Intraoperative thyroid frozen section: indications, results and consequences.

Background: Frozen section (FS) analysis is strongly influenced by the experience of surgeons and pathologists. We analyzed its performance in a secondary care hospital with surgical and pathologic experience transferred from a university hospital. Methods: Indications, results, and consequences of all thyroid FS performed between January 1, 2021 and December 31, 2022 were critically reviewed. Results: FS was performed in 90 (26.5%) of 340 procedures. Indications consisted in a suspicious fine needle biopsy in 28 (31.1%) cases, (99m) Technetium-Methoxy-Isobutyl-Isonitrile (MIBI) retaining hypofunctional nodules in 25 (27.8%), the intraoperative appearance in 18 (20%), the sonographic appearance in 18 (20%) and a positron emission tomography (PET) positive result in 1 case (1.1%). Malignancy was diagnosed in 21 (23.3%) and confirmed by final histology in all cases (100%). In the remaining 69 (76.7%) FS displaying no positive malignancy criteria, final histology delivered benign in 62 (89.8%) and malignant diagnoses in 7 cases (10.1%). 25% of thyroid carcinomas could not be diagnosed by FS. FS sensitivity was consequently 75% (95% CI: 55.1-89.3%). All missed malignancies were papillary thyroid carcinomas of follicular variant (fvPTC). FS sensitivity was lowest in MIBI positive hypofunctional nodules (33%) and Bethesda III (50%) as opposed to Bethesda V (92.9%) and to those cases with suspicious sonographic or intraoperative appearance (71.4%). Two-staged surgery was necessary in 10 (15.8%) of carcinomas. Conclusions: Sensitivity of FS in a secondary care hospital offering surgical and pathologic experience from a specialized university center is 75% and mainly reduced by the prevalence of fvPTC. Omitting FS in Bethesda III and MIBI positive hypofunctional nodules might improve FS performance.

The Impact of the COVID Pandemic on Adrenal Surgery at an Academic Endocrine Surgery Unit.

COVID-19 has severely affected the delivery of surgical care worldwide. The aim of the present study was to evaluate its impact on adrenal surgery at our academic endocrine center. All primary adrenal surgeries performed at the University Hospital of Cologne, Germany between 01.01.2019 and 31.07.2022 were included. This time frame was divided into pre-Covid (before 02/20), acute Covid (until 05/21), and post acute period (after 05/2021). Demographics, clinic-pathologic characteristics and treatment of these patients were analyzed. One hundred adrenalectomies were included: 22 before, 30 during, and 48 after the acute phase. The percentage of Conn adenomas and pheochromocytomas decreased during the acute phase (from 45.4 to 26.6% and from 18 to 10%, respectively) in favor of Cushing adenomas and suspicious tumors (from 4.5 to 20% and from 31.8 to 36.6%). About 90.9% of tumors resected for suspicion of malignancy were confirmed malignant by final histopathology, as opposed to 71.4% and 52.6% before and after the acute phase. The operative technique was similar during the three phases (63% retroperitoneoscopic, 34% laparoscopic and 2% open resections), with a significantly shorter operative time for retroperitoneoscopy (p=0.04). ICU monitoring demand increased during the acute phase (from 13.6% to 43.3%), according to the increase in Cushing adenomas and malignant tumors. During the acute phase of COVID-19 pandemic adrenal surgery for Cushing and malignant tumors increased, while a delay in pheochromocytoma surgery to the post acute phase was observed. The suspicion of malignancy formulated by the endocrine tumor board was accurate in 90.9% of cases.

What's the Catch? The Significance of Catch Bonds in T Cell Activation.

One of the main goals in T cell biology has been to investigate how TCR recognition of peptide:MHC (pMHC) determines T cell phenotype and fate. Ag recognition is required to facilitate survival, expansion, and effector function of T cells. Historically, TCR affinity for pMHC has been used as a predictor for T cell fate and responsiveness, but there have now been several examples of nonfunctional high-affinity clones and low-affinity highly functional clones. Recently, more attention has been paid to the TCR being a mechanoreceptor where the key biophysical determinant is TCR bond lifetime under force. As outlined in this review, the fundamental parameters between the TCR and pMHC that control Ag recognition and T cell triggering are affinity, bond lifetime, and the amount of force at which the peak lifetime occurs.

