The incidence and magnitude of indicators of radiographic response of pancreatic cancer to systemic chemotherapy and (chemo)radiation administered prior to anticipated pancreatectomy are unclear.Sequential computed tomography scans of 226 patients with localized pancreatic cancer who received chemotherapy consisting of 5-fluorouracil, leucovorin, oxaliplatin, and irinotecan (FOLFIRINOX) or gemcitabine and nanoparticle albumin-bound paclitaxel (GA) with or without (chemo)radiation and who subsequently underwent surgery with curative intent from January 2010 to December 2018 at The University of Texas MD Anderson Cancer Center and Verona University Hospital were re-reviewed and compared.p = 0.04), and the median tumor volume loss after chemotherapy was significantly greater than that after (chemo)radiation .Overall, 141 patients (62%) received FOLFIRINOX, 70 (31%) received GA, and 15 (7%) received both; 164 patients (73%) received preoperative (chemo)radiation following chemotherapy and prior to surgery; and 151 (67%), 70 (31%), and 5 (2%) patients had Response Evaluation Criteria in Solid Tumors (RECIST) stable disease, partial response, and progressive disease, respectively. The tumors of 29% of patients with borderline resectable or locally advanced cancer were downstaged after preoperative therapy. Radiographic downstaging was more common with chemotherapy than with (chemo)radiation radiation experienced either RECIST partial response or radiographic downstaging prior to surgery. The incidence of tumor downstaging was higher and the magnitude of tumor volume loss was greater following chemotherapy than after (chemo)radiation. More than 30% of all patients who present with pancreatic ductal adenocarcinoma (PDAC) do so with infiltrative, borderline resectable (BR), or locally advanced (LA) tumors without distant metastases. Margin-negative (R0) pancreatectomy, the treatment modality most likely to lead to long-term local control and survival in patients with PDAC, is unlikely for most of such patients.Historically, anatomic downstaging was distinctly rare following the administration of gemcitabine, an agent associated with a radiographic response rate lower than 10% in patients with metastatic disease.8The primary aim of this study was to characterize the changes in primary pancreatic tumor size and/or anatomic extent that occur in response to systemic treatment with FOLFIRINOX and GA as well as subsequent (chemo)radiation. To that end, we evaluated the computed tomography (CT) scans of PDAC patients who underwent surgery with curative intent following therapy at two institutions.The Institutional Review Boards of The University of Texas MD Anderson Cancer Center (IRB #PA18-1093) and Verona University Hospital  approved this retrospective study. Individual informed consent was waived. The two centers’ prospectively maintained pancreatic tumor databases were used to identify consecutive patients who (1) received at least three cycles of preoperative chemotherapy with FOLFIRINOX and/or GA as their first line of therapy; (2) underwent surgery with curative intent for localized PDAC from January 2010 to December 2018; and (3) had both pretreatment and preoperative CT scans available for review. Among 240 patients who met these criteria, 14 patients were subsequently excluded from analysis: 5 patients who had a final diagnosis of PDAC arising in an intraductal papillary mucinous neoplasm, 4 who had a baseline CT scan showing severe acute pancreatitis or no visible mass, and 5 in whom surgical resection was aborted for a reason other than oncologic .Prior to initiation and following the completion of preoperative chemotherapy and (chemo)radiation, anatomic disease staging for all patients was accomplished using multidetector CT or magnetic resonance imaging (MRI) and standard protocols optimized for imaging pancreatic tumors. Multiplanar reconstructions were used as necessary to visualize the vascular anatomy of each tumor.All treatment decisions were made by the multidisciplinary teams at both centers. Systemic chemotherapy was routinely recommended as primary therapy to patients with a BR or LA tumor, and was also generally recommended to all patients with a resectable tumor at MD Anderson Cancer Center. At Verona University Hospital, it was administered more selectively to such patients, primarily to those with a radiographic interface between their tumor and superior mesenteric vein or portal vein. However, at both institutions, systemic chemotherapy was routinely administered to patients with a resectable tumor and one or more of the following: (1) imaging studies demonstrating findings suspicious but not diagnostic for extrapancreatic disease; (2) a depressed performance status or significant comorbidity profile; and (3) a carbohydrate antigen (CA) 19-9 level (in the absence of jaundice) suggestive of disseminated cancer.Systemic chemotherapy consisting of FOLFIRINOX and/or GA was administered to all evaluated patients. (Chemo)radiation therapy was administered selectively at both centers and was delivered more frequently to patients with tumors who had any degree of mesenteric vascular involvement. Radiation therapy consisted of external-beam radiation therapy  with concurrent administration of 5-fluorouracil, capecitabine, or gemcitabine or stereotactic body radiation therapy (SBRT) delivered over 5 days without a radiosensitizer. Within 8 weeks after completing preoperative therapy, the patients’ disease was clinically and radiographically restaged. Patients without evidence of disease progression and with adequate performance statuses were considered for surgical resection. Pancreatoduodenectomy, distal pancreatectomy, or total pancreatectomy was performed using standardized techniques at both centers.10Gastrointestinal pathologists used standardized protocols to evaluate all surgical specimens.CT images of all patients were reviewed for this study by two research associates (GP and AC) blinded to treatments and outcomes.To evaluate the cumulative response to preoperative therapy, the images of each patient obtained before preoperative therapy and before surgery were compared. In addition, the radiographic changes associated with chemotherapy and (chemo)radiation were assessed independently. To evaluate the changes associated with chemotherapy alone, the pretreatment images were compared with the post-chemotherapy images (obtained prior to surgery or prior to radiation therapy). To evaluate the changes associated with radiation therapy alone, the post-chemotherapy images were compared with the preoperative images Fig. .Fig. 1RaL) and shortest (W) axial diameters and the craniocaudal diameter (H), and the volume of each tumor was calculated using the formula for a typical ellipsoid: volume = π/6 × L × W × H.Each tumor was staged radiographically as potentially resectable, BR, or LA according to two different classification systems—the MD Anderson Classification and the National Comprehensive Cancer Network (NCCN) guidelines.Serum CA 19-9 levels  were measured prior to and following treatment. Patients in whom the CA 19-9 level was lower than 1 U/mL both prior to and following treatment were defined as nonproducers.t test if normally distributed and a nonparametric Mann–Whitney U test if not. Categorical variables were compared using a Pearson Chi square test (or Fisher’s exact test when appropriate). Statistical analyses were performed using the SPSS software program version 24.0 , and p values < 0.05 were considered significant. All p values were two-sided.Continuous data were expressed as medians and ranges, whereas categorical data were expressed as frequencies and percentages. Continuous variables were compared using a We analyzed a total of 226 patients who underwent surgery following at least three cycles of FOLFIRINOX or GA with or without subsequent (chemo)radiation. Clinical characteristics of the study population are listed in Table Changes in radiographic characteristics that occurred in association with preoperative chemotherapy and (chemo)radiation are reported in Table The MD Anderson radiographic stage of 197 tumors (87%) did not change following the administration of preoperative therapy, whereas five tumors 2%) were upstaged due to local progression. The tumors of 24/82 patients (29%) who had BR or LA cancer were downstaged after preoperative therapy. We observed radiographic downstaging of BR and LA tumors after chemotherapy in 20 patients (24%) and after subsequent (chemo)radiation in 4 patients (6%) [Fig. % were upp = 0.5).According to RECIST 1.1, 151 (67%), 70 (31%), and 5 (2%) patients had SD, PR, and PD, respectively, after preoperative therapy. We observed no significant differences in responses assessed using RECIST 1.1 after (chemo)radiation or chemotherapy (p = 0.7); however, the median tumor volume loss after chemotherapy was significantly greater than that after (chemo)radiation . Notably, volume loss following chemotherapy was similar between patients who received only chemotherapy and those who received chemotherapy followed by (chemo)radiation . In addition, there was no difference in terms of median volume loss between 50.4 Gy and 30 Gy (chemo)radiation , and between ‘conventional’ (chemo)radiation and SBRT .The volume of 180 primary tumors (80%) decreased after preoperative therapy. Patients experienced a median baseline tumor volume loss of 36% over the entire preoperative treatment course. Loss of volume was equally common following chemotherapy and (chemo)radiation  or a local tumor anatomy that precluded resection . Patients who did not undergo resection were similar to those who did in terms of treatment response (according to RECIST 1.1), decrease in tumor volume, increase in tumor attenuation, changes in tumor-vessel interfaces, and other potential indirect radiographic signs of tumor response to preoperative therapy  [Table p = 0.04).After receiving chemotherapy with or without (chemo)radiation, 193 patients (85%) underwent resection of their primary tumors and regional lymph nodes, whereas 33 (15%) did not due to intraoperative identification of metastases radiation with each putative metric of treatment response. We found a greater magnitude of tumor volume loss and a higher incidence of radiographic downstaging following chemotherapy than following subsequent (chemo)radiation. Specifically, the tumors in only 6% of the patients with LA or BR cancer were further downstaged by (chemo)radiation administered after chemotherapy Fig. . AlthougIn a secondary analysis of the CT scans of these patients, we could not identify any radiographic measures that could be reliably used to indicate occult metastases in patients with otherwise localized cancer. Therefore, at present, surgery with curative intent is a reasonable approach for physiologically robust patients without radiographic evidence of disease progression during preoperative therapy. Novel biomarkers may help avoid unnecessary surgical exploration or even pancreatectomy in patients unlikely to benefit from surgery, and are clearly needed. In the meantime, proposed strategies to reduce the rate of unnecessary laparotomies due to unanticipated metastases include staging laparoscopy prior to laparotomy (at least in patients with CA 19-9 levels or radiographic findings suspect for disseminated disease) and/or routine preoperative MRI.21The primary limitation of this study was that all patients underwent laparoscopy or laparotomy and thus were already selected on the basis of their radiographic response. Certainly, some patients not included had radiographically evident metastatic disease during preoperative therapy. However, the primary findings of this study, i.e. that PDAC in fewer than one-third of patients was downstaged after treatment with FOLFIRINOX or GA with or without (chemoradiation) and that radiographic measures of response were generally more robust following chemotherapy than after (chemo)radiation, would only be more pronounced if we had included patients with metastatic progression. In addition, we evaluated radiographic response to (chemo)radiation only in patients who had already received preoperative chemotherapy. The responses to (chemo)radiation delivered de novo may have been more robust; however, (chemo)radiation is typically delivered to patients with localized PDAC only after induction chemotherapy to improve patient selection for this local treatment modality.We found that despite the use of FOLFIRINOX or GA, radiographic downstaging of PDAC occurred in less than one-third of patients administered systemic chemotherapy prior to anticipated pancreatectomy. Furthermore, the magnitude of tumor volume loss was greater and the incidence of tumor downstaging was higher following chemotherapy than following subsequent (chemo)radiation. These findings should be used to set expectations with respect to the possible effects of chemotherapy and (chemo)radiation in PDAC patients prior to pancreatectomy.


