Biochemical markers of bone turnover were markedly increased . The high bone formation and increased skeletal calcium acquisition resulted in profound hypocalcemia  and hypocalciuria . Positron emission tomography (PET) with 2‐deoxy‐2‐[fluorine‐18] fluoro‐D‐glucose (FDG) confirmed diffuse osteosclerosis without focal areas of abnormal FDG uptake in the skeleton or elsewhere to suggest either an underlying primary malignancy or metastatic disease. Bone biopsy showed markedly sclerotic woven and lamellar bone. The marrow space was devoid of typical bone cells and adipocytes and instead was filled by fibromyxoid stroma, infiltrated by small clusters of tumor cells. Bone histomorphometry and micro–computed tomography demonstrated an elevated trabecular bone volume and trabecular plate thickness. The bone disorder in this case is unique and raises the possibility of a new yet undefined novel anabolic paracrine factor (or factors) secreted by an adenocarcinoma of unknown primary that resulted in dramatic increases in BMD, HBM, and radiological osteosclerosis. The differential diagnosis and potential mechanisms responsible for the HBM are discussed. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.A 71‐year‐old man was referred for evaluation of incidental generalized osteosclerosis. He was found to have a high bone mass (HBM) with an elevated lumbar spine bone mineral density (BMD) Z‐score of +5.3. Over an 18‐month period, his lumbar spine BMD measured by dual energy X‐ray absorptiometry (DXA) had increased by +64% from 1.09 to 1.79 g/cm Lumbar spine and femoral neck BMD showed marked increases in the 18 months prior to presentation with incidental osteosclerosis. Iliac crest biopsy reveals dense trabecular bone reflecting sclerosis at the time of presentation. Thirty‐five percent of referrals are usually for suspicious malignant bone disease, and 22% of patients have an underlying medical disorder. Fifty percent are usually due to artifact elevations in BMD  Z‐scores, there are no data relating to dual‐energy quantitative computed tomography (QCT) Z‐scores, which are more accurate for quantitating “true” vertebral body cancellous BMD and eliminating artifact elevations.High bone mass (HBM) is defined as a bone mineral density (BMD) Z‐score ≥+2.0 and occurs with an incidence of three per 100,000 individuals.1 Thir They comprise loss‐of‐function SOST mutations (Slereosteosis and Van Buchem's disease) or gain‐of‐function LRP4, LRP5, and LRP6 mutations. Other genetic disorders called osteopetrosis result from a decrease in osteoclast bone resorption. Acquired HBM is encountered with toxic fluoride or heavy metal exposure, mastocytosis, and as a tumor manifestation. HBM is commonly seen in prostate cancer but has also been reported to occur with breast and pancreas; mucinous adenocarcinoma of the gastrointestinal tract; transitional cell carcinoma; carcinoid tumor; and myeloma, lymphoma, and neuroblastoma.HBM can be present at birth or be acquired. There are rare genetic disorders that affect osteoblast activity, resulting in an increase in bone formation via the Wnt/ß‐catenin signaling pathway, and are termed sclerosing bone dysplasias.3, 4We report on an unusual case of acquired generalized osteosclerosis in a man who was found to have HBM with an elevated lumbar spine DXA Z‐score of +5.3 and a QCT Z‐score of +11.0 as a consequence of rapid bone mass accrual most likely from an adenocarcinoma of an unknown primary.2. There were no signs of tetany, bone deformities, exostoses, or torus palatinus, and his dentition was normal. There were several 1‐ to 3‐mm darkly pigmented verruca‐looking warts on the plantar surfaces of his hands and temple regions of his face. Cardiorespiratory and neurological examinations were normal. Abdomen was normal to palpation, there was no lymphadenopathy or hepatosplenomegaly, and his rectal examination revealed a prostate apparently devoid of lesions/nodularity on manual exploration. Systematic review confirmed long‐standing gastroesophageal reflux managed on esomeprazole and hypertension treated with amlodipine/valsartan combination. He was retired, living with his wife, a nonsmoker who rarely drank alcohol. He had no fracture history and had never been treated with bisphosphonates or bone anabolic agents. There was no previous history of exposure to heavy metals, fluoride, or herbal preparations and no intravenous drug use. There was no family history of osteosclerosis or bone disorders.The patient was a 71‐year‐old man referred for incidental osteosclerosis noted on a recent abdominal CT scan. He had been previously well until 6 months prior to his presentation in 2016, when he developed abdominal discomfort, mild fatigue, and low‐grade back pain. He was investigated by gastroscopy and colonoscopy