Differentiated Thyroid Cancer in Adolescents: Single Center Experience and Considerations for Surgical Management and Radioiodine Treatment

Objective: Differentiated thyroid cancer (DTC) in adolescents rare but with a favorable outcome, despite higher rates of cervical lymph node and pulmonary metastasis compared to adults. The aim of this study was to critically evaluate treatment of adolescents with DTC at a single center. Methods: Patients receiving postoperative radioiodine treatment (RAIT) for DTC between 2005 and 2020 at our institution were screened to identify adolescents according to the World Health Organization definition (10-19 years of age). Demographics, clinico-pathological characteristics, treatment and outcome were analyzed. Results: Among 1,897 DTC patients, 23 (1.3%) were adolescents with a median (range) age of 16 (10-18) years. The female to male ratio was 3.6:1. Sixty percent had classic papillary thyroid cancer, with follicular variant in 40%, which was higher than previously reported (15-25%) for this age group. pT-status was pT1 in 9 (39.2%), pT2 in 8 (34.8%), pT3 in 3 (13%) and pT4 in 3 (13%) patients. In 19 (82.6%) patients, central lymphadenectomy was performed and metastasis was seen in 57%. All patients received RAIT with initial activities of 1.2 (n=1, 4.3%), 2 (n=12, 52.2%) or 3.7 GBq (n=10, 43.5%). Eighteen (78.2%) patients were free of biochemical and radiologic disease at a median follow-up of 60.7 months. Second-line surgery for lymph node relapse was necessary in 3 (13%) cases. There was one disease-associated death. Conclusion: Despite high rates of metastasis, most patients were cured, and second-line surgery was rarely required. Further prospective studies are needed to determine whether less aggressive surgical management or omitting adjuvant RAIT are feasible in patients with limited stages at diagnosis.

Preoperative Short-Term Restriction of Sulfur-Containing Amino Acid Intake for Prevention of Acute Kidney Injury After Cardiac Surgery: A Randomized, Controlled, Double-Blind, Translational Trial.

Background Acute kidney injury (AKI) is a major risk factor for chronic kidney disease and increased mortality. Until now, no compelling preventive or therapeutic strategies have been identified. Dietary interventions have been proven highly effective in organ protection from ischemia reperfusion injury in mice and restricting dietary intake of sulfur-containing amino acids (SAA) seems to be instrumental in this regard. The UNICORN trial aimed to evaluate the protective impact of restricting SAA intake before cardiac surgery on incidence of AKI. Methods and Results In this single-center, randomized, controlled, double-blind trial, 115 patients were assigned to a SAA-reduced formula diet (LowS group) or a regular formula diet (control group) in a 1:1 ratio for 7 days before scheduled cardiac surgery. The primary end point was incidence of AKI within 72 hours after surgery, secondary end points included increase of serum creatinine at 24, 48, and 72 hours as well as safety parameters. Quantitative variables were analyzed with nonparametric methods, while categorical variables were evaluated by means of Chi-square or Fisher test. SAA intake in the group with SAA reduced formula diet was successfully reduced by 77% (group with SAA reduced formula diet, 7.37[6.40-7.80] mg/kg per day versus control group, 32.33 [28.92-33.60] mg/kg per day, P<0.001) leading to significantly lower serum levels of methionine. No beneficial effects of SAA restriction on the rate of AKI after surgery could be observed (group with SAA reduced formula diet, 23% versus control group, 16%; P=0.38). Likewise, no differences were recorded with respect to secondary end points (AKI during hospitalization, creatinine at 24, 48, 72 hours after surgery) as well as in subgroup analysis focusing on age, sex, body mass index and diabetes. Conclusions SAA restriction was feasible in the clinical setting but was not associated with protective properties in AKI upon cardiac surgery. Registration URL: https://www.clinicaltrials.gov; Unique Identifier: NCT03715868.

Subacute thyroiditis after SARS-Cov2 vaccination: A review of the cases being described and personal experience.