Alterations in hemodialysis patients’ serum trace metals have been documented. Early studies addressing associations levels of serum trace metals with erythropoietic responses and/or hematocrit generated mixed results. These studies were conducted prior to current approaches for erythropoiesis stimulating agent (ESA) drug dosing guidelines or without consideration of inflammation markers (e.g. hepcidin) important for regulation of iron availability. This study sought to determine if the serum trace metal concentrations of incident or chronic hemodialysis patients associated with the observed ESA response variability and with consideration to ESA dose response, hepcidin, and high sensitivity C-reactive protein levels. Inductively-coupled plasma-mass spectrometry was used to measure 14 serum trace metals in 29 incident and 79 prevalent dialysis patients recruited prospectively. We compared these data to three measures of ESA dose response, sex, and dialysis incidence versus dialysis prevalence. Hemoglobin was negatively associated with ESA dose and cadmium while positively associated with antimony, arsenic and lead. ESA dose was negatively associated with achieved hemoglobin and vanadium while positively associated with arsenic. ESA response was positively associated with arsenic. Vanadium, nickel, cadmium, and tin were increased in prevalent patients. Manganese was increased in incident patients. Vanadium, nickel, and arsenic increased with time on dialysis while manganese decreased. Changes in vanadium and manganese were largest and appeared to have some effect on anemia. Incident and prevalent patients’ chromium and antimony levels exceeded established accepted upper limits of normal. In practice the method removes both low and medium molecular weight compounds including normal biologics as well as uremic toxins. Many factors affect the equilibration of solutes across the dialyzer membrane including concentration, size, charge, and compartmentalization. Chronic hemodialysis as a medical tool has been used as supportive care for individuals who have lost the function of their kidneys. The largest fraction of dialysis care is provided to adults 65 years old or greater and in this population HD is associated with an average life expectancy of 5 years2. Multiple co-morbid conditions contribute to the reduced life expectancy including infection, cardiovascular disease, hypertension, hyperlipidemia, diabetes, and anemia. Protein energy wasting is a prevalent finding in maintenance hemodialysis patents and may be associated with diminished levels of iron, selenium, vitamin C and other micronutrients. These data suggest that micronutrient loss including trace metals may contribute significantly to hemodialysis patient comorbidities and mortality. Thus we hypothesize an improved understanding of associations between trace metals and the anemia management may support identification of salutatory trace metals supporting a longer and an improved quality of life8.Hemodialysis (HD) is a blood purification method intended to remove metabolic waste products (‘uremic toxins’) by primarily equilibrating the composition between the patients sera (higher concentrations) and the dialysate (lower concentrations) across a semipermeable dialyzer membrane9. The goal of HD is to clear low molecular weight uremic toxins but it also affects beneficial low molecular weight substances including amino acids, salts, vitamins and serum trace minerals10. In fact, clearance of non-protein bound, low molecular weight solutes tends to be enhanced unless specifically supplemented into the dialysate fluid. Although the generation of water for dialysate purposes is heavily regulated to minimize microbial toxins, the potential still remains for trace metal contamination resulting in toxicity to the HD patients14. An example would be aluminum toxicity associated with contamination of dialysate water and/or oral phosphate binders resulting in softening of the bones , anemia, or dialysis encephalopathy in hemodialysis patients16.HD patients are medically complex with many suffering from metabolic syndrome, diabetes and dyslipidemia while others suffering from protein-energy wasting syndrome, loss of appetite and reduced intake of nutrients such as trace minerals17. The levels of selenium, zinc, and manganese were lower in hemodialysis patients than controls. These same authors recently reported on 1278 patients with 2-year prospective follow up measuring 25 trace elements and outcomes including death, cardiovascular events, systemic infection and hospitalization. Cardiovascular disease, the main cause of mortality in hemodialysis patients, was associated with higher zinc levels at 2 years of hemodialysis. Lower concentrations of selenium were independently associated with high risk of cardiovascular disease, all-cause hospitalization or death3. In an exploratory analysis, copper and cadmium concentrations were associated with a higher risk of death. Ari et al. report a strong positive correlation between high concentration of cadmium, lead, magnesium, and copper-to-zinc ratio with increased carotid artery intima-media thickness and hence with carotid artery atherosclerosis18.A meta-analysis of 128 studies on the trace element status of hemodialysis patients identified levels of cadmium, chromium, copper, lead and vanadium higher in hemodialysis patients than compared to normal controls19. However Prodanchuk et al. compared 17 serum trace metal concentrations in existing hemodialysis and hemofiltration patients and documented significantly higher serum levels of cadmium, cobalt, copper, and lead in hemodialysis patient serum samples20. A meta-analysis of clinical trials for the replacement of therapeutically low trace metals such as zinc via dietary supplementation has shown beneficial results. Zinc supplementation positively affected serum zinc levels as well as dietary protein intake, increased levels of the antioxidant superoxide dismutase, and decreased inflammatory markers such as C-reactive protein (CRP)22.Studies on serum trace metal levels affected by applied dialytic modality have yielded mixed results. Cross and Davenport reported no difference in observed serum zinc and selenium concentrations for maintenance hemofiltration patients that cross-over to hemodialysis24 and erythropoiesis26. Trace metals such as manganese, zinc, chromium, and selenium can interact with and regulate iron available for erythropoiesis at the levels of iron uptake, transport, and iron storage regulatory proteins27. Experimentally trace metal  intake or trace metal interactions with cognate-iron binding partners have been demonstrated to modify globin gene synthesis, erythrocyte production and/or metabolically reprogramming cellular differentiation pathways30. Lastly, zinc  supplementation may have a direct (progenitor cell differentiation) or indirectly to enhance iron availability by reduction of inflammatory modulators (CRP) of red blood cell productions (erythropoiesis)34.The kidney is responsible for the production and releasing of erythropoietin (EPO) as a function of the blood oxygen levels. The anemia of chronic kidney disease and ESKD may be associated with failed EPO production, diminished sensitivity to exogenous EPO or compounding effects of dysregulated trace metal metabolism. In the absence of the sufficient kidney function these minerals build-up in the serum as a function of dietary intake and consumption or through environmental exposure. Studies on trace metals, such as iron, copper, cobalt and zinc play essential roles in many facets of biology relevant to the complications and comorbidities associated with hemodialysis including the function of the hypoxia inducible factor-1 (HIF-1) pathway37. Patients that are hypo-responsive to EPO may be at risk for increased morbidity and mortality38. In the clinical setting of ESKD and hemodialysis there are a multitude of factors which may contribute to anemia and hypo-responsiveness to ESA including inadequate ESA dose, iron deficiency, blood loss, infection, inflammation, secondary hyperparathyroidism, hemolysis, malignancies, hematologic disorders, acquired immune-deficiency syndrome (AIDS), pregnancy, vitamin deficiency, and aluminum toxicity. The critical central role that iron maintains in erythropoiesis itself may be modified by relative concentrations of other trace metals. However other trace metal differences may exist in patients with poor response to ESAs that may be detectable in the serum.Central to the current discussion of anemia management with EPO and biosimilar drugs is the identification of those patients that respond normally to pharmacologic concentrations of EPO or erythropoiesis stimulating agents (ESAs) and those patients that do not. Many patients respond well to low doses of drug while other patients may require much larger doses41.HD may contribute to trace metal dysregulation directly via loss or gain by exchange with the dialysate. Erythropoiesis requires iron and other trace metals as important co-factors in many cellular erythropoietic processes including globin gene synthesis and maturation. As such we hypothesized that ESA responsiveness would be impacted by differing concentrations of metals. We studied both incident and prevalent patients on hemodialysis from a single dialysis provider over a six-month period of time and measured ESA dose, hemoglobin, critical markers of inflammation, and 14 trace metals. Our primary objective was to analyze the relationship between ESA response and trace metal concentration. Our secondary objectives were to examine the changes in trace metal concentration between incident and prevalent patients, and between male and female given known baseline sex-dependent differences in Hb levelsA total of 100 prevalent and 33 incident subjects were enrolled and sufficient sample was available for analysis in 108 subjects . The results for primary metals measurements have been uploaded into the metabolomics workbench of the NIH Common Fund Metabolomics Program Data Repository and Coordinating Center (DRCC) as study ST000565. The demographic information on the population are shown in Table To determine if there were a linear relationship of trace metals and anemia metrics, a multivariate regression of prior month ESA dose, Hb concentration and 6-month average ER and all of the trace metal concentrations was conducted. Starting with all trace metals, the final regression model was determined after sequentially removing individual trace metals that did not contribute significantly. The model results are presented in Figs. 40. Therefore an analysis of variance for trace metal concentrations by prevalence  and sex  was performed. As shown in Table 46 are shown in Fig. The results of the inductively coupled plasma-mass spectrometry (ICP-MS) analysis for each measured metal in prevalent and incident subjects are shown in Fig. 48. Since trace metals are important co-factors in many cellular processes including erythropoiesis and dialysis patients can have altered concentrations of trace metals we looked to see if ESA responsiveness was associated by differing concentrations of metals. We examined at three measures of ESA response: ESA dose, Hb achieved, and a factor that averages these measures over a 6 month period, the ER49. Of the trace metals tested, arsenic was the only one to be positively correlated within all three linear regression models for measures of ESA response. Arsenic was infrequently detected in the patient samples and at non-toxic concentrations that approached the maximum contaminant level for drinking water. Arsenic may be encountered in either inorganic or organic forms and with different valence states; arsenites/As(III) and arsenates/As(V)50. While our approach did not differentiate the serum samples arsenic valence it is known that arsenic trioxide (arsenite) impacts erythropoiesis and has been used to treat several blood malignancies52. Zhang et al. have demonstrated a biphasic effect of low dose arsenic trioxide on red blood cell and Hb expression. In studies using CD34 + cells isolated from chronic myeloid leukemia patient bone marrow, sub-toxic levels of arsenic trioxide enhanced or primed erythroid colony forming activity. Additionally the same study demonstrated sub-toxic levels of arsenic trioxide inhibited globin gene expression in K562 cells, a chronic myelogenous leukemia patient derived erythroid-myeloid precursor cell line53. While the effect of arsenic appears biphasic in cell culture this cannot be assumed to be the case in the setting of chronic hemodialysis. Hence the positive correlation of arsenic with the anemia end-points  may reflect both the combined effects of sub-toxic levels of arsenic and increased erythroid colony formation with increased need for ESA dosing to achieve Hb target levels.Anemia