. Additional patients will be enrolled to replace patients who drop out during baseline until the end of the inclusion period. Patients who unexpectedly get claustrophobic during brain scanning may be included in the dietary intervention without functional magnetic resonance imaging (fMRI) examination.2.3The study is an open, single-center, case–control characterization study, followed by open label dietary intervention for a subgroup of subjects with IBS-D. Table 33.1Primary outcomes included proportion of patients with treatment success in the lowFODMAP dietary intervention. Treatment success is defined as an improvement of ≥50 points on the IBS symptom severity score (IBS-SSS) at 12 weeks after treatment start compared with the score at baseline.3.2≥50 points decrease in the IBS symptom severity score (IBS-SSS) from the scores 4 weeks after treatment compared to baseline.≥13 points increase in the IBS-Qualiy of Life (IBS-QoL) week 4 and/or week 12 after treatment when compared to the scores at baselineTwenty percent reduction on visual analog scale on nausea, pain, and bloating measured post lactulose-test at week 12, compared with baseline scores.Proportion of patients with change dysbiosis index at week 4 and week 12 according GA-map technology, compared with baseline and/or healthy controls.3.3Changes in structural and functional brain connectivity signatures, including visceral pain provocation and psychophysiological measures at 12 weeks compared with baseline, and healthy controls.Changes in resting state regional brain interactions at 12 weeks compared with baseline and healthy controls, as measured with fMRI.Changes in cortical thickness  as measured with DTI (MRI)Differences in taxonomy and function of the microbiome, the immune system, metabolome and gut epithelial barrier in IBS patients at 12 weeks compared with baseline measures, and healthy controls.Changes in levels of SCFA composition, compared with baseline, and heathy controls.Changes in cognitive and emotional function, personality traits, and behavior at 12 weeks compared with baseline, and healthy controls.Improvements in The Conner's Continuous Performance Test (CPT-3) at 12 weeks in IBS patients, compared with baseline.3.42) is medium (0.20), desired statistical power level is 0.8 (80%) with type-I error of 5% (α = 0.05). On the basis of these assumptions, the minimum required samples size is 59 in each group for multiple regression analyses. Previous experience with this patient group has shown that we may expect a high level of missing data. To allow diagnostic subclassification, we therefore have increased the baseline sample size to n = 100, and the dietary intervention samples size n = 60 in patients with IBS-D and n = 40 for healthy controls. However, to identify biomarkers and patient subpopulations, comprehensive analysis methods will be employed to combine neuroimaging and neuromics data with clinical patient data. Although IBS is a highly heterogeneous patient group, we believe machine learning techniques and possibly pre-trained models will enable us to make inferences about an IBS population from the given sample, considering the limitation of cost, time, and convenience of data collection.Generally, for samples size calculation and statistical power, we would make the following assumptions: Anticipated effect size ; T1-w 3D MPRAGE , T2-w 3D TSE , GRE field map ; rs-fMRI epi2d moco , rs-fMRI epi2d moco , dMRI MMDW /1000 (n = 30)/2500 (n = 30) s/mm2; TA 8:34, 8 PA epi2d diff , a 12-channel RF head coil and paralell imaging. The following MRI sequences will be applied: Scout  involves measuring of water diffusion in brain tissue. This provides information on a voxel-by-voxel basis about white matter microstructure, properties of with matter fiber bundles in the brain.–15 For this study, we do not plan to use the opportunity of truly simultaneous acquisition of structural and functional information of the same brain regions from both MRI and PET. Our cerebral MRI protocol is a partly compliant with and motivated by the standardized Pain and Interoception Imaging Network protocol.Functional MRI, using blood-oxygenation-level dependent (BOLD) contrast, will be used to record neuronal activity  in both IBS patients and healthy controls. Resting-state fMRI is a special “task-free” variant in which the subject is asked to lie as still as possible in the head coil while being continuously scanned, to investigate spontaneous brain activity (resting state networks).3.5.2 The test battery includes 12 subtests giving measures of attention, language, visuospatial/constructional abilities, and immediate and delayed memory function, in addition to a composite score and estimates of intellectual function according to available Scandinavian norms. In the present study, the results will be used