Objective. The present study evaluates the occurrence of subacute thyroiditis in temporal connection with SARS-Cov2 vaccinations described in the literature last year and confirmed by our clinical routine. Methods. Systematic literature search in Medline for studies reporting diagnosis of subacute thyroiditis in temporal connection with vaccinations against Covid 19. Results. The literature search yielded 24 relevant references out of which 22 were "case reports" and two "Letters to the Editor" and encompassed 37 patient cases, in total. They had received a SARS-Cov2 vaccination shortly before the diagnosis (median interval to vaccination six days). In none of these cases, infection of the upper respiratory tract had previously been identified as a classic trigger of the disease. Newly occurring hyperthyroidism and increased laboratory signs of inflammation were described in 78% and 74% of cases, respectively. Atypical clinical pictures (asymptomatic, euthyroid, no inflammation marks) have been observed in both the literature and our patients suspected of thyroid cancer referred to surgery. Conclusions. In times of pandemics and the resulting vaccination, new rapidly occurring sonographic changes in the thyroid gland should be revaluated after 2-3 weeks, or recommended to undergo a fine-needle biopsy, in order to avoid unnecessary surgical interventions.

Hürthle Cell Carcinoma: Single Center Analysis and Considerations for Surgical Management Based on the Recent Literature.

Background: Hürthle cell carcinoma (HCC) of the thyroid is rare. There are contrasting data on its clinical behavior. The aim of this study was to describe clinic-pathological features and outcomes of HCC patients at our institution, in order to adapt our surgical management. Methods: We retrospectively studied 51 cases of HCC treated at the interdisciplinary endocrine center of the University Hospital of Cologne, Germany between 2005 and 2020. Results: Patients median age was 63 years (range 29-78) with 64.7% of cases being female. Primary treatment included surgery and postoperative radioiodine therapy with 3.7 GBq in all patients. Surgery consisted of total thyroidectomy in all cases and additional central lymphadenectomy in 90.2% of cases. The median number of harvested lymph nodes was 11 (range 2-31). Lymph node involvement was found in two (4.3%) pT4a tumors. In all other cases (95.7%), central lymphadenectomy was prophylactic and lymph nodes were free of metastasis in final histopathology. Twelve (23.5%) patients with incomplete biochemical response to primary treatment were diagnosed with structural relapse during the course of disease, for which seven (58.4%) underwent resection of isolated cervical metastasis. Histopathology revealed soft tissue implants in all cases and cervical surgery led to biochemical and radiologic cure in only two (28.5%) cases. Five (41.6%) patients developed metastatic disease, followed by systemic therapy in two patients. Vascular invasion of the primary tumor was significantly associated with relapse (p<0.01). Conclusions: Recurrence of HCC was common in this study. Given the low rate of lymph node metastases both in this study and in recent literature and the nature of relapse (soft tissue instead of nodal metastasis), the benefit of routine prophylactic central lymph node dissection for HCC remains unclear, especially in the absence of vascular invasion from the primary tumor.

Adrenal Surgery in the Era of Multidisciplinary Endocrine Tumor Boards.

Work up of adrenal masses includes assessment of endocrine activity and malignancy risk. There is no indication for surgical removal of nonfunctional adrenal adenomas, according to the guidelines. In the present study, we aimed at evaluating the impact of a university endocrine tumor board on the quality of the indications for adrenal surgery at our institution. One hundred consecutive patients receiving primary adrenal surgery at the University Hospital of Cologne, Germany were included. Their demographics, clinic-pathologic characteristics, treatment and outcome were analyzed. In 55 (55%) cases, indication for surgery consisted in functional benign tumors, including Conn, Cushing adenomas and pheochromocytomas. Forty (40%) tumors were referred to surgery for malignancy suspicion and 5 (5%) myelolipomas were removed due to their size. Eighty-nine percent of surgeries were performed as minimally invasive procedures. Overall morbidity included two (2%) self-limiting pancreatic fistulas after left laparoscopic adrenalectomy for pheochromocytoma. All functional tumors were confirmed benign by final histology. Only 33 (82.5%) of 40 suspicious cases turned out to be malignant. Consequently, nonfunctional benign adenomas were "unnecessarily" removed in only 7 (7%) patients, with 6 (85.7%) of them having a history of extra-adrenal cancer and all of them fulfilling criteria for surgery, according to the international guidelines. In conclusion, the endocrine tumor board provided an excellent adherence to the guidelines with most surgeries being performed either for functional or malignant tumors. In nonfunctional tumors with history of extra adrenal cancer, CT guided biopsy might be considered for obviating surgery.