as well as trace metal derangements are common complications of advanced CKD and ESKD54. Lead poisoning interferes with the heme synthesis pathway and can at high enough levels cause iron deficiency 55. While there is no safe level of lead in the body, the maximum lead concentration observed in our study was 2.08 µg/dL. This is well below the action level of 5 µg/dL level if detect in children or the concentration of 10–25 µg/dL in adults indicating elevated exposure levels have been encountered. The positive correlation of lead with Hb may reflect ESA dosing overcoming lead impairment of erythropoiesis.Lead was detected infrequently in patient samples and when observed it was noted the mean concentration decreased from incident to prevalent patients. Similar to arsenic the relative lead concentrations were below what would be considered toxic. Lead was found to be a final component of the linear regression models associated with prior ESA dose and Hb concentration but not ER. Lead is a major public health issue and can cause adverse hematological effects in exposed workers56. Manganese and iron uptake shares a common transporter for dietary uptake (DMT1) and storage (transferrin)57. Blood manganese and Hb values both positively correlate with renal function59. Functional iron deficiency defined as insufficient iron incorporation into erythroid precursors despite adequate iron levels is a frequent cause of anemia and often may be associated with high manganese levels60. Hence the relationship between manganese concentration, Hb and anemia is complicated. Our data showed a significant reduction in manganese from incident (3.0 µg/L) to prevalent (1.1 µg/L) patients. Multivariate linear regression identified six features that best explained the relationship of Hb to observed trace metal concentrations and ESA dose includes manganese. It is difficult to extrapolate the effects of single variables from logistic regression models but these data may point to trace metal interactions that collectively associated with lower Hb values or diminished ER.Manganese is an abundant trace metal with many important extra- and intra-cellular functions61. Pan et al. report from the National Health and Nutrition Examination Survey (NHANES) data on the association between trace elements  and Hb levels in patient with normal renal function or CKD59. Regardless of renal function status, Hb concentrations were associated positively with cadmium and negatively with copper. Bayhan et al. report that increased but sub-toxic levels of blood cadmium associate with ineffective erythropoiesis in thalassemia major, thalassemia intermedia and congenital dyserythropoietic anemia 62. These data suggest that sub-toxic levels of blood cadmium in individuals with a chronic hypoxic state are susceptible to the contribution of cadmium toward ineffective erythropoiesis.The final trace metals found significant in the tests on anemia were cadmium, antimony, vanadium, and chromium. All of these metals are found to be higher in multiple cohorts of dialysis patients 63 or direct effects to diminished bone marrow populations of cells in the erythroid lineage, larger circulating populations of immature erythrocytes, and/or smaller populations of mature erythrocytes concomitant with diminished oxygen carrying capacity 65.In our study, cadmium was observed in a majority of patient samples and the median concentrations were higher in prevalent patients. While the cadmium levels were detected below the threshold suggesting toxicity, a regression analysis identified a moderate negative association of cadmium with Hb concentrations Table . Publish20. The biochemistry of vanadate and vanadyl ions is complex. The vanadium oxoanions can interfere with the functions of enzymes such as tyrosine kinases, cAMP-dependent kinases, ceramidases, and endothelial cell nitric oxide synthases71. As such, vanadate is commonly included in sample lysis buffer recipes to inhibit tyrosine phosphatases. Collectively the presence of vanadium compounds presence results in altered enzyme function and second messenger signaling74. It is noteworthy that vanadium compounds are actively taken up by red blood cells to compete with iron binding to hemoglobin76, influences HIF-1α signaling77 and also can lead to increased erythrocyte death78 with shorten red blood cell life span..Vanadium showed the greatest accumulation between incident and prevalent patients and between patients on the lowest and highest ESA dose. Vanadium accumulates biologically through inhalation and ingestion as either the vanadate ion (V(V)) or vanadyl ion (V(IV)). Vanadium accumulation in dialysis patient blood has been demonstratedSignificant differences exist in trace metal concentrations between incident and prevalent patients Fig. . Vanadiu79.The largest changes between incident and prevalent patients were in vanadium and manganese concentrations. The impact of vanadium accumulation on enzymatic processes and the reported relationship between manganese and anemia may explain the observation in the group of patients studied that a greater proportion of subjects requiring an ESA in the prevalent group and that anemia might worsen as dialysis vintage increases. We have observed the increase in ESA requirements in a separate population of patients that were followed over a 4 year period what ESA requirements increased by approximately 400 units/week/year as dialysis vintage increased from 4.6 to 7.3 years3. Our study examined total serum trace metal levels but not total blood trace metals that would be inclusive of the cellular blood compartment. Some metals exist as organo-metallic compounds  such as Sn(II) or the more stable Sn(IV) whose toxicities are associated with the specific organic moiety, or as oxo-acids whose toxicity is associated with a specific oxidation state (e.g. arsenic or chromium)82. Therefore, absent sample handling to stabilize and separate these charge forms prior to ICP-MS  we cannot report if the observed associations are with a specific organo-metalloid or charge state (e.g. vanadate versus vanadyl ion). As shown in the Pearson Correlation table  as well as chronic dialysis patients. Additional strengths are the use high sensitivity clinical assays that phenotype for important markers of inflammation (hsCRP) and iron availability (hepcidin) and lastly a dialysis population using dynamic ESA dosing protocols delivered to maintain a target Hb value. Limitations of the current study included confounding factors that cannot be addressed due the cross-sectional nature of this study. These confounders significantly included a lack of dietary information for vitamin or mineral supplement usage that might have contributed to observed trace metals, lack of dialysate water sampling, point of source water measurements from patient homes, and due to the study vintage a loss of patients for follow-up due to the low life expectancy of hemodialysis patients. Also the trace metals analysis constrained to a subset of trace metals n = 14) that did not include some  previously shown to be associated with death two years after dialysis initiation4 that diIn conclusion, we report on 14 trace metals and their association with anemia treated with an ESA in both incident and prevalent dialysis patients. Linear regression modeling identified groups of metals that together were significantly associated Hb, ESA dose, and the Hb-dose averaged response of patients (ER). Hb was negatively associated with cadmium while positively associated with antimony arsenic and lead; (b) e amount of ESA dose was negatively associated with vanadium while positively associated with arsenic. The Hb-dose averaged response of patients (‘ER’) to ESAs most strongly positively associated with arsenic. No sex-dependent trace metal differences were observed in incident or prevalent populations. Other observations included significant accumulation of vanadium and reduction in manganese concentration as a function of incident versus prevalent status. These data support future studies for a broader comprehensive metabolomic study into anemia of ESKD and ESA hypo-response with a goal to define targets for interventional studies addressing ESA response and anemia management in hemodialysis populations.The research protocol conformed to the Declaration of Helsinki and was approved by the Universities of Louisville and Washington Institutional Review Board. Subjects were enrolled following an informed consent process and all data were de-identified. One hundred prevalent  and 33 incident  dialysis dependent hemodialysis subjects were enrolled at the University of Washington. Subjects donated 20 mL of blood, processed as both plasma and serum, and stored as 0.5 mL aliquots at − 70∘ until analyzed. These samples were collected using the same protocol and the same serum and plasma collection tubes . Additionally, information on age, gender, race, and hemodialysis vintage as well as up to 6 months of data on EPO  dose (units), hemoglobin , ferritin (µg/L), and transferrin saturation (TSAT) were collected.Serum samples (0.5 mL) were digested with a mixture of concentrated nitric acid, hydrochloric acid, and hydrogen peroxide, then analyzed alongside calibration standards, blanks, and appropriate quality control samples to evaluate assay performance. Elements (antimony (Sb), arsenic (As), cadmium (Cd), chromium (Cr), cobalt (Co), copper (Cu), lead (Pb), manganese (Mn), molybdenum (Mo), nickel (Ni), selenium (Se) , tin (Sn), vanadium (V) and zinc (Zn)) were monitored using a Thermo Element 2 high resolution sector field ICP-MS in multiple resolution modes to provide optimal analytical sensitivity and interference resolution for each element. See The hepcidin-25 EIA kit S-1337 was purchased from Peninsula Laboratories . The hepcidin-25 standard was serially diluted. Serum samples were diluted 1:50. Samples and standards were brought to 50 μL and added to an antiserum pre-coated plated for competitive immunoassay according to kit instructions. A uniform volume of 25 μL biotinylated tracer was added to each well of the plate after 2 h and incubated for 1 additional. Captured biotinylated tracer was subsequently bound by streptavidin-conjugated horseradish peroxidase. A color correlated to the sample hepcidin concentrations was produced after TMB substrate was added. The absorbance was read at 450 nm and a four-parameter polynomial regression was used for the standard curve fitting. The coefficient of variation (CV) using a hepcidin-25 concentration of 1.56 ng/mL was 3.49% intra-assay and 3.43% inter-assay. All samples were measured in duplicates and the valid OD was kept correspondent to hepcidin standard between 0.78 ng/mL and 6.25 ng/mL for best reproducibility .CRP values were determined using the high sensitivity CRP (hsCRP) latex immunoturbidometric assay kit from Roche Diagnostics . Data were collected on an Roche Diagnostics Cobas Integra 800 using patient serum samples according to the manufacturer’s instructions. Briefly human CRP agglutinates with latex particles coated with monoclonal anti-CRP antibodies. The precipitate is determined turbidimetrically at 552 nm. All samples were measured in duplicates and the valid OD was kept correspondent to the linear range of the hsCRP standards .Statistical analysis was performed using SAS version 9.4 and SPSS version 24. Measured serum concentrations of metals below the limit of detection (LOD) were imputed as ½ LOD if < 25% of the observations were below the LOD and then transformed using the log of the concentration base 2 to address non-normality in the dataset. For metals with a large number of missing values (> 25% of observations below LOD), comparisons of abundances were made based on detection and non-detection events using a chi-square test due to non-normal in the event distribution. Total Epoetin alfa dose (U) was categorized into three groups: 0; 1000 to 29,200; and 30,000 to 136,200. Erythropoietin response (ER) was normalized to achieved hemoglobin over 6 months as ESA Dose/1000 × Hb and categorized into three groups: 0; 0.02 to 2.55; and 2.60 to 11.81. Comparison of means between incident and prevalent hemodialysis groups was performed by t-test for metals < 25% LOD and chi-square for > 25% LOD . Metals were included in Pearson correlation calculations when observed in at least 75% of all samples. The effects sex and dialysis prevalence was tested using two-way ANOVA to determine main effects and interaction. The effect of metal concentration on erythropoietin response was tested using multivariate regression analysis using backward selection of the metal concentration vs. ESA dose, ER, and current Hb. Regression analysis was performed as linear regression. Post hoc analysis was done by one-way analysis of variance using Student–Newman–Keuls.Supplementary Information.