for defining the cognitive function of the participants, as a covariate in the statistical analyses, and for establishing norms for patients with IBS.The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) is a brief, individually administered test-battery designed as a screening instrument to evaluate neuropsychological status of adults, ages 20 to 89 years. The scale includes 86 items in 8 clinical scales  and 2 validity scales (Inconsistency and Negativity). These scales form 2 global indexes  representing overall EF.The Behavior Rating Inventory of Executive Function (BRIEF-A) will be used to assess executive functions (EFs), as they are experienced in the daily life of the participants. The test is characterized by high signal-to-noise ratio by including 90% target stimuli and 10% no-target stimuli (x). The test incudes 360 trials presented across 6 blocks, with 3 subgroups with different interstimulus intervals. The test durance is 14 minutes, and the results generate measures of reaction time, accuracy, variability, consistency, and vigilance. BRIEF and CPT-3 will be used as a measure of attention and EF in patients with IBS, as a predictor of treatment response (baseline); and as an outcome variable (change from baseline to follow-up).The Conners’ Continous Performance Test – third edition (CPT-3) is a computerized test wherein the participants are instructed to push the spacebar as soon as a target appears on the screen (a letter), except when the letter is X.,21 The instrument is well validated for use in several countries. Measures will include the 5 factors as well as profiles of factors for individual adults. In the present study, the personality assessment will be part of the phenotyping of adults with IBS and controls; one of the predictors of treatment response, a more general predictor of quality of life, pain, and other self-reported challenges associated with IBS, and related to the BGM axis.The NEO-FFI-3 is a 60-item version of the NEO-PI-3 providing a quick and reliable measure of the Five-Factor model of personality .3.5.3–24 We will characterize the patient's gastric accommodation, emptying, and quantify antral contractions. Antral biopsies will be investigated for density of interstitial cells of Cajal, correlated to functional observations during the UMAT.Gastric motility will be characterized using transabdominal ultrasound and the meal accommodation test (UMAT). The UMAT involves consumption of a standardized 500 mL, pre-boiled, low-caloric soup after which transabdominal ultrasound allows visualization and measurement of the proximal and distal compartments of the stomach.3.5.4Pulse oximetry data will be acquired during the resting-state fMRI sequence in order to collect (heart) interbeat intervals (IBIs), with a photoplethysmograph placed on the right index finger (50 Hz). Pulse oximetry data offers an especially accurate approximation of interbeat intervals and is associated with less artifacts in a MR-environment. In order to control for the effect of respiration in the pulse oximetry data, a chest strain gauge will be applied to measure respiratory frequency. Heart rate variability (HRV) and breathing pattern is registered during resting state, that is, the time the patient is in the MR machine in supine position with spontaneous breathing (approximately 20 minutes). The pulse oximetry data will be used to calculate HRV. In order to estimate the parasympathetic modulation for heart rate, an estimate of fast IBI changes in the time scale of milliseconds will be obtained, that is, faster changes that sympathetic modulation for heart rate. Thus, high frequency HRV > 0.14 Hz and time-domain measures reflecting such fast changes ] provide a readily available, estimate of vagal activity.3.5.5A subgroup of patients with IBS-D (n = 60) will undergo a strict 12-week low-FODMAP dietary intervention guided by clinical dietitian. Here, patients will exclude FODMAPs from their diet without reintroduction during the entire study period. Patients will meet with their registered dietitian four times . At baseline, the patients will be informed about the diet intervention and the low-FODMAP-diet. They will fill out questionnaires and records of the diet (3 days) are collected at week 2, 4, 6, 8, 10, and 12  will be collected for analysis of GI hormones, serotonin levels, gut integrity markers, and pro-inflammatory cytokines and interleukins). Stool samples will be collected for gut microbiota profiling and estimates of abundance and functions.3.5.7 Irritable Bowel Syndrome – Symptom Severity Score (IBS-SSS), Hospital Anxiety and Depression scale (HADS), Bristol Stool Scale (BSS), Visceral sensitivity index (VSI), Gastrointestinal Symptom Rating Scale for Irritable Bowel Syndrome (GSRS-IBS), IBS symptom questionnaire (IBS-SQ),,32 Chalder fatigue scale (CFQ-11), Wender Utah Rating Scale for ADHD in adults (WURS), Autism-spectrum quotient (AQ), and Bergen Insomnia Scale (BIS), Hospital Anxiety and Depression scale (HADS), Bristol Stool Scale (BSS), Visceral sensitivity index (VSI), Gastrointestinal Symptom Rating Scale for Irritable Bowel Syndrome (GSRS-IBS), IBS symptom questionnaire (IBS-SQ),,32 Chalder fatigue scale (CFQ-11), Adult ADHD self-report scale (ASRS), Wender Utah Rating Scale for ADHD in adults (WURS), Autism-spectrum quotient (AQ), and Bergen Insomnia Scale (BIS).During baseline and multiple time-points throughout the dietary intervention Table , standar3.5.8TM DNA Flex Library Preparation kit  from 350 to 450 ng of gDNA. A low cycling indexing protocol (5 PCR cycles) and Unique Dual Indices will be used to minimize amplification bias and to prevent index hopping respectively. Every final library will be individually quantified on the Qubit 3.0 fluorometer and run on a Bioanalyzer HS DNA chip  to verify quality and size distribution. The 2 x 150nt paired-end sequencing will be conducted on a NovaSeq 6000 sequencer . All samples will be accommodated in one S4 flow cell to minimize the bias of batch sequencing. Paired-end fastq files will be used to perform a phylogenetic marker gene-based operational taxonomic units (mOTUs) analyses, enabling the taxonomy profiling of >7700 microbial species. In addition, reconstruction of metagenome assembled genomes (MAGs) will permit assess gut microbiota functionality with different representation across the intervention time points.The DNA will be extracted from stool samples using the QIAamp Fast DNA Stool Mini Kit , according to the manufacturer's instructions, with a prior step of bead beating in 2 mL microcentrifuge tubes containing 0.1 mm diameter glass beads, ∼ 150 mg faeces, and 1 mL InhibitEX buffer . The faecal DNA concentration will be measured by fluorescence-based methods such as Qubit 3.0 and the Qubit dsDNA HS Assay Kit . Library preparation for shotgun metagenomic sequencing will be performed using the Nextera2SO4; 2.5 mL of the homogenate will be vacuum distilled, according to the method of Zijlstra et al, as modified by Høverstad et al. The distillate will be analyzed with gas chromatography , using a capillary column , and quantified using internal standardization. Flame ionisation detection will be employed. In addition, we will calculate the proportional distribution of individual SCFA to total SCFA.Feces will be analysed for short-chain fatty acids (SCFA). About 0.5 g of the fecal material will be homogenised after addition of distilled water containing 3 mmol/L of 2-ethylbutyric acid  and 0.5 mmol/L of HAnalysis of salivary microbiota composition will be performed using PCR. A total of 5 mL unstimulated saliva is collected from each patient and healthy control using a 50 mL sterile Falcon tubes. Each sample is then immediately placed in an ice bag and stored at –80°C until extractions. DNA from the collected saliva samples will be extracted using a TIANamp Bacteria DNA Kit  following the manufacturer's instructions. Genomic DNA will be used as the template for bacterial 16S rRNA gene amplification with the barcoded primers 338F (50-ACTCCTACGGGAGGCAGCAG-30) and 806R (50- GGACTACHVGGGTWTCTAAT-30), which target the V3–V4 hypervariable region. A real-time PCR system combination with the Illumina MiSeq platform (16S rRNA)  will be used for sequencing of oral microbiome profile.3.5.9https://checkware.com), security level 4 . All patient data will be plotted into an electronic database, FileMakerPro 17 Advanced, stored at Haukeland University Hospital Research server (2015–01621). All data are registered using the patient's study-ID, and the study participant-identifying key is stored in a separate location at the research server. No patient data are located in proximity to the study participant-identifying key. Biological material such as GI biopsies, blood , stool samples, and saliva is collected in 2D barcode labeled tubes (Matrix), scanned, and stored in Biobank #2176 at Haukeland University Hospital.Data will be collected during consultations at Haukeland University Hospital (HUH). Specific patient-reported outcome data  are collected electronically using CheckWare (https://www.python.org) and Jupyter notebooks (https://jupyter.org) to support open science and reproducible research.,42 For implementing machine learning procedures and pipelines, we will use Pandas Numpy, Scikit-learn (https://scikit-lern.org). For deep learning approaches, especially related to the MRI acquisitions, we will consider the Pytorch (https://pytorch.org) framework and the high-performing fastai (https://docs.fast.ai) libraries using deep learning best practices allowing models to train fast with limited resources. We will address the importance of separate