and diagnosed with chronic gastritis. On evaluation he appeared otherwise well, with ormal blood pressure 110/70 mm Hg, heart rate 76 beats per minute, and body mass index 24.6 kg/mTen years previously he had presented with incidental hypercalcemia  and hypercalciuria  due to primary hyperparathyroidism (serum parathyroid hormone [PTH] 7.6 pmol/L). He was successfully treated by subtotal  parathyroidectomy. Histology confirmed primary hyperparathyroidism‐related multiglandular parathyroid disease. The postoperative serum calcium (2.33 mmol/L) and PTH levels (1.5 pmol/L) normalized. There was no history of tetany or documented hypocalcemia. Genotyping for menin and RET variants were negative. Gene studies for underlying multiple endocrine neoplasia (MEN) 4, MEN5, and hyperparathyroidism‐jaw tumor were not performed. However, whole genomic sequencing of peripheral blood mononuclear cells failed to demonstrate any specified mutational abnormalities associated with genetic hyperparathyroidism syndromes (data not shown).2 (T‐score: −2.7) to 1.09 g/cm2 (T‐score: 1.1) and femoral neck BMD from 0.81 g/cm2 (T‐score: 2.0) to 0.83 g/cm2 (T‐score: 1.8).For the next 10 years, the patient regularly attended his local practitioner. His biochemistry remained normal  and his lumbar spine BMD measured by DXA using a lunar scanner  progressively increased from 0.92 g/cmWhole blood, serum, and an early‐morning 2‐hour urine void were collected after an overnight fast at presentation and again at 6 months. Samples were sent for routine biochemistry and hormonal profiles. PTH was measured using the Elecsys PTH (1–84) assay  and serum 1,25 dihydroxyvitamin D by the LIAISON® XL 1,25 dihydroxyvitamin D assay (DiaSorin). Serum propeptide of type 1 collagen (P1NP) was performed using an electrochemiluminescence immunoassay . The precision error for the assay is 4.05% for a mean of 30.7 mcg/L and 3.16% for a mean value of 200.2 mcg/L. The age‐specific reference range for a man is 16.9–42.4 ng/mL. Urinary deoxypyridinoline excretion (uDPyD) was measured using an enzyme‐linked chemiluminescent competitive immunoassay  . The precision error for the assay is 8% for a mean value of 98 nmol/L. The age‐specific reference range  for a man is 2.3–5.4 nmol/mmol of creatinine. Urine cross‐linked N‐terminal telopeptide (NTX) of type 1 collagen was measured by enzyme‐linked immunosorbent assay (ELISA) . Specimens with values exceeding 3000 nmol/mmol creatinine were diluted 1:5 using a urine diluent of known Osteomark® value (300 nmol/mmol creat) and retested. The precision error for the assay was 5% for a mean of 417 nmol/mmol creatinine and 5% for a mean value of 2640 nmol/mmol creatinine. The age‐specific reference range  for a man is 21–83 nmol/mmol of creatinine.M technetium‐methylene diphosphonate whole‐body bone scan combined with single‐photon emission tomography (SPEC)/CT using commercially available E.software  and a F18FDG PET CT using a GE Healthcare Discovery MI were used to assess skeletal activity and exclude the presence of a primary neoplasm or metastatic activity.Radiographs and CT scans of the thoracolumbar spine and pelvic regions were performed to assess for the presence of vertebral fractures, lytic lesions, and skeletal abnormalities. A thoraco‐abdominal CT was used to assess for cardiorespiratory pathology. A radionuclide 99The BMD of the lumbar spine and hip was determined by DXA using a lunar scanner  and following the manufacturer's guidelines for patient positioning and scan acquisition. The machine's calibration is checked on a daily basis prior to each scanning session using the GE Lunar Calibration Phantom and Aluminum Spine Phantom. The precision error for the spine (L1–L4) is 1.0%, for the total femur 1.2%, and for the femoral neck 1.5%. All scans were evaluated retrospectively by the “compare” method by a single operator in a blinded fashion to eliminate bias and to ensure equivalent data acquisition.3 and for the hip in g/cm2, as well as the derived BMD T‐ and Z‐scores. The in vitro volumetric BMD precision was performed weekly on the scanner and calculated for the solid phantom with aqueous K2HPO4 of 0.7% at nominal density of 50–200 mg/cm3. The in vivo BMD precision is 2.4% for the spine at a nominal BMD of 124 mg/cm3 and 1.4% for the hip at a nominal BMD of 1.0 g/cm2. Using a digital ruler, the height, width, and depth of L2 and L3 vertebrae were determined by repeated measures on the images acquired on the QCT scout films at baseline and 6 months. The mean vertebral body bone volume was calculated to determine whether there was a significant change in the vertebral body size. Scan measurements were analyzed retrospectively and in a blinded fashion by an independent operator. The intra‐observer precision was 0.4–0.6% for