GCK) variants causing mild fasting hyperglycemia are at risk of macrosomia if they do not inherit the variant. Conversely, babies who inherit a pathogenic hepatocyte nuclear factor 4a (HNF4A) diabetes variant are at increased risk of high birth weight. Noninvasive fetal genotyping for maternal pathogenic variants would inform pregnancy management.Babies of women with heterozygous pathogenic glucokinase (GCK or HNF4A variant and to determine fetal fraction by measurement of informative maternal and paternal variants. Droplet numbers positive for the reference/ alternate allele together with the fetal fraction were used in a Bayesian analysis to derive probability for the fetal genotype. The babies’ genotypes were ascertained postnatally by Sanger sequencing.Droplet digital PCR was used to quantify reference and variant alleles in cell-free DNA extracted from blood from 38 pregnant women heterozygous for a GCKor HNF4A variants were validated for testing in all 38 pregnancies. Fetal fraction of ≥2% was demonstrated in at least 1 cell-free DNA sample from 33 pregnancies. A threshold of ≥0.95 for calling homozygous reference genotypes and ≤0.05 for heterozygous fetal genotypes allowed correct genotype calls for all 33 pregnancies with no falsepositive results. In 30 of 33 pregnancies, a result was obtained from a single blood sample.Droplet digital PCR assays for This assay can be used to identify pregnancies at risk of macrosomia due to maternal monogenic diabetes variants. Prenatal diagnostic testing has been transformed through the discovery in 1997 of cell-free fetal DNA (cffDNA) in maternal plasma during pregnancy , fetal sNIPT for maternally inherited variants that cause autosomal dominant disorders presents a particular challenge because only a small proportion of the total cfDNA in maternal blood is derived from the fetus during early pregnancy . In suchGCK) variant causing mild fasting hyperglycemia from birth. These variants account for approximately 1% of gestational diabetes  monogenic diabetes variant from their mother are at increased risk of high birth weight. Currently, the risk and management of macrosomia is based on tight maternal glycemic control and serial ultrasound scans from 26 weeks, with the option of preterm delivery or elective cesarean or the induction of labor at 38 weeks. Detection of macrosomia by ultrasound analysis relies on measurement of fetal metrics  to derive a proxy for fetal weight by combination into ≥1 formula -funded genetic diabetes nurses, endocrinologists, or self-referral. All participants gave informed consent for testing before samples were taken, and the study was approved by the North Wales Research Ethics Committee. Sample collection was facilitated through the Genetic Beta Cell Research Bank . This process was set up to ensure effective guardianship of samples obtained during routine clinical diagnostic procedures, where consent for genetic testing included permission to store excess samples for research into improvements in diagnosis, care, and treatment of genetic diabetes.Patients with known monogenic forms of diabetes caused by single-nucleotide variants (SNVs) or 1- or 2-bp insertions or deletions (indels) in Participants had a venous blood draw of 20 mL into cell-stabilizing blood collection tubes (Streck), and plasma was separated within 7 days. Plasma was stored at –80 °C before extraction of cfDNA using the QIAamp Circulating Nucleic Acid Kit (Qiagen), with replicate extractions from the same plasma sample pooled for ddPCR analysis.Fetal genotype was determined postnatally by molecular genetic analysis of DNA extracted from cord blood (n = 26), saliva (n = 9), venous blood (n = 1), or chorionic villus sampling (n = 2). Maternal cell contamination in cord blood and chorionic villus sampling was excluded by microsatellite analysis using a PowerPlex 16 Kit (Promega) with a threshold of <5% maternal contamination.GCK or HNF4A, custom hydrolysis probe assays were designed using online tools available via the websites of Thermo Fisher Scientific and Bio-Rad Laboratories; primer and probe sequences are available on request. We initially attempted design for 34 unique SNVs or 1- or 2-bp indels in GCK or HNF4A. Two of these, for GCK variants c.1343G>T, p. and c.1175G>C, p.(Arg392Pro), either failed the suppliers’ design and manufacture processes or yielded assays that were unable to provide sufficient detection or discrimination of the target alleles; samples with these variants were excluded from the cohort. A complete list of the 32 remaining variants for which working assays were obtained is shown in GCK or HNF4A variant was then verified by testing heterozygous maternal gDNA samples carrying the appropriate variant. Of the 32 custom assays used , 31 showed the expected 50:50 allelic balance, and a single assay  showed a slight apparent bias for the reference allele  and the same number of template-free control wells as used for cfDNA samples. Raw data were collected and analyzed using QuantaSoft analysis software (Bio-Rad), with data from replicate wells merged as appropriate.ZFX or ZFY, respectively)  . For casctively)  to identctively)  and inclctively) . VariantA Bayesian Markov chain Monte Carlo (MCMC) analysis was conducted using JAGS . The modBoth ddPCR and calculation of probability for homozygous reference genotype were performed blind to the known genotype, which was determined at completion of each pregnancy.GCK or HNF4A variant and the informative SNP identified for determination of fetal fraction . In most cases (30/ 33), probability scores lay very close to upper or lower limits, indicating a high likelihood of fetal homozygosity or heterozygosity, respectively, and in these 30 samples, there was 100% concordance between the probability score and the actual genotype (p≤0.95), we carried out “follow-up” testing of a second sample from the same pregnancy. In all 3 cases, these samples yielded high probability scores concordant with the actual genotype. Furthermore, testing of all available samples from all pregnancies showed that for samples generating probability scores of ≤0.05 or ≥0.95, there was complete concordance of predicted genotype both between samples from the same pregnancy and with the actual genotype , there was considerable variation between samples at any given time point . TherefoAlthough other studies have also made use of ddPCR for NIPT, our method offers the potential advantage of providing an objective, probabilistic score for fetal genotype based on both allelic balance of the variant in question and the fetal fraction. As such, we were able make high-confidence calls in samples with fetal fraction as low as 2.5%. By comparison, the lowest fetal fraction supporting genotyping of X-linked and recessive disorders in a ddPCR-based assay was 3.6% , whereasGCKand HNF4A pathogenic or likely pathogenic variants, but larger indels (≥3bp) and copy number variants account for approximately 9.7% or 8.3% of GCK or HNF4A variants, respectively, in the Human Gene Mutation Database , which allowed design of probebased hydrolysis assays in which both alleles could be detected simultaneously. Such variants comprise the majority of reported Database . In suchase (GCK . Neverthase (GCK  and multIn summary, we describe the development of a rapid, sensitive, and precise method for the prediction of fetal genotype in maternally inherited monogenic diseases. This approach has potential applicability to the management of macrosomia risk in monogenic diabetes pregnancies and offers considerable improvement in prediction of fetal genotype compared with the current ultrasound scanning approach, which has accuracy of only 56%–72%. Further work is required to investigate the wider applicability of the ddPCR assay for the early detection of fetal genotype and the management of clinical care both during and after pregnancy in other monogenic diseases.Supplemental material is available at Clinical Chemistry onlineSupplemental material


Chronic wasting disease (CWD) is a prionopathy affecting wild and farmed cervids. This disease is endemic in North America and has been recently identified in Europe. Ante-mortem CWD tests of pre-clinical cervids may be an important tool in helping control the spread of this disease. Unfortunately, current CWD diagnostic methods are not suitable for non-tissue type samples. We reported that CWD prions can be detected in blood of pre-clinical CWD-infected white-tailed deer (WTD) with high sensitivity and specificity using the Protein Misfolding Cyclic Amplification (PMCA) assay. However, that report only included animals homozygous for codon 96G, the most common polymorphic version of the prion protein within this animal species. Here, we report CWD prion detection using blood of naturally infected WTD coding one or two copies of the PrP-96S polymorphic variant. Our results, from a blinded screening, show 100% specificity and ~ 58% sensitivity for animals harboring one 96S codon, regardless of their stage within the pre-clinical phase. Detection efficiency for PrP-96S homozygous animals was substantially lower, suggesting that this allele affect peripheral prion replication/tropism. These results provide additional information on the influence of codon 96 polymorphisms and the ability of PMCA to detect CWD in the blood of pre-clinical WTD. Screening and containment programs aimed at halting CWD spread have been established with variable success6. Unfortunately, the lack of practical diagnostic methods has been one of the factors stopping the implementation of more efficient containment practices. At present, the gold-standard method for CWD diagnosis is the immunohistochemical (IHC) analysis of disease-associated prion protein (PrPSc) accumulation in medial retropharyngeal lymph nodes and obex . The fact that several bodily fluids and excreta from CWD infected animals carry prion infectivity9 provides potential targets for animal diagnostic testing. Our lab and others have shown that in vitro prion replication techniques (such as the protein misfolding cyclic amplification (PMCA)10 and real-time quaking induced conversion (RT-QuIC)11) have the potential for use in pre-clinical diagnosis using a wide variety of biological samples. In a previous publication12, we communicated the results of CWD prion screening using blood from a group of farm raised pre-symptomatic white-tailed deer (WTD). Our results showed 96% sensitivity at late pre-symptomatic stages, and 53% at early stages. The specificity of our assay was 100%. These results suggested that PMCA can identify a fraction of CWD positive animals.Chronic wasting disease (CWD) is the only prion disease found in free-ranging animalsC) sequence only. PrPC polymorphisms are seen in several animal species, including cervids16. In WTD, the most commonly found polymorphism occurs at position 96 where a glycine (G) is substituted for a serine (S)17. The presence of at least one S at position 96 is associated with longer incubation periods compared to animals expressing the most common  genotype18. Importantly, animals carrying PrP 96S allele(s) have been associated with lower prion shedding, suggesting lower accumulation of PrPSc in peripheral tissues compared to their wild-type counterpart20. This variable could have important implications for pre-clinical diagnosis when using peripheral tissue biopsies, bodily fluids and excreta. In this work, we