Lymphatic Vessel Invasion in Routine Pathology Reports of Papillary Thyroid Cancer.

Purpose: It is not mandatory to report lymphatic vessel invasion in pathology reports of papillary thyroid cancer (PTC) according to the current Union for International Cancer Control (UICC) TNM (tumor, nodes, and metastases) classification. However, there is some evidence for its correlation with lymph node metastasis (LNM) and prognosis. The aim of this study was to explore the clinical implication of lymphatic vessel invasion documentation of PTC because pathology reports play a pivotal role in postsurgical clinical decision-making in endocrine tumor boards. Methods: Patients undergoing postoperative radioiodine treatment for PTC at the University Hospital of Cologne, Germany between December 2015 and March 2020 were identified. Pathology reports were screened for documentation of lymphatic vessel invasion. Demographics and clinicopathologic data of patients documented, including lymphatic vessel invasion and lymph nodal involvement were analyzed. Results: A total of 578 patients were identified and included. Lymphatic vessel invasion was reported in pathology reports of 366 (63.3%) and omitted in 112 (36.7%) patients. Positive lymphatic vessel invasion (L1) was diagnosed in 67 (18.3%) of 366 patients and was documented as absent (L0) in 299 (81.7%) patients. Lymph nodal (N) status was positive (N+) in 126 (45.6%) and negative (N0) in 150 (54.3%) of these patients. In 54 (80.6%) L1 cases N+ status and in 137 (65.6%) L0 cases N0 status was diagnosed. In 13 (19.4%) cases with L1 status, there were no LNMs (L1 N0). In total, 72 (34.4%) patients had LNM despite L0 status (L0 N+). The sensitivity and specificity of LVI reporting for LNM were 0.42 and 0.91, respectively. Conclusion: In routine pathology reports of PTC used for indication to postoperative radioiodine treatment by a German endocrine tumor board, lymphatic vessel invasion was found to be reported inconsistently and mostly as L0. L1 diagnoses, however, reliably correlated with reported LNM and might, thus, be relevant for clinical decision-making. For this reason, we advocate for standardized pathologic reassessment of lymphatic vessel invasion, in particular for cases where lymph nodes are not included in the pathologic specimen and if L0 is documented.

Radioiodine Refractory Follicular Thyroid Cancer and Surgery for Cervical Relapse.

Compared to its more common counterpart papillary thyroid cancer (PTC), follicular thyroid cancer (FTC) has a less favorable outcome, due to its higher incidence of distant metastases and advanced stages at diagnosis. Despite radioiodine (RAI) avidity, metastatic FTC often progresses after radioiodine treatment (RAIT). We aimed at evaluating the indications and outcomes of surgery for cervical relapse of radioiodine refractory FTC. Patients receiving RAIT between 2005 and 2015 at the University Hospital of Cologne, Germany, were screened. Patients with FTC were identified. Demographics, clinic-pathologic characteristics, treatment, and outcome of patients diagnosed with RAI refractory FTC, who underwent cervical surgery in the course of disease, were analyzed. FTC accounted for 8.8% of all thyroid carcinomas undergoing RAIT. In 35.2% of FTC patients, disease persisted or recurred despite a cumulative mean RAI activity of 18.7 GBq ± 11.6 (follow-up 83.5 ± 56.7 months). Distant metastases were diagnosed in 75% of these patients, as bone (57.6%), lung (54.6%), and liver metastases (12.1%). Cervical relapse occurred in 63.6% of these patients and was treated in 57.1% with surgery with, and without, external beam radiation therapy (EBRT). Despite surgery and EBRT, in 75% of patients, cervical relapse recurred again. In conclusion, surgery for cervical radioiodine refractory FTC relapse is often performed in metastatic setting. With and without EBRT, cure is rare, although metastases can appear radioiodine avid. Early biological marker and systemic treatments for these patients are still needed.