height, width, and depth measurements.Vertebral body and hip BMD were measured by QCT using Siemens Somatom Definition Flash CT Scanner and calculated using single‐dose dual‐energy technology, an In‐Table phantom, and QCT software. Measurements are calculated for the three lumbar spine vertebrae L2–L4 and expressed in mg/cm Undecalcified methylmethacrylate embedded sections were stained with toluidine blue, acid phosphatase reagents, and Goldner's trichrome to assess osteoid and tartrate‐resistant acid phosphatase (TRACP), as previously described to examine osteoclast activity. Dynamic bone histomorphometry to assess bone formation parameters was not performed as the patient did not receive tetracycline labeling at the time. Decalcified, paraffin‐embedded, hematoxylin and eosin (H&E)‐stained sections were examined to determine the cellular pathophysiology underlying the changes in bone mass, and polarized light was used to assess lamellar and woven bone matrix. Immunohistochemistry was performed to assess the expression of CK7, CK20, TTF‐1, Caudal type homeobox transcription factor ‐2 (CDX2) (primary and metastatic lung cancer markers), NKX3.1, SOX‐10 (metastatic prostate cancer markers), and CD117 and tryptase (mast cell markers). Fine needle aspiration of the pleural fluid was assessed for whole protein levels. The pleural fluid cytospin was assessed by a pathologist following staining for D periodic Acid Schiffs (DPAS)‐positive intracytoplasmic mucin cytokeratin ‐7, Cytokeratin8/18, and carcinoembryoinc antigen (CEA) , TTF1, PAX8 , prostate‐specific antigen (PSA) (prostate cancer marker) and synaptophysin, neuron‐specific enolase (NSE), and chromogranin A (CgA) (neuroendocrine marker).Iliac crest bone biopsy was performed using a trephine needle by a vertical approach, which enabled the assessment of subcortical and deep cancellous bone. Cortical bone could therefore not be assessed on this sample. Following fixation in paraformaldehyde, transiliac cores were scanned using a Bruker 1172 scanned at a resolution of 4.37 um using a 0.5‐mm aluminum filter, and scan settings and trabecular bone parameters were assessed using standard approaches.11 Und3 concentrations were elevated. The normal serum 25‐hydroxyvitamin D3 excluded a nutritional/deprivational cause as a contributing factor of the hypocalcemia and hyperparathyroidism. Biochemical markers of bone formation  and bone resorption  were markedly raised. Full blood count, liver and kidney functions, electrolytes, immunoglobulin electrophoresis, free light chains, angiotensin converting enzyme level, serum tryptase activity, and serum fluoride levels were normal. Hepatitis B and C serology were negative and tumor markers including PSA, carbohydrate antigen (CA) 19‐9, CA 125, and CA 72‐4, carcinoembryonic antigen, and neurone‐specific enolase were in the normal range. Age‐specific serum testosterone and insulin‐like growth factor 1 levels were mildly reduced.Serum and 24‐hour urine calcium levels were reduced even after correction for low albumin, while serum PTH and 1, 25‐dihydroxyvitamin DThoracic, lumbar spine, and pelvic X‐rays Fig. A demons2 (Z‐score + 5.3) and 22% increase in the femoral neck BMD to 1.01 g/cm2 (Z‐score + 0.8)  over the time interval .DXA scans were repeated and compared to scans that were performed 18 months prior to presentation. There was a striking 64% increase in the lumbar spine to 1.79 g/cmval Fig. . Lumbar CT of chest, abdomen, and pelvis demonstrated bilateral small pleural effusions measuring about 1–2 cm in depth, normal cardiac dimensions and no pulmonary infiltrate, mediastinal lymphadenopathy, hepatosplenomegaly, or intra‐abdominal tumors Fig. .18F‐FDG PET CT  Fig. B confir Detailed bone histomorphometry  and a lactate dehydrogenase fluid/serum ratio (2.7), suggestive of an exudate (32 g/L). The pleural fluid cytospin confirmed metastatic adenocarcinoma with atypical cells showing hyperchromatic overlapping nuclei with prominent nucleoli and thickened nuclear membrane. The cell block showed cells with DPAS‐positive intracytoplasmic mucin and positive staining for CK7, CK8/18, and CEA but negative for TTF1, PAX8, PSA, synaptophysin, NSE, and CgA Fig. . Excisio whereas the dramatic increases seen over 18 months prior to presentation was more likely due to a different cause associated with high bone formation, increased skeletal calcium uptake, and hypocalcemia.This case presented with generalized diffuse osteosclerosis, HBM, and rapid bone mass accrual. The elevated lumbar spine DXA (+5.3) and QCT Z‐scores (+11.0) confirmed a true HBM phenotype. The initial increases in BMD could be attributed to successful parathyroid surgery,15 whe and a number of acquired disorders  cause generalized osteosclerosis and HBM. In our patient, osteosclerosis