evaluated the diagnostic efficiency of the PMCA technology using blood specimens collected from farmed/non-symptomatic WTD carrying one or two PrP 96S allele(s).A drawback from the study mentioned above is that data was collected using specimens from animals harboring the most common prion protein (PrP21). Procedures for animal handling were approved by the Institutional Animal Care and Use Committee at UTHealth.Blood samples from male and female WTD were collected from farmed herds depopulated due to CWD by the USDA. These samples were sent to Dr. Morales’ group for prion screening using the PMCA technology. The investigators at Dr. Morales’ lab were blinded to the identity of the samples when performing the PMCA assay. All work manipulating these samples was previously approved by the Institutional Biosafety Committee of The University of Texas Health Science Center at Houston . PMCA substrate was derived from brains of transgenic mice expressing the WTD prion protein expressing glycine at position 96 . All animals were devoid of classical CWD clinical signs  as assessed by Dr. Tracy Nichols and/or other qualified USDA personnel. These animals were sacrificed immediately after blood collection. CWD status was assessed by the presence of protease-resistant PrP aggregates by IHC in the obex and retropharyngeal lymph nodes (RPLN). CWD staging was assigned as follows: obex (+) and RPLN (+): late pre-symptomatic; obex (−) and RPLN (+): early pre-symptomatic; obex (−) and RPLN (−): non-detect. In summary, blood samples from 42 animals  were used in this study. The specific data for blood donors is displayed in Table Blood was collected from the jugular vein of chemically immobilized animals using commercially available blood collection tubes containing EDTA as anticoagulant. Specimens were frozen and stored at − 80 °C at USDA facilities . These samples were overnight-transferred to UTHealth in containers filled with ice packs. Once at UTHealth, samples were stored again at − 80 °C until use.Sc concentration by ultracentrifugation as previously described22. Briefly, the 200 µL blood aliquots were mixed with equivalent volumes of a 20% w/v sarkosyl solution (prepared in PBS) for 1 h at room temperature and then centrifuged at 100,000×g for 1 h at 4 °C. Supernatants were carefully discarded without disturbing pellets, 400 µL of PBS added to them, and centrifuged using the same protocol described above but for just 30 min. Resulting pellets were resuspended in PMCA conversion buffer and transferred to 200 µL PCR tubes.Blood specimens were thawed at room temperature and vortexed. One aliquot (10 µL) of each of these samples was directly mixed with PMCA substrate, while a second aliquot (200 µL) was processed for PrP23. Briefly, each blood sample was run in quadruplicates considering the modalities explained above: direct spiking and concentration . PMCA products were treated with 100 µg/mL proteinase K (PK) at 37 °C and constant shaking for 1 h. PK reaction was stopped by adding LDS sample buffer and exposure to 95 °C for 10 min. Later, samples were fractionated by SDS-PAGE and transferred to nitrocellulose membranes as described10. Membranes were probed with either 6H4 .Overall correct diagnosis was calculated using the following formula:  × 100. Estimated predictive values for sensitivity and specificity were calculated using the MedCalc software .The experiments listed in this manuscript did not involved animal experimentations. Blood samples from WTD were collected previously for other purposes by TN. We have USDA approval to receive and work with these samples. As substrate for PMCA, we used brains from transgenic mice expressing deer PrPOriginal files for the figures included in this article can be found at Supplemental Figure 25. For that reason, we first evaluated the efficiency of our previously published cervid PMCA system23 that utilized substrate homozygous for Glycine at codon 96 in the PRNP gene. This experiment was done as this substrate is not homologous to the PRNP polymorphisms of the WTD samples used in this study. Specifically, brain extracts from experimentally infected PrP 96GG and PrP 96GS WTD (kindly provided by Dr. Edward Hoover) were used to compare in vitro amplification rates. After normalizing the amount of PK-resistant PrP in both inocula by western blot, serial dilutions in PMCA substrate were performed. The PMCA reaction using the PrP 96GG inoculum provided amplification up to 1 × 10−6–1 × 10−7 dilutions in the first PMCA round. At the third PMCA round, positive prion replication was reproducibly observed up to 1 × 10−11 dilution for this particular inoculum  late pre-symptomatic and 19 (61%) early pre-symptomatic cases  direct spiking of blood in PMCA substrate, or (ii) pellets derived from the concentration of larger blood volumes Fig. . From thante-mortem diagnostic assays would be highly useful in identifying early cases of CWD, providing a useful tool for disease management. The development of in vitro prion conversion assays (PMCA10 and RT-QuIC11) provides hope towards the development of ante-mortem CWD tests for easily accessible samples such as blood, feces, urine and saliva28. Additionally, these techniques could be utilized to detect CWD prions longitudinally in experimentally infected WTD using the above mentioned sample types. Blood is an ideal sample type for several reasons: (i) it provides the lowest chances of cross-contamination due to the aseptic nature of collection, (ii) it is routinely taken for other testing on farmed animals, and (iii) it is known to contain seeding competent prions in experimental and natural conditions30.CWD is problematic due to its deadly outcome and its ability to efficiently spread across wild and farmed cervid populations. Sensitive 31. Our previously published data12 show that we can detect CWD prions, to various degrees, in blood of both early and late pre-symptomatic WTD. That report focused on WTD that were G homozygous at PRNP codon 9620. We described a mean correct diagnostic identification of 80.6% in animals known to be CWD positive by IHC, including 100% specificity, 95.9% sensitivity in late pre-symptomatic animals, and 52.9% in early pre-symptomatic animals12. However, WTD have different PRNP polymorphisms at codon 96, where GG is considered to be the wild type, and GS and SS animals are less common20. Several reports suggest that presence of S at position 96 can modulate susceptibility to prion infection20, as well as peripheral replication and shedding of infectious particles20. In that sense, polymorphic variations in the prion protein may greatly affect blood-based diagnosis of field samples.Previous reports show that prion-seeding activity and PrP deposition can be identified in a variety of biological samples from experimentally infected WTD shortly after infectionPRNP 96S homozygous and heterozygous WTD. First, we tested whether our WTD PMCA (utilizing PrP 96G susbtrate) was able to replicate CWD prions from animals carrying at least one 96S allele. Our results . Unfortunately, accurate diagnosis for the late pre-symptomatic PrP 96GS deer reached a similar level of accuracy (58.3%) when compared to subjects at the early pre-symptomatic phase. These values contrasted with the ones found for the PrP 96GG cohort where correct diagnosis at late pre-symptomatic stages reached 95.9%12. If we consider that just 1 out of 5 (20%) of the CWD-infected PrP 96SS deer was accurately diagnosed, we can assume that the PrP 96S polymorphism modulates the presence of CWD prions in peripheral compartments, such as blood, in a dose dependent manner, especially at late stages of the CWD incubation periods. In addition, our data suggest that the presence of CWD prions in peripheral compartments reach their intrinsic maximum levels early during the incubation periods when the PrP 96S allele is present. Future research using controlled experimental conditions may confirm or refute the previously mentioned assumption.Considering the results described above, we tested blood samples derived from WTD carrying one or two PrP 96S alleles for their presence of seeding competent CWD prions. The overall diagnostic power for this new WTD cohort substantially decreased when compared to a similar population of animals homozygous for the PrP 96G haplotype12, some of the tested samples did not result in positive PMCA detection all the times they were tested. In that sense, our current protocol require several replicates in order to provide accurate results. Additional tests assessing for the reproducibility of our assays were not possible due to the limited amount of sample available. Nevertheless, several optimization studies are currently ongoing in our laboratory using other blood sources and biological specimens. One explanation for the lower detection efficiency in samples from deer carrying at least one PRNP 96S allele may be found in potential incompatibilities between the PrPSc CWD inocula and PMCA substrates. Compelling evidence in several animal species, including WTD, show that polymorphic variation in the prion protein may modulate the susceptibility of disease transmission34. Although the use of PrP 96GG PMCA substrate may have had an effect on our results, the data presented in Fig. Sc particles present in brain and blood of infected animals. In that line, the concentration of infectious prion proteins present in peripheral versus brain compartments may be different between PrP 96GG and PrP 96GS animals and could have contributed to the lower diagnostic sensitivity observed in this study. If we additionally consider the possibility that these animals are naturally infected with different strains of the CWD agent  the scenario becomes even more complex. Studies in these directions may help to not only further our understanding on natural CWD transmission, but also contribute in refining diagnostic methods.Similarly as described in our previous report29 for different polymorphic groups may also help not only to improve detection efficiency, but also to provide information on peripheral prion replication for WTD harboring different PrP polymorphic variants. In addition, bioassays would further confirm whether prion infectivity in blood is correlated with in vitro prion detection. Nevertheless, this data suggest that the PRNP codon 96 polymorphisms in WTD influence the ability to detect CWD in blood samples and needs to be considered in advance when pursuing ante-mortem diagnostic tests.Our results suggest that blood detection using the current PMCA protocol is limited, especially in early stages of the CWD incubation period. Nevertheless, practices like repeated sampling at different times and improvements in the PMCA protocol (at the sample processing or the prion replication stages), enrichment of certain blood components , testing PrP polymorphic -specific PMCA substrates, among others, may improve the numbers presented in this article. Another limitation of this study involves the limited sample size interrogated by PMCA. This is more pronounced for the PrP 96 SS samples as just 5 specimens comprising only the early pre-symptomatic stage were tested. In that sense, we acknowledge that further studies utilizing larger cohorts from all polymorphic groups and using complementary techniques  are needed to confirm our findings. The evaluation of CWD-prions content in different blood fractionsSupplementary Information.