Targeted Therapy of Papillary Thyroid Cancer: A Comprehensive Genomic Analysis.

Background: A limited number of targeted therapy options exist for papillary thyroid cancer (PTC) to date. Based on genetic alterations reported by the "The Cancer Genome Atlas (TCGA)", we explored whether PTC shows alterations that may be targetable by drugs approved by the FDA for other solid cancers. Methods: Databases of the National Cancer Institute and MyCancerGenome were screened to identify FDA-approved drugs for targeted therapy. Target genes were identified using Drugbank. Genetic alterations were classified into conferring drug sensitivity or resistance using MyCancerGenome, CiViC, TARGET, and OncoKB. Genomic data for PTC were extracted from TCGA and mined for alterations predicting drug response. Results: A total of 129 FDA-approved drugs with 128 targetable genes were identified. One hundred ninety-six (70%) of 282 classic, 21 (25%) of 84 follicular, and all 30 tall-cell variant PTCs harbored druggable alterations: 259 occurred in 29, 39 in 19, and 31 in 2 targetable genes, respectively. The BRAF V600 mutation was seen in 68% of classic, 16% of follicular variant, and 93% of tall-cell variant PTCs. The RET gene fusion was seen in 8% of classic PTCs, NTRK1 and 3 gene fusions in 3%, and other alterations in <2% of classic variant PTCs. Ninety-nine of 128 (77%) FDA-approved targetable genes did not show any genetic alteration in PTC. Beside selective and non-selective BRAF-inhibitors, no other FDA-approved drug showed any frequent predicted drug sensitivity (<10%). Conclusion: Treatment strategies need to focus on resistance mechanisms to BRAF inhibition and on genetic alteration-independent alternatives rather than on current targeted drugs.

Potassium Iodide Tablets Instead of a Saturated Solution Preoperatively for Reaching Euthyroidism Quickly in Refractory Graves' Disease.

The optional use of a saturated solution of potassium iodide in the immediate preoperative period to reach euthyroidism is included both in the American Thyroid Association (ATA) and in the European Thyroid Association (ETA) guidelines for the treatment of Graves' disease (GD). The recent literature though, shows that it does not translate to more clinically meaningful differences in surgical outcome. In our experience, potassium iodide should not be seen as a means for reducing operative time or complications; it is an effective way for reaching euthyroidism quickly. Herein, we describe three selected cases in which concentrated potassium iodide 65 mg tablets - instead of a saturated solution - were administered for thyroid blocking preoperatively, as recommended in the event of a nuclear emergency. One of the patients was pregnant. After oral treatment with potassium iodide 130 mg daily (two pills), euthyroidism was reached in all three cases within 24 hours. There were no side effects and surgery was performed without complications. Although the current literature did not report a significant benefit concerning operative time and complications, in our opinion preoperative potassium iodide plays an important role in selected cases for reaching euthyroidism preoperatively quickly. Potassium iodide 65 mg tablets, which are recommended in case of a nuclear emergency, are a very effective alternative to saturated solutions, which are not always quickly available and generally need to be administered over seven to 10 days.

Avoidance of iodine deficiency/excess during pregnancy in Hashimoto's thyroiditis.