was seen in both the axial and appendicular skeleton. Without knowledge of the prior BMD measurements, a differential diagnosis of a heterozygous sclerosing bone mass disorder could be considered. However, the clinical presentation of accelerated bone mass accrual (in 18 months) and the associated findings of adenocarcinoma in the pleural fluid, skin lesions, and bone marrow stroma favored the possibility of malignancy in the pathogenesis of the patient's bone disorder. While fluorosis, hepatitis C‐associated osteosclerosis, and hematological disorders present with a very similar pattern of bone disease, these were excluded by appropriate testing.Two distinct genetic (sclerosing bone dysplasias and osteopetrosis)4, 53 (Z‐score + 11.0) to 545 mg/cm3 (Z‐score + 15.6), femoral neck by 28% from 0.97 g/cm2 (Z‐score + 1.7) to 1.24 g/cm2 (Z‐score + 3.8), and total hip by 49% from 1.16 g/cm2 (Z‐score + 1.7) to 1.73 g/cm2 (T‐score + 10.8). The increases in lumbar spine BMD occurred without apparent changes in vertebral body size or volume . At no time did he receive bone active agents. His response to chemo−/immunotherapy was poor, and his condition progressively deteriorated with recurrent pleural effusions, new onset ascites, and pancytopenia due to bone marrow failure Table . His bonume Fig. . At 9 mo−39 percent of the general population. The extent to which the patient's BMD increased over the last 6 months of his life is unique and, to our knowledge, of such a magnitude that had never previously been reported. These increases at all skeletal sites (30%–50%) were significantly higher than those seen in SOST‐related sclerosing bone dysplasias and patients receiving anabolic agents for osteoporosis and greater than that seen during maximal skeletal growth from adolescence to adulthood.The severity of the osteosclerosis and HBM (QCT Z‐score + 15.6) seen in this case is exceptionally rare and estimated to occur in 1 in 10pulation.16 The through the paracrine action of either single or multiple growth factors  secreted by circulating tumor cells in the fibro myxoma. The very high bone formation is probably a consequence of an increase in individual osteoblast activity at the cellular BMU. Together these mechanisms are responsible for the positive bone balance and HBM seen in this case.Numerous reports exist of histological localised sclerosis in patients with various cancers, predominantly breast and prostate, but our report is the first detailing the striking sequential changes in both DXA and QCT measurements. The presumed pathophysiological mechanisms leading to osteosclerosis in this case were complex and guessed at in Fig. 20 thrAlthough our patient initially presented with primary hyperparathyroidism, this was surgically corrected and with no clinical recurrence at 10 years after surgery. Detailed exploration for an occult neuroendocrine tumor, as part of a MEN syndrome, and secretion of an anabolic peptide was never forthcoming. A plasma neuroendocrine hormonal screen was negative. PET/CT Ga68‐DOTATE and/or 111‐labeled somatostatin analogue scintigraphy were not performed because a primary solid tumor was never discovered. The circulating tumor cells found in the pleural fluid, ascites, and bone marrow demonstrated “classic” cytological features of adenocarcinoma. Specific immunoperoxidase staining  excluded a possible neuroendocrine tumor. and, more recently, in a case of metastatic gastric carcinoma and in another individual with prostate carcinoma. Our patient demonstrated some of these features of HBS. Although he had evidence of hypocalcemia, this was chronic and without symptoms of tetany. The compensatory mechanisms to prevent his hypocalcemia, which included secondary hyperparathyroidism, reduced urinary calcium excretion, elevated 1,25 dihydroxyvitamin D synthesis to promote gastrointestinal calcium absorption, and osteocytic osteolysis, failed to restore normal calcium homeostasis. Although increases in bone formation were striking, there were also significant increases in bone resorption. There was an increase in the density of perilacunar spaces surrounding TRACP+ active osteocytes within the trabecular bone, indicative of osteocytic osteolysis. Unfortunately, quantitative back‐scattered electron imaging was unavailable to confirm osteocyte viability and mineralization of the osteocyte lacunar area. Osteoclast parameters were within normal ranges, but TRACP+ multinucleated cells were seen in increased numbers lying dormant in the bone marrow stroma and adjacent to bone surfaces . This condition was initially described following parathyroidectomy in patients with severe primary hyperparathyroidism23 and However, the mechanisms of osteoblastic bone metastases in cancers such as prostate and breast carcinoma are