The neuropeptide Calcitonin Gene-Related Peptide (CGRP) is a 37-amino acid peptide, with a wide-range of biological activities including vasodilation , neurogeEvidence that CGRPα may have dedicated function in the pancreas comes from a wide number of early studies demonstrating a potent role in antagonizing insulin release from pancreatic β-cells –16. In tRecently, monoclonal antibodies have been developed against CGRP to treat migraines and effectively reduce the frequency of attacks in chronic and episodic migraine patients . This pedb mutation (db/db) lack the leptin receptor and display hyperphagia, obesity and type 2 diabetes. In the C57BL/6J background, these mice manifest transient hyperglycemia, pancreatic β cell hypertrophy and hyperinsulinemia [ob mutation), which lack leptin and develop hyperglycemia and β cell hypertrophy. In accordance with a β cell-dependent inhibition of insulin release by the CGRP peptide, monoclonal CGRP therapy failed to restore normoglycemia in streptozotocyn-mediated loss of pancreatic islets. Direct evidence of CGRP ability to block GSIS was provided in functional assays to measure GSIS in primary mouse islets, where recombinant CGRP peptide blocked GSIS compared to vehicle treatment.Earlier findings highlighting the ability of CGRP to reduce insulin secretion are further supported by data from the recent report of Halloran et al. demonstrating that monoclonal therapy against CGRP specifically improved glucose tolerance and insulin resistance in monogenic models of diabetes in mice . Mice houlinemia . Mechanidb and wild-type BL6 mice. β-cell hypertrophy is highly dependent on the mouse strain and intervention performed. It is well established that weight gain due to high fat diet feeding leads to increase insulin secretion combined with fast expansion of β-cell mass to ramp-up insulin production [db glycemia but had minor effects on body weight (unpublished observations). The Leprdb mutation leads to different forms of diabetes depending on the animal’s genetic background and is characterized by severe depletion of the pancreatic β-cells in the BKS mice and a more severe diabetic outcome, whereas compensatory hyperplasia is seen in BL6 mice together with increased survival [Calcrl) and receptor activity-modifying protein 1 (Ramp1), which encode for the CGRP receptor, are enhanced in isolated Leprdb islets. The presence of a maladaptive CGRP-dependent signaling in response to weight gain, aging or other conditions associated with impaired insulin secretion from β-cells could promote hyperglycemia [A key observation in the study by Halloran et al. was the inability of the CGRP mAb to ameliorate glucose tolerance and insulin sensitivity in mice fed a high fat diet and manifesting diet-induced obesity. This drastic difference in response, compared with the monogenic models of diabetes, may arise from divergent pancreatic islet plasticity between Leproduction ,27. β-ceoduction . Interessurvival . The abiglycemia ,29–31. MAdditionally, the peripheral mechanism of action of CGRP mAb to reduce glucose levels and weight gain may be due to combined modulation of GSIS as well as loss of adiposity in the mice. High fat feeding-dependent weight gain and increased adiposity was significantly reduced upon CGRP mAb treatment. Halloran et al. provide further evidence that CGRP modulates adipose tissue energy utilization in accordance with a previous study demonstrating a causal link between CGRP and lipolysis in soleus muscle . Gene exCan monoclonal antibodies against CGRP be considered in the treatment of type 2 diabetes? This prospect needs to be considered in the context of the pharmacokinetics of CGRP mAbs and their potential side effects on liver and cardiovascular physiology. Blocking CGRP in migraine patients is well tolerated, but the abundant presence of CGRP receptors in the vasculature may pose a risk for subjects presenting comorbidities, such as type 2 diabetes and cardiovascular diseases. Initial studies on inhibiting the CGRP pathway with small molecules induced liver toxicity, but mAbs do not produce toxic metabolites ,33. The Despite the theoretical harmful inhibition of vasodilation due to CGRP inhibition, no cardiovascular concerns have yet been disclosed with any of these mAbs. Remarkably, CGRP-positive perivascular nerves innervate mostly small blood vessels, and are weakly present around epicardial coronary veins of the heart ,36. CGRPAn additional drawback of antibody therapies is the risk of developing antidrug antibodies, which impairs efficacy. Indeed, antidrug antibodies immunogenicity was detected with all four antibodies but these did not seem to affect efficacy ,40,41. NFinally, when contemplating a new medication to a disease with already many therapeutic options, one must evaluate if the CGRP mAb could provide any benefit to already existing drugs. Many pharmaceutical options are available to lower blood glucose and achieve their effects through successful modulation of one or several biological pathways leading to hyperglycemia. Recognized molecular targets for type 2 diabetes treatment include elevating insulin secretion from pancreatic β-cells; increasing glucagon secretion from pancreatic α cells; boosting liver glucose production; management of brain insulin resistance; increasing lipolysis; enhancing renal glucose reabsorption; lowering incretin effect in the small intestine; and mitigating the impaired or diminished glucose uptake in peripheral tissues such as skeletal muscle, adipose tissue and liver . AccordiOut of the major classes of oral antidiabetic medication, drugs capable of overlapping action are incretin mimetics, such as glucagon-like peptide (GLP-1) and its analogs . The GLPTaken together, the current evidence supports the use of CGRP mAb in pre-clinical model of type 2 diabetes, with successful management of blood glucose in animal models of monogenic diabetes. Further studies are necessary to evaluate the ability of this treatment to lower blood glucose in human type 2 diabetes. In particular, it will be fundamental to clarify the significance of this treatment in cardiovascular complications and potential side-effects that may arise in diabetic subjects. Type 2 diabetes mellitus is a chronic condition with multiple treatment options, combined with lifestyle interventions. Improving treatment efficiency, cost and frequency of administration is a priority with the number of patients steadily increasing world-wide.