PURPOSE: Hashimoto's thyroiditis is a common disease that also affects pregnant women. We analyzed to what extent the inflammatory process of Hashimoto's thyroiditis changes with iodine prophylaxis in pregnant women. The target for immunologic activity was levels of thyroid antibodies (TPO). METHODS: The data were collected retrospectively from 20 consecutive, pregnant patients who had been diagnosed with Hashimoto's thyroiditis between 01.12.2012 and 01.12.2014 and had received iodine supplementation with 100 µg (n = 1) or 150 µg (n = 19). At least two measurements of TPO antibody levels taken during pregnancy and one after pregnancy were evaluated for each patient in the study group. RESULTS: At the end of pregnancy, the average TPO antibody level for the 20 patients studied was 137 IU/ml (+/-214 IU/ml), the lowest being 16 IU/ml and the highest 1000 IU/ml. Despite iodine prophylaxis, levels of TPO antibodies decreased in 18 patients during pregnancy, falling below the reference value of 35 IU/ml in 5 cases. However, in one patient TPO antibodies increased from 60 IU/ml to 237 IU/ml during pregnancy while in another, levels remained constant at 1000 IU/ml. CONCLUSION: Iodine prophylaxis in pregnant women, taken at a dose of 100 or 150 µg daily was shown to have no negative systemic effects on Hashimoto's thyroiditis in a group of pregnant women with increased TPO levels, and can therefore be recommended for expectant mothers, including those with this disease.

Peptide receptor radionuclide therapy as a two-step strategy for initially unresectable liver disease from neuroendocrine tumors: a single-center experience.

PURPOSE: In this study, we describe our experience with peptide receptor radionuclide therapy (PRRT) for initially unresectable liver disease as a two-steps therapeutic strategy, first in neoadjuvant intention before surgery and then later on in case of disease relapse. METHODS: We performed a retrospective evaluation of four cases of unresectable liver metastases of NET of different origins treated with neoadjuvant Lu-177-DotaTATE for conversion into resectability first and as rechallenging treatment after disease relapse. RESULTS: After treatment with Lu-177-DotaTAE, resectability was reached in three of four cases. In one case, SIRT was additionally performed preoperatively. Relapse occurred in three of four cases after 32, 34, and 37 months, respectively, and was managed with Re-PRRT-treatment. CONCLUSION: Although more data are needed, our retrospective study suggests that treatment with Lu-177-DotaTATE is an important adjunct to surgery not only in neoadjuvant intention but also for treating disease relapse. A register study might deliver more evidence for supporting this strategy.

Benign Thyroid Diseases: Are There Gender-Specific Differences for Diagnosis and Treatment of Nontoxic Thyroid Nodules? Results from a 4-Year Retrospective Analysis of an Endocrine Tumor Board.

INTRODUCTION: Benign nodular goiters are endemic in Germany and diagnostic thyroidectomy is one of the most frequent surgical procedures. Less than 10% result in a malignant finding which is a poor ratio in comparison to other European countries. There is a female preponderance in almost all thyroid pathologies according to the current literature but not much is known concerning gender-specific differences in thyroid nodules. OBJECTIVE: We aimed at finding gender-specific differences in diagnosis and treatment of nontoxic thyroid nodules and focused on preoperative workup as well as cytological and histological findings of patients with solitary and multinodular nontoxic goiters. METHODS: We retrospectively analyzed 392 cases out of 693 thyroid cases managed interdisciplinarily by the endocrine board of a university center between January 2015 and December 2018 (4 years). RESULTS: The combination of fine needle biopsy and interdisciplinary case discussion resulted in a rate of malignancy of 28.9% in patients undergoing surgery for solitary and multinodular nontoxic goiter. Although there was no significant gender-specific difference in the distributions and malignancy rates of Bethesda categories, male patients had a significantly higher malignancy rate of 40% in our collective (p = 0.04). CONCLUSIONS: Surgical treatment for male patients with suspicious hypofunctioning thyroid nodules should be favored. However, well-designed prospective studies are required to investigate gender-specific recommendations for the treatment of benign thyroid diseases in the future.

Thyroid hemiagenesis is combined with a variety of thyroid disorders.