complex and less well understood. Sclerotic bone lesions are common in advanced prostate cancer patients and present many challenges. A number of bone anabolic pathways have been implicated in the pathology of sclerotic/osteoblastic prostate carcinoma lesions. These include bone morphogenic proteins (BMPs), osteopontin, and the Wnt/β catenin pathway, each of which stimulates downstream Runt‐domain transcription factor signaling and aberrant osteoblast activation. Overactive bone formation enhances tumor growth and further compromises the bone marrow compartment. Preclinical models have demonstrated that inhibition of BMP signaling in particular can limit the osteoblastic response and, to some extent, indirectly suppress tumor growth; however, to date these approaches have not translated to the clinic. Osteocytes have also been implicated in the regulation of bone response to tumor, but this has been less well explored due to complexities involved in assessing osteocyte function in vivo. Stimulation of tumor cell growth, migration, and invasion has been shown in vitro using osteocyte‐conditioned media. The simulation of mechanical stimuli in 3D osteocyte organ chips was further increased by the invasion of prostate cancer cells. We were unable to find reports relating to osteoblast/osteocyte responses in patients presenting with malignant osteosclerosis, such as in this case. Nevertheless, we cannot rule out the possibility of a direct or indirect effect of malignant tumor cells, via paracrine or endocrine mechanisms, on osteoblast or osteocyte function in this patient.Tumor‐induced changes in osteoclast activity leading to lytic bone lesions have been thoroughly explored, and it has been revealed that lesion progression can be supressed via antiresorptive therapies.25 HowOur case outlines the dynamic changes in bone homeostasis that can occur in malignancy and raises the possibility of perhaps some other novel paracrine anabolic factor (or factors) secreted by an adenocarcinoma of unknown primary that resulted in dramatic increases in BMD, HBM, and radiological osteosclerosis. An alternative explanation for our findings may be that of a novel endocrine (circulating) anabolic factor secreted by a distant occult neuroendocrine tumor. More detailed studies are planned to unravel the mystery of this patient's osteosclerosis and the mechanisms responsible for it.Terrence H. Diamond: Conceptualization. Carl Bryant: Data curation. Richard Quinn: Data curation; formal analysis. Sindhu T. Mohanty: Generated MicroCT data and figures. Fiona Bonar: Formal analysis. Paul A. Baldock: Contributed to data interpretation and MicrCT analysis. Michelle M. McDonald: Contributed to conceptualization, provided specialized histology techniques and analysis, data interpretation and contributed to manuscript production.https://publons.com/publon/10.1002/jbm4.10734.The peer review history for this article is available at Appendix S1. Supporting informationClick here for additional data file.Figure S1. (A) Lumbar spine CT: coronal views demonstrating diffuse sclerosis with a heterogenous “mottled appearance” and no lytic lesions or fractures. (B) Lumbar spine CT: sagittal views demonstrating diffuse sclerosis with a heterogenous “mottled appearance” and no lytic lesions or fractures. (C) Chest CT scan: axial views demonstrating left pleural effusionClick here for additional data file.Figure S2. Changes in lumbar spine and femoral neck BMD measured by DXA prior to and at 10 years after parathyroidectomy and again at 18 months when he presented with incidental osteosclerosisClick here for additional data file.Figure S3. (A) Pleural fluid H&E section of cell block with gland forming malignant cells consistent with adenocarcinoma. (B) Papanicolauo stain of highly atypical malignant cells with mitosis. (C) Periodic acid‐Schiff stain showing focal cytoplasmic positivity in keeping with adenocarcinoma. (D) Strong expression of CK7 in tumor cells.Click here for additional data file.


Skip metastasis is a special type of lateral lymph node metastasis, which is not classified definitely by the eighth edition of the AJCC TNM staging system. The aim of the research was to study the prognosis of skip metastasis in PTC patients, and carry out a more appropriate N staging for skip metastasis.Study subjects were 3167 patients with papillary thyroid carcinoma (PTC), who underwent thyroidectomy at three clinical centers from 2016 to 2019. We identified two well‐balanced cohorts matched on the basis of propensity score.p < 0.001). After propensity‐score matching, recurrence rate was significantly lower in the skip metastasis group than in the LLNM group (p = 0.039), whereas the rate was similar in the skip metastasis groups and the CLNM group (p = 0.29).During a median follow‐up of 42 months, recurrence occurred in 68 (4.3%) patients with lymph node metastasis. 