Hair cells are sensitive to many insults including environmental toxins such as heavy metals. We show here that cadmium can consistently kill hair cells of the zebrafish lateral line. Disrupting hair cell mechanotransduction genetically or pharmacologically significantly reduces the amount of hair cell death seen in response to cadmium, suggesting a role for mechanotransduction in this cell death process, possibly as a means for cadmium uptake into the cells. Likewise, when looking at multiple cilia-associated gene mutants that have previously been shown to be resistant to aminoglycoside-induced hair cell death, resistance to cadmium-induced hair cell death is only seen in those with mechanotransduction defects. In contrast to what was seen with mechanotransduction, significant protection was not consistently seen from other ions previously shown to compete for cadmium uptake into cells or tissue including zinc and copper. These results show that functional mechanotransduction activity is playing a significant role in cadmium-induced hair cell death. Hearing loss is one of the most common sensory disorders affecting upwards of 20% of Americans over the age of 12  larvae. Experiments were carried out with either *AB wild type zebrafish, cdh23tj264  4, 150 μM KH2PO4, 42 μM Na2HPO4, 1 mM CaCl2, 500 μM KCl, 15 mM NaCl, and 714 μM NaHCO3. They were housed in an incubator maintained at 28.5°C. The Lafayette College or the University of Washington Institution Animal Care and Use Committee approved all experiments.Larvae were raised in Petri dishes containing embryo media (EM) consisting of 1 mM MgSOFish were treated with cadmium chloride hemipentahydrate  dissolved in EM for 3 h at 28.5°C. Some fish were additionally treated with benzamil hydrochloride hydrate , zinc sulfate heptahydrate , or copper sulfate pentahydrate . A stock of benzamil was made at a concentration of 40 mM in dimethyl sulfoxide . Control animals for the benzamil experiment were therefore treated with DMSO diluted in EM at a 1:200 ratio to match the DMSO levels in the benzamil group. Stocks of all other compounds were made in water and DMSO was not used for those experiments. For all treatments, fish were put into six well plates containing net-well inserts and the net-wells were moved to plates containing the different solutions the fish were exposed to. Following treatment, animals were washed three times in EM, euthanized with MS-222 and immediately fixed for immunostaining.Fish used for immunohistochemistry were fixed for either 2 h at room temperature or overnight at 4°C in 4% paraformaldehyde. Antibody labeling was carried out as previously described  mutants, also known as sputnik. Cadherin 23 is one of the proteins that make up the tip links that link neighboring stereocilia in hair cells. Mutants no longer have tip links and thus do not have functional mechanotransduction activity (Nicolson et al., Multiple hair cell toxicants have been shown to enter hair cells in a mechanotransduction-dependent manner (Marcotti et al., ift88tz288, and cc2d2aw38, both mutants contain premature stop codons in their respective genes (Tsujikawa and Malicki, ift88 mutants have previously been shown to have reduced responses to water jet stimulation (Kindt et al., cc2d2a mutants have been previously shown to have normal neomycin uptake and mechanotransduction activity (Owens et al., ift88 mutants (cc2d2a mutants in response to cadmium chloride (We next tested whether genetic mutants identified through a screen looking for mutants resistant to aminoglycoside-induced hair cell death (Owens et al.,  mutants , furtherchloride .cdh23 mutants, which completely lack mechanotransduction, we wanted to test if there were alternative means by which cadmium could enter hair cells. Previous experiments have shown that cadmium enters other cell types through zinc transporters (Dalton et al., As we still saw some hair cell death in While cadmium is not believed to travel through copper transporter family gene products, it has been shown that copper cotreatment can impair cadmium uptake in fish (Komjarova and Bury, Here, we show that cadmium can consistently cause hair cell death in the lateral line system of zebrafish larvae. This is consistent with prior work that has shown cadmium can cause hair cell death in fish (Faucher et al., via a genetic mutation in cdh23 mutants or pharmacologically via benzamil lead to a significant reduction in cadmium-induced hair cell death. While cdh23 mutants still showed significant cadmium-induced hair cell death at higher doses of cadmium, benzamil appeared to show an almost complete elimination of hair cell death across the dose range tested. This is different from what was previously shown for copper, where amiloride could protect against only lower doses of copper (Olivari et al., cdh23 mutants vs. benzamil treatment may be that unlike cdh23, benzamil is likely not blocking mechanotransduction selectively. Amiloride and benzamil are both considered nonselective epithelial sodium channel blockers and have been shown to be capable of blocking a number of channel types including some Ca2+, TRP, and K+ channels (Bielefeld et al., cdh23 mutants. The small remaining level of hair cell death seen in cdh23 mutants in the absence of mechanotransduction activity is in contrast to other toxicants that kill hair cells in a mechanotransduction-dependent manner, such as cisplatin and aminoglycosides, where toxicity can be completely blocked (Wang and Steyger, In this study, we attempted to elucidate how cadmium enters hair cells. We did this by blocking potential uptake mechanisms and looking for a decrease in cadmium-induced hair cell toxicity. We showed that blocking mechanotransduction activity either To test other potential uptake routes for cadmium into hair cells, we next investigated whether zinc or copper could protect hair cells from cadmium-induced hair cell death potentially by competing for entry. We failed to see consistent protection from either of these ions; however, these experiments were complicated by the fact that both copper and zinc can kill hair cells on their own (Hernández et al., While we failed to show consistent protection against cadmium-induced hair cell death from zinc, it has previously been shown that zinc can protect against cadmium-induced hearing loss in rats (Agirdir et al., ift88, did lead to resistance in cadmium-induced hair cell death similar to what had previously been seen with aminoglycosides. Ift88 is a component of the IFT-B complex a group of proteins important for anterograde intraflagellar transport in cilia and also for cilia formation (Pazour et al., cdh23 mutants where no response is seen (Nicolson et al., ift88 mutant’s resistance to cadmium-induced hair cell death further supports the role of mechanotransduction in this process. They are not as resistant as cdh23 mutants, which fits the fact that mechanotransduction is not as impaired. We failed to see significant resistance to cadmium-induced hair cell death in mutants of the other gene tested, cc2d2a. Cc2d2a is part of a complex of proteins located at the base of the cilia known as the transition zone that is believed to function as gatekeepers for cilia (Garcia-Gonzalo et al., ift88 mutants, these mutants do not show any impairment in FM1-43 uptake, suggesting normal mechanotransduction activity (Owens et al., ift88 mutants is due to the reduced mechanotransduction in these mutants rather than a general role of cilia in cadmium-induced hair cell death.We also investigated whether two cilia-associated gene mutants that are resistant to aminoglycoside-induced hair cell death were similarly resistant to cadmium-induced hair cell death. Mutations in one of those genes, The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by University of Washington IACUC, Lafayette College IACUC.TS designed this study. Experiments were performed and data analyzed by TS, CS, IA, JB, and KT. The article was written by TS and CS. All authors contributed to manuscript revision 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.


Newborn neurons assemble a new primary cilium, leading to a change in the mode of Shh signaling that now directs axon extension. + particle, which elongates and initiates intraflagellar trafficking as it transits toward the cell body, indicating primary cilium remodeling. Notably, disrupting cilia during and after remodeling inhibits axon extension and leads to axon collapse, respectively. Furthermore, cilium remodeling corresponds to a switch from a canonical to noncanonical cellular response to Shh. This work transforms our understanding of how cells can rapidly reinterpret signals to produce qualitatively different responses within the same tissue context.Cellular differentiation leads to the formation of specialized cell types and complex morphological variations. Often, differentiating cells transition between states by switching how they respond to the signaling environment. However, the mechanisms regulating these transitions are poorly understood. Differentiating neurons delaminate from the neuroepithelium through the regulated process of apical abscission, which mediates an acute loss of polarity and primary cilium disassembly. Using high-resolution live-cell imaging in chick neural tube, we show that these cells retain an Arl13b The early embryonic neuroepithelium consists of elongated neural progenitor cells that form membrane contacts with neighboring cells at both their apical and basal poles . This retained Arl13b+ particle remained at the tip of the apical process and progressively elongated as apical process withdrawal proceeded and was then closely associated with the neuronal cell body as axonogenesis initiated, indicating that the Arl13b+ particle acts as a site for primary cilium reassembly . Cells in early stages of apical process retraction were classified as proximal to the apical surface, while those in later stages of retraction were classified as being distal to the apical surface . To confirm these observations in cells that are not misexpressing Arl13b, which can lead to inconsistencies in cilium length , and this structure progressively elongated in distal cells , localizing at the tip of the apical process. To further characterize primary cilium reassembly, we labeled unmanipulated embryos with Tuj1 and polyglutamylated tubulin to visualize the ciliary axoneme , which progressively elongated in distal cells . Furthermore, the ratio between the length of the axoneme and Arl13b remained constant throughout apical process retraction, indicating that the axoneme is reassembled proportionally to the ciliary membrane (fig. S1B). Together, these results show that primary cilia of newborn neurons undergo disassembly during apical abscission, followed by rapid reassembly during apical process retraction. Furthermore, the reassembled primary cilium is maintained in cells undergoing axonogenesis.To investigate cell behavior following primary cilium disassembly by apical abscission , indicating active IFT in these cells. In contrast, IFT88 did not localize to the tip of the retained Arl13b+ particle but was frequently detected only in the basal region in cells proximal to the apical surface . This IFT88 partially colocalized with the basal body labeled with PACT-TagRFP , suggesting inactive IFT at these stages, with a reservoir of IFT88 at the base of the cilium to facilitate rapid reactivation of IFT , suggesting reinitiation of IFT in reassembled primary cilia. This pattern of IFT88 localization was maintained in cells undergoing axonogenesis , suggesting maintenance of IFT in this cell population. This progressive restoration of ciliary IFT88 accumulation as neuronal differentiation progresses was confirmed by three-dimensional (3D) Airyscan microscopy  is required for trafficking of proteins involved in assembly and maintenance of the primary cilium and to transport proteins that transduce cilium-mediated signaling . This revealed that in proximal cells, IFT88 was relatively static and remained localized to the base of the Arl13b+ particle but did not colocalize with Arl13b, indicating inactive IFT. Halfway through retraction, as the retained particle elongated, IFT88 trafficking to the tip of the primary cilium was reestablished, indicating that primary cilium–mediated signal transduction is also reactivated at this point, as IFT complexes are required for proper dynamics of diverse signaling components within the primary cilium . Consistent with these observations, quantification of IFT88 present within