AIM: Thyroid hemiagenesis (TH) is a rare congenital anomaly in which one thyroid lobe fails to develop. We describe our experience with at least 13 patients presenting with TH at our department. METHODS: We retrospectively analysed patients with TH, who had been referred primarily to our clinic between 2004 and 2010. In patients with TH, thyroid function parameters and thyroid autoantibodies were examined. 99mTc-pertechnetate thyroid scintigraphy and sonography were performed in all patients and confirmed the diagnosis of TH. RESULTS: We identified 13 patients (11 women, 2 men) with TH in our patient collective and calculated an estimated prevalence of TH of 0.08 %.We found TH to occur more frequently in the left lobe and also more frequently in females than in males. 9 patients presented with a total absence of one thyroid lobe and 4 patients presented with severe hypoplasia of one thyroid lobe with an isthmus appearing as a "hockey stick sign" on scintigraphic imaging. Associated thyroid diseases could be observed in the remaining lobe in all patients and included hyperthyroidism, hypothyroidism, nodular goiter, toxic goiter, hypofunctioning nodules, Graves' disease and Hashimoto-thyroiditis. The most frequent thyroid disease in our patients with TH was nodular goiter. We did not find any association of TH with malignancy. CONCLUSION: TH is mostly detected incidentally as the prevalence of TH is extraordinary low. The fact that all of our patients with TH were also affected by other forms of thyroid disease is reasonable since the patients were not referred to the diagnostic centre due to TH but rather due to the associated thyroid disease. Possibly there are different groups of TH: the symptomatic hypothyroid children, the lifelong euthyroid adults who are diagnosed incidentally through another thyroid disease and the patients with a molecular failure of proper thyroid development.

Current EU research activities on combined exposure to multiple chemicals.

Humans and wildlife are exposed to an intractably large number of different combinations of chemicals via food, water, air, consumer products, and other media and sources. This raises concerns about their impact on public and environmental health. The risk assessment of chemicals for regulatory purposes mainly relies on the assessment of individual chemicals. If exposure to multiple chemicals is considered in a legislative framework, it is usually limited to chemicals falling within this framework and co-exposure to chemicals that are covered by a different regulatory framework is often neglected. Methodologies and guidance for assessing risks from combined exposure to multiple chemicals have been developed for different regulatory sectors, however, a harmonised, consistent approach for performing mixture risk assessments and management across different regulatory sectors is lacking. At the time of this publication, several EU research projects are running, funded by the current European Research and Innovation Programme Horizon 2020 or the Seventh Framework Programme. They aim at addressing knowledge gaps and developing methodologies to better assess chemical mixtures, by generating and making available internal and external exposure data, developing models for exposure assessment, developing tools for in silico and in vitro effect assessment to be applied in a tiered framework and for grouping of chemicals, as well as developing joint epidemiological-toxicological approaches for mixture risk assessment and for prioritising mixtures of concern. The projects EDC-MixRisk, EuroMix, EUToxRisk, HBM4EU and SOLUTIONS have started an exchange between the consortia, European Commission Services and EU Agencies, in order to identify where new methodologies have become available and where remaining gaps need to be further addressed. This paper maps how the different projects contribute to the data needs and assessment methodologies and identifies remaining challenges to be further addressed for the assessment of chemical mixtures.

Oral nitrite restores age-dependent phenotypes in eNOS-null mice.

Alterations in the synthesis and bioavailability of NO are central to the pathogenesis of cardiovascular and metabolic disorders. Although endothelial NO synthase-derived (eNOS-derived) NO affects mitochondrial long-chain fatty acid ß-oxidation, the pathophysiological significance of this regulation remains unclear. Accordingly, we determined the contributions of eNOS/NO signaling in the adaptive metabolic responses to fasting and in age-induced metabolic dysfunction. Four-month-old eNOS-/- mice are glucose intolerant and exhibit serum dyslipidemia and decreased capacity to oxidize fatty acids. However, during fasting, eNOS-/- mice redirect acetyl-CoA to ketogenesis to elevate circulating levels of ß-hydroxybutyrate similar to wild-type mice. Treatment of 4-month-old eNOS-/- mice with nitrite for 10 days corrected the hypertension and serum hyperlipidemia and normalized the rate of fatty acid oxidation. Fourteen-month-old eNOS-/- mice exhibited metabolic derangements, resulting in reduced utilization of fat to generate energy, lower resting metabolic activity, and diminished physical activity. Seven-month administration of nitrite to eNOS-/- mice reversed the age-dependent metabolic derangements and restored physical activity. While the eNOS/NO signaling is not essential for the metabolic adaptation to fasting, it is critical for regulating systemic metabolic homeostasis in aging. The development of age-dependent metabolic disorder is prevented by low-dose replenishment of bioactive NO.