34 cases recurred in 1120 patients with central lymph node metastasis (N1a), and 34 recurred in 461 patients with lateral lymph node metastasis (N1b), among which 73 patients were diagnosis with skip metastasis. The RFS of N1a was significantly lower than that of N1b (In conclusion, our study indicated that, among patients with LLNM, those with positive skip metastasis showed significantly lower recurrence, exhibiting a similar rucurrence tendency as patients with CLNM. Thus, skip metastasis could be categorized into N1a stage rather than N1b stage based on the AJCC TNM staging system. The downstaging of skip metastasis may reveal more conservative treatment strategy. Our study provided the largest number of skip metastasis cases among current studies. Our series of studies on skip metastasis have progressed step by step, from the characteristics of primary tumor site to the possible factors of skip metas occurrence leading to the correlation between skip metas and prognosis in the present study. We proposed adjustments to the current AJCC TNM staging system. We provided potential treatment strategy for skip metas in PTC patients. This study analyzed 3167 patients from three clinical centers that underwent thyroidectomy for PTC within the period 2016–2019.We focused on skip metastasis, a class of lymph node metastases with a low incidence based on our previous systematic researches . The study indicated that, among patients with LLNM, those with positive skip metastasis showed significantly lower recurrence, exhibiting a similar recurrence tendency as patients with CLNM. Thus, skip metastasis could be categorized into N1a stage rather than N1b stage based on the AJCC TNM staging system. The downstaging of skip metastasis may reveal more conservative treatment strategy.1Papillary thyroid carcinoma (PTC), the most common type of endocrine malignancy with rapidly increasing incidence, but stable high survival rate of exceeding 98 percent in 5‐year follow‐up.Regional LNM occur first in the paratracheal and prelaryngeal/tracheal lymph nodes of the central compartment and subsequently in the lateral compartment.In recent studies, clinicians and researchers have a more positive attitude about the less extensive surgeries, which parallel the underlying theme of “less is more” presented in the 2015 ATA guidelines.22.1n = 90) or histologically proven poorly differentiated papillary cancer (PDTC) (n = 24), (3) no lymph nodes removed (n = 23), (4) history or coexistence of other head and neck cancer (n = 6), (5) initial diagnosis staging TxNxM1 (n = 16). After exclusion, 3167 patients that had pathological PTC, and received thyroidectomy between January 2016 and March 2019 were studied for enough follow‐up duration.A retrospectively collected, multicenter database from Shanghai, China was analyzed over a 5‐year period for patients who underwent first‐time thyroidectomy for PTC. Clinical centers involved in the study included Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, ENT Institute and Department of Otorhinolaryngology, Eye and ENT Hospital of Fudan University and Department of General Surgery, Civil Aviation Shanghai Hospital. A total of 3814 patients were enrolled in the study. The exclusion criteria were as follows: (1) no follow‐up or unavailable follow‐up data (n = 511); (2) no histologically proven PTC  and fine‐needle aspiration (FNA) were collected from Electronic Medical Records System for further analysis. Serum index examination included blood routine, liver and renal function, electrolyte, DIC, and thyroid function in all enrolled patients, CEA and other tumor markers, parathyroid function in some patients. Preoperative US and US‐guided FNA were performed strictly according to Thyroid Imaging Reporting and Data System (TI‐RADS).0‐4N0‐1bM0. Surgery was performed by thyroid specialists of high operation volume at Ruijin Hospital, Civil Aviation Shanghai Hospital and Eye and ENT Hospital.According to 2015 Tumor Node Metastasis (TNM) staging system of American Joint Committee on Cancer (AJCC), 8th edition, all patients enrolled were identified as TAll acquired specimens were examined by two or more board‐certified pathologists from Shanghai Ruijin Hospital and Shanghai EENT Hospital. Pathological features analyzed were pathological type of tumor, type of the surrounding thyroid tissues, tumor size, multifocality , and lymph node metastasis.2.3Propensity‐score matching (PSM) has been widely used in the clinical research due to its advantage of minimal bias in comparative studies. A retrospectively maintained database parameters from our previous experience and other studies was searched to conduct the case‐match analysis.2.4Patients