the primary cilium revealed a progressive increase in the area occupied by IFT88 in the primary cilium as well as a progressive increase in the number of IFT88 particles present in the primary cilium as cells retracted their apical processes (fig. S2). These results confirm that IFT is progressively restored as newborn neurons delaminate from the neuroepithelium and indicate that the molecular machinery required for signal transduction is reestablished during the later stages of apical process retraction.To confirm progressive reinitiation of IFT during primary cilium remodeling, we then monitored the temporal dynamics of IFT88 localization during apical process retraction. Neuroepithelial cells were cotransfected with IFT88-mNeonGreen, Arl13b-TagRFP, and mKate2-GPI, and cells that had recently undergone apical abscission were rapidly imaged for 3 min every hour, allowing us to capture the key stages of apical process retraction . Furthermore, this was accompanied by a 60% reduction in Arl13b protein measured using Arl13b antibody  compared with cells not subjected to green light irradiation . We further demonstrated that the centrosome remains unaltered , and EB3-GFP comets continued to extend from this organelle following CALI  but were unable to initiate axonogenesis , suggesting that the remodeled primary cilium directs this process. We then disrupted the remodeled primary cilium in cells undergoing axonogenesis. Following green light irradiation, most cells briefly continued to extend an axon (~3 hours), which subsequently suffered marked collapse , suggesting that sustained primary cilium–mediated signaling through the remodeled cilium is also required for maintenance of axonogenesis. In contrast, cells transfected only with GFP-GPI, which were subjected to green light irradiation, extended axons normally, demonstrating that this procedure does not generate phototoxicity in the absence of SuperNova . Furthermore, the majority of cells transfected with GFP-GPI and Arl13b-SuperNova, which were not subjected to sustained green light irradiation, extended axons normally , demonstrating that SuperNova does not have phototoxic effects in the absence of sustained green light irradiation. As these experiments were carried out on a wide-field imaging system, there is a possibility that the phenotype observed could be due to depletion of both cilium-localized and undetectable cytoplasmic Arl13b-SuperNova. To discount this possibility, we used a targeted 561-nm laser to disrupt only the ciliary Arl13b of cells expressing Arl13b-SuperNova. Following laser-targeted CALI, we again observed notable axon collapse in an average of 3.5 hours following 561-nm laser irradiation . This was in contrast to cells where the laser was targeted to a cytoplasmic region away from the primary cilium, which continued to extend axons normally . Together, these results strongly indicate that primary cilium–mediated signaling is required for initiation and maintenance of axonogenesis.To investigate whether reestablishment of primary cilium–mediated signal transduction is required for neuron repolarization following loss of polarity by apical abscission . This was in contrast to control embryos electroporated with a construct targeting firefly luciferase where the number of primary cilia was unaffected . We then cotransfected cells with the Arl13b knockdown constructs, GFP-GPI and pCIG-Neurog2, to promote neuronal differentiation. The effect of Arl13b knockdown on axon extension was then monitored by performing time-lapse imaging of these cells. We observed a range of axonogenesis defects in the majority of these cells (n = 30 of 41 cells). A subset, which had retracted their apical process, were unable to initiate axonogenesis and remained as round cells with an unpolarized appearance . Another subset of cells, which had initiated axonogenesis, briefly continued to extend their axons, but this was followed by a marked collapse . A further subset of cells had initiated axonogenesis, but these axons partially collapsed, stalled, and did not extend further (n = 5 of 41 cells). Only a small proportion of cells were able to extend axons normally for the 10-hour imaging period (n = 11 of 41 cells). This was in contrast to cells expressing the control luciferase targeting construct, the majority of which continued to extend their axons throughout the duration of imaging . Together, with the CALI-mediated disruption of ciliary function, these results are consistent with a key role for the remodeled primary cilium in directing initiation and maintenance of axonogenesis in newborn neurons.To confirm that primary cilium–mediated signaling is directing axon extension following apical abscission, we then disrupted primary cilium function by performing RNA interference (RNAi)–mediated knockdown of Arl13b. Neuroepithelial cells were electroporated with a mix of two short hairpin RNA–expressing constructs targeting different regions of the Arl13b sequence, which also expressed red fluorescent protein (RFP) as a marker for transfected cells (pRFPRNAi Arl13bA and pRFPRNAi Arl13bB). After 26 hours of incubation, embryos were fixed and labeled for Arl13b to assess knockdown efficiency. This revealed a 40% reduction in the number of primary cilia on the electroporated side of the spinal cord versus the unelectroporated side , consistent with previous experiments . In contrast, Smo did not accumulate in the retained Arl13b+ particle of proximal cells . Notably, ciliary Smo accumulation was reestablished in distal cells , and this was maintained during axonogenesis , indicating reactivation of Shh signaling, which is now transduced by the remodeled primary cilium.Given the role of the primary cilium in transducing canonical, Gli transcription factor–dependent Shh signals in neural progenitor cells . Conversely, although Gli activity was initially reduced in the progeny of mitotic cells, this was quickly reestablished to premitotic levels , confirming that the reduction in Gli activity observed during apical process retraction was not due to GFP photobleaching. The rapid loss of Gli activity during apical abscission indicates an acute cessation of canonical Shh signaling following primary cilium disassembly. Consistent with this, immunostaining for the negative canonical Shh signaling regulator GPR161 , which carries eight copies of the Gli-binding site from the FoxA2 enhancer upstream of nuclear GFP . This was in contrast to cells imaged in control medium containing vehicle only (ethanol), which continued to extend axons during the 6-hour imaging period . To further confirm the noncanonical mode of Shh signaling in cells extending axons, we then disrupted the function of the Src family kinases (SFKs), which mediate the cytoskeletal rearrangements downstream of noncanonical Shh signaling, using the chemical inhibitor PP2. We observed that cells extending axons imaged in medium containing PP2 also abruptly collapsed their axons , in contrast to cells imaged in medium containing vehicle control dimethyl sulfoxide (DMSO) , which continued to extend axons during the 8-hour imaging period. Together, these results indicate that cilium disassembly provides a pause in Gli-dependent Shh signaling as the newborn neuron exits the cell cycle. Subsequent cilium remodeling in the newborn neuron, which has now transitioned from a cycling progenitor to a postmitotic neuron but is encountering the same signaling cues, facilitates entry of GPR161 and Smo to the remodeled cilium, leading to a switch to Smo-dependent Shh transduction in the absence of Gli-mediated transcription to direct axon extension.The correlation between cessation of Gli-mediated transcription during apical process retraction and the subsequent initiation of Smo and GPR161 accumulation in the remodeled primary cilium suggests that the remodeled cilium is now transducing Shh through a Gli-independent noncanonical mechanism, which is likely to direct axon extension  leads to loss of axon extension provide an unexpected role for the primary cilium in controlling initiation and maintenance of axon extension through transduction of noncanonical Shh signaling. Although it is widely accepted that axon extension is modulated by noncanonical Shh signaling, the precise molecular mechanisms of this signal transduction pathway have remained elusive. Our work provides a starting point for future studies into the molecular cascades downstream of the remodeled primary cilium and how these directly facilitate the cytoskeletal remodeling events that drive the process of axonogenesis in early differentiating neurons.On a wider scale, dynamic changes in the molecular configuration of primary cilia may also regulate similar switches in interpretation of other signaling molecules during neuronal differentiation and in other developmental processes such as gastrulation and neural crest delamination. Furthermore, abnormal activation of canonical Shh signaling through the primary cilium is associated with a number of cancer types  eggs were obtained from Henry Stewart & Co. Ltd.  and incubated at 38°C to Hamburger and Hamilton stage HH10-12. Spinal cords were electroporated as described previously (Fertilized chicken (Chick embryos [between embryonic day 3 (E3) to E4] were fixed in 4% formaldehyde and dehydrated in 30% sucrose overnight. These were then mounted in 1.5% Luria-Bertani (LB) agar, dehydrated in 30% sucrose again, and then snap frozen on dry ice. Cryosections of 20-μm thickness were then collected using a Leica CM3050s cryostat and then processed for immunofluorescence. For immunofluorescence, samples were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) and blocked in 1% donkey serum (Sigma-Aldrich). Primary antibodies were used at the following dilutions: Tuj1  1:1000, Arl13b , Smo  1:50, IFT88  1:200, and GPR161. All secondary antibodies were Alexa Fluor conjugates (Life Technologies) and used at 1:500. Images were acquired using a 60× 1.40 numerical aperture (NA) objective on a Zeiss Cell Observer Z1 microscope system (Carl Zeiss). 3D images of the primary cilium were acquired using a Zeiss LSM 880 Airyscan system equipped with a 60× 1.40 NA objective.2. Image acquisition was carried out with a 40× 1.2 NA silicone immersion objective (Carl Zeiss), a Colibri 7 light-emitting diode (LED) light source (Carl Zeiss) and a Flash4 v2 sCMOS camera (Hamamatsu). Image stacks were acquired using minimal exposure times (20 to 50 ms each channel). Stacks consisted of 40 to 50 optical sections (spaced 1.5 μM) using intervals of 10 min between exposures. Imaging was carried out for 4 to 24 hours. To evaluate IFT88 dynamics, differentiating neurons were rapidly imaged at acquisition speed of one z-stack per 10 s for 3 min every hour. For CALI experiments, photobleaching was carried out by irradiating cells with 594-nm illumination from the Colibri 7 LED light source for 15 min. Targeted photobleaching was carried out using a targeted 561-nm laser on a 3i spinning disk confocal enclosed in an environment chamber maintained at 37°C and 5% CO2.Embryos were electroporated at Hamburger and Hamilton stage HH10-12, incubated for 16 hours, sliced, and embedded in type I collagen (Corning) on a glass-bottomed dish , as described previously (We used a previously described RNAi system (2) in the middle of the cell body and measuring the GFP intensity mean value in every frame. Ciliary IFT88 measurements were carried out using the threshold tool in ImageJ 1.52p  and counting the number of particles and area occupied by IFT88 using the analyze particle function. For cilium number quantification in RNAi experiments, the same tool described above was used to count particles between 0.1 and 1 μm2. Arl13b-SuperNova intensity was measured by manually drawing cilia contours and quantifying the intensity mean value in the area selected using Zen 2.3 . Deconvolution and postprocessing were performed in Zen 2.3 . Images acquired at 1 × 1 binning were deconvolved using the constrained iterative algorithm for a maximum of 40 iterations or until a 0.1% quality threshold was achieved. Statistical analyses were performed in GraphPad Prism version 8.3.0 . EB3 comet displacement was quantified using the manual tracking option in Imaris version 9.2.1 (Bitplane).Cilium length measurements for Arl13b and PolyGluTub were carried out using the line tool in Zen 2.3 . This measurement was performed using single z-sections selecting only cilia that were completely captured in a single plane. GFP intensity was measured by selecting a circular area (3 μm