training, validation and test datasets, and employ cross-validation techniques to reduce overfitting and improve generalization to any data from the problem domain. Before employing machine learning techniques, for processing the acquired neuroimaging data, we will consider using Freesurfer (http://surfer.nmr.mgh.harvard.edu), FSL (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/), and AFNI (https://afni.nimh.nih.gov/). Freesurfer will mainly be used for cortical reconstruction and volumetric segmentation to extract the region of interest (ROI). We have planned to used FSL and AFNI to obtain diffusion and functional maps, respectively.In this large and heterogeneous data set collected from multiple sources from the individual participant in the study, we will employ exploratory data analysis (EDA) to get insight, and state-of-the-art supervised and unsupervised machine learning techniques for predictions, and detection of IBS subgroups and identification of diagnostic biomarkers Fig. . More spWe would like to thank all the staff and patients involved with the study at the Department of Medicine, Section for Gastroenterology, Department of Radiology and the Center for Nuclear Medicine and PET, Department of Pathology, Mohn Medical Imaging and Visualisation Center, at Haukeland University Hospital for their ongoing support.All authors made a significant contribution to the conception and design of the protocol. BB made major contributions to the design of this study, ethical approval application, development of the protocol, and drafting the manuscript. GAL, EKS, BB, TH, EPT, ERH designed the study and dietary intervention, EKS, OHG, TH designed the UMAT test, BO designed the protocol for vagal activity measures, GAL and IB designed the biochemistry analysis plan, YA, ABP, JV designed the protocol and plan for microbiota analysis, EAM, AG and AL developed and designed the advanced neuroimaging protocol, AL and BHN developed the big data statistical analysis and machine-learning plan. AJL designed the protocol for all psychometric tests. AL, TH, OHG, JGH, GAL, and BB secured funding. All authors contributed to refinement of the study protocol and the final manuscript.


The understanding of food cue associated neural activations that predict future weight variability may guide the design of effective prevention programs and treatments for overeating and obesity. The current study investigated the association between brain response to different food odors with varied energy density and individual changes of body mass index (BMI) over 2 years. Twenty-five participants received high-fat (chocolate and peanut), low-fat (bread and peach) food odors, and a nonfood odor (rose) while the brain activation was measured using functional magnetic resonance imaging (fMRI). BMIs were calculated with participant’s self-reported body weight and height collected at the time of the fMRI scan and again at 2 years later. Regression analyses revealed significant negative correlations between BMI increase over 2 years and brain activation of the bilateral precuneus and the right posterior cingulate cortex (PCC) in response to high-fat vs. low-fat food odors. Also, brain activation of the right supplementary motor area (SMA) in response to food vs. non-food odor was negatively correlated to subsequent BMI increase over 2 years. Taken together, the current findings suggest that individual differences in neural responsivity to  food odors in brain regions of the default mode and motor control network serve as a neural marker for future BMI change. Bodyweight variability in humans is usually regulated by many factors including diet and lifestyle  2-years later and were included in the current analyses. Normal olfactory functions of them were ascertained using the threshold and identification test of the “Sniffin’ Sticks” battery , a high-fat non-sweet peanut odor , a low-fat sweet peach odor , and a low-fat non-sweet bread odor . Also, a rose-like odor  was chosen as non-food olfactory stimuli. The odorants were diluted in propylene glycol to the following finalized concentrations to reach the proper and equal intensity according to daily life experiences: chocolate 1% (v/v) for chocolate odor; 20% (v/v) for peanut odor; 1% (v/v) for peach odor, 5% (v/v) for bread odor, and 0.5% (v/v) for rose odor.All participants were refrained from consuming food or drinks (except water) for 2 h before taking part in the experiment. On arrival and before the fMRI test, participants rated their hunger/fullness levels on a 9-point scale (from 0 very hungry to 9 very full). The hunger ratings were used to check compliance with the fasting instructions and were used as a covariate during the correlation analysis between brain activation and future body weight changes, as suggested by a previous study , pleasantness , sweetness  of the odor stimuli, and their desirability to eat food with a similar odor  h Kobal, , and werh Kobal, .Please estimate the fat content of the odor-associated food,” and the question “Please estimate the calorie density of the odor-associated food,” respectively. Ratings were performed on a 9-point scale for macronutrient content (range from 1 = very low to 9 = a large amount) and calorie density . Notably, participants were not aware of the aforementioned rating task before and during the fMRI scan. This was meant to avoid biased brain activation results caused by any selective attention to such food attributes during the fMRI test.To verify the significant differences regarding the energy density, taste quality, and major macronutrient content between the selected odors, participants completed an estimation task for the fat content and calorie density for the odor related food items after the fMRI session. We asked participants to rate the fat content and calorie density of the odor-associated food with the question “We collected the whole-brain functional and structural images with an 8-channel head coil on a 3 Tesla MRI scanner . For each participant, functional images were collected using a T2-weighted echo-planar imaging (EPI) sequence . A high-resolution T1-weighted anatomical image was acquired using a standard MPRAGE (magnetization prepared rapid acquisition gradient echo) sequence .fMRI data were analyzed using SPM12  run with MATLAB . For image preprocessing, we used the standard pipeline batch in (\spm12\batches\preproc_fmri.m). In short, the preprocessing included realignment to the first image of the first run and unwrapped, co-registration with anatomical image, segmentation and spatial normalization of the standard EPI MNI template, and spatial smoothing with an 8-mm full width at half maximum (FWHM) isotropic Gaussian kernel. Finally, we used the ArtRepair tool  to examine the post-preprocessing brain volumes to detect and repair the artifact volumes due to excessive head motion. Brain volumes with image-to-image motion larger than 0.5 mm/TR and total volumes repaired exceed 20% of the total volumes were excluded from the analysis. Data from 17 participants were repaired with no participant was excluded at this stage.For the baseline period, we discarded the first 5 s due to the potential carry-over effect from the odor period. We performed the analysis in a two-step manner. First, we modeled the following contrasts for each participant: food > nonfood odor; high-fat > nonfood odor; low-fat > nonfood odor; high-fat > low-fat odor; low-fat > high-fat odor, sweet > savory odor, savory > sweet odor. Then, on the group level, we entered the contrasts from all individual participants into a linear regression model to explore the association between brain responses and 2-year BMI changes. Subjective hunger ratings and baseline BMIs were entered as covariates as suggested in previous studies  empirically determined by 1,000 Monte Carlo simulations, using AlphaSim as implemented in the REST toolboxp < 0.001 would provide sufficient cluster-level Family-Wise Error correction of p < 0.05 across the whole brain. Mean responses  of the significant clusters were extracted using the Marsbar toolbox. Specific brain regions were identified using the Automated Anatomical Labeling (AAL) toolbox .Participants rated the chocolate and peanut odors as significantly higher in terms of the fat content and calorie density as compared to bread and peach odors .No significant correlation was found between olfactory performance , TFEQ scores, psychophysical odor ratings , and increases of BMI or body weight over the 2 years follow up  that predicted 2-year BMI or weight gain. A recent study using a bootstrap sampling approach had evaluated the reliability of the food-related neural responsivity as predictors for weight change among 10 bootstrap samples. An elevated response in the precentral gyrus/Rolandic operculum to images of appetizing foods turned out to be a moderately reliable predictor of future BMI gain  were not followed during the 2-year follow-up, which affects the results cannot be ruled out. Besides, the dynamic changes of fat mass or lean tissues in the process of weight gain cannot be distinguished by the BMI values although BMI is widely used to assess excess adiposity and shows high test-retest reliability, it does not distinguish between increased fat mass and increased lean tissue mass, and hence can lead to significant misclassification . The patients/participants provided their written informed consent to participate in this study.PH designed the study, performed the experiment, analyzed the data, and wrote the manuscript. TH and HC reviewed and revised the manuscript critically. 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.