who underwent surgery were assessed for perioperative and postoperative short‐term complications during hospitalization and recorded in the progress notes. Patients enrolled were treated with postoperative TSH suppression therapy and RAI (Radioactive Iodine) therapy according to the 2015 ATA Guidelines. Patients were followed up regularly while the first, second, and third follow‐up were scheduled in the first, second,and third months after the operation, and the follow‐up visits were made at intervals of 6 months. Follow‐up methods included outpatient records, electronic medical record (EMR), and telephone, online follow‐up. Thyroid function examination including thyroglobulin (Tg) and other serum index were reexamined within half a year after operation, while US together with serum examination were performed every 6 months starting from half an year after operation.In this study, recurrence, excluding new lesions in residual thyroid gland, included recurrence in thyroidectomy bed, lymph nodes, and distant site. Recurrence was defined as structural recurrence after completion of initial treatment, identified using imaging modalities, that is, US examination and/or radioactive iodine‐131 (RAI) whole‐body scan imaging, followed by cytological or histological confirmation, regardless of serum levels of Tg.2.5Written informed consent was obtained and the research was approved by the local Ethics Committee and the Institutional Review Board, including the Institutional Ethics Committee of the Eye and ENT Hospital of Fudan University, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, and Civil Aviation Shanghai Hospital, and was also approved by Chinese Clinical Trial (ChiCTR2100043353). All patients were fully informed of the experimental procedures and patient data including demographics, operative procedures, pathology, and complications, were retrospectively collected.2.6p‐value <0.05 was considered as statistical significance. Hazard ratio and the relative 95% confidence interval (CI) were also calculated. Propensity‐score matching (PSM) was used to balance the patients' characteristics, with the aim of collecting bias of the pairwise comparison between the three groups: LLNM Group, CLNM Group, and Skip Group. A caliper width of 0.1 of standard deviation for the nearest neighbor matching and the 1:1 matching method were adopted using the MatchIt package of R .Chi‐square and Student's t‐test were conducted for comparing categorical and continuous variables of baseline and clinicopathological characteristics, respectively. Kaplan–Meier method and log‐rank test were used to compare recurrence‐free survival estimates. All the above‐mentioned statistical analyses were conducted using the SPSS 24.0 package (SPSS Inc.), and a two‐sided 33.1This study included 3167 PTC patients, 1031 males 32.6%) and 2134 females (67.4%). The mean age was 41.6 years old with a range of 14–78, and 980 patients (30.9%) were over 55 years old. The clinical characteristics of patients were summarized in Table .6% and 23.2Contribution of 3167 patients T, N, and M to AJCC‐staging was shown in Table Detailed distribution of LNs revealed that, of the patients with lateral lymph neck dissection, the mean numbers of total LNs positive/harvested in LLNM and skip metastasis patients were 9.37 (2–41)/31.2 (21–56) and 3.51 (1–12)/29.7 (17–49), respectively. More importantly, the mean numbers of central LNs positive/harvested in LLNM were 4.61 (1–13)/8.33 (3–19), whereas, those in skip metastasis patients were 0/8.21 (4–14). No significant difference in harvested central LNs number in these two groups revealed that comprehensive central LNs dissection is necessary to define skip metastasis. The mean size of the largest metastatic LN in LLNM and skip metastasis patients were 1.16 (0.3–3.1) and 1.26 (0.2–3) cm, respectively.3.3Postoperative temporary and permanent hypocalcemia occurred in 445 (14.1%) and 22 (0.7%) patients, respectively. The mean parathyroid hormone (PTH) level of hypocalcemia patients was 5.79 (0.5–15.9) pg/ml, the mean PTH level of temporary hypocalcemia patients after 3 month was 33.2 pg/mL. 182 (5.7%) patients had varying degrees of hoarseness or cough after surgery: of these, 163 (5.1%) had temporary vocal fold paralysis and 19 (0.6%) had permanent vocal fold paralysis. In addition, 12 (0.4%) patients underwent secondary surgery due to postoperative bleeding, and 13 (0.4%) patients had chyle leakage. The risk of surgical complications was significantly different between thyroidectomy+CLND and LLND. Patients underwent neck radical dissection (LLND) had longer inpatient days, higher risk of temporary and permanent hypocalcemia, vocal fold paralysis, bleeding, and chyle leakage comparing to those underwent thyroidectomy+CLND. Since LLND procedure was performed in 73 skip LN metastases, incidences of complications were similar to other LLNM, and significantly higher than those in CLNM Table .3.41 patients. The mean recurrence time was 27.8 (8–60) months after initial treatment. In detail, 34 (3.0%) cases recurred in 1120 CLNM (pN1a) patients. 32 (8.2%) cases recurred in 388 patients with LLNM and CLNM (pN1b), 2 (2.7%) cases recurred in 73 patients with skip metastasis (pSkip). In Figure 1a and pN1b patients , as well as no statistically significant difference between pN1b and pSkip patients . To standardize the enormous difference in the number of cases between pSkip and the other two groups, we used the method of propensity‐score matching to balance other clinical characteristics.During the period of follow‐up, 68 (4.3%) patients were diagnosed with recurrence in pN3.51b patients (cohort 1), pSkip patient with pN1a patients (cohort 2), respectively (a 1:1 match). After propensity‐score matching was completed, there were 62 matched pairs of patients in cohort 1 , as well as no statistically significant difference between pSkip and pN1a patients   for patients and clinicians. In contrast with predicting DSS, lymph node metastasis plays a more important role in assessing the risk of recurrence after initial treatment.131I, complete TSH suppressive therapy and elongated duration of follow‐up.The latest edition of AJCC/UICC TNM system downstaged a significant number of patients by removing regional lymph node metastases from the definition of T3 disease, which represented the N stage might play a limited role in predicting disease mortality. However, the staging system reserved the classification of the involved lymph nodes location (N1a vs. N1b), for the usefulness in assessing the risk of recurrence and early response to therapy.Interestingly, the presence of skip metastasis, a special type of LLNM, played a different role in conventional LNM. The incidence of skip metastasis in LNM was relatively low, and the frequency was reported to vary between 6.8% and 21.8% in N1b patients.Surprisingly, the recurrence risk of skip metastasis was not consistent with that of N1b patients as expected. On the contrary, skip metastasis and N1a patients have similar recurrence risk, both of which much lower than the recurrence probability of N1b patients. Thus, skip metastasis had a different mechanism from that of conventional LLNM, which should not be roughly classified as the second‐tier LNM (N1b). Although skip metastasis was defined as N1b in the current staging system, the risk of recurrence, staging and treatment mode were inconsistent with those of conventional LLNM.The exact cause of skip metastasis was not clear, of which the lymph drainage and biological behavior might be different from the common LLNM. Our previous studies indicated that skip metastases was not an accidental event in large samples, but a special type of lymph node metastases related to tumor location and specific lymphatic drainage pathways: factors including upper pole locations, capsular invasion/extracapsular spread (ETE), multifocality, tumor size <10 mm were considered to have a certain correlation with skip metastases, which was consistent with others findings.N1b patients, those with LLNM, total thyroidectomy (TT), and postoperative RAI and more strict TSH suppression therapy are still the standard therapy as recommended in all guidelines.The limitation in our current study was associated with retrospective design. In addition, our median follow‐up time was only 34 months, lacking of a long‐term follow‐up of PTC patients after surgery compared with other studies. However, researches showed a recurrence time of PTC was mostly within 36 months after initial treatment,5In conclusion, our study indicated that, among patients with LLNM, those with positive skip metastasis showed significantly lower recurrence, exhibiting a similar rucurrence tendency as patients with CLNM. Thus, skip metastasis could be categorized into N1a stage rather than N1b stage based on the AJCC TNM staging system. The downstaging of skip metastasis may reveal more conservative treatment strategy.Zheyu Yang: Conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; project administration ; supervision ; validation ; writing – original draft ; writing – review and editing . Yu Heng: Conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; supervision ; validation ; visualization ; writing – original draft ; writing – review and editing . Qiwu Zhao: Conceptualization ; data curation ; methodology ; writing – original draft . Hao Ding: Conceptualization ; data curation ; supervision ; writing – original draft . Lei Tao: Conceptualization ; data curation ; visualization . Xiaxing Deng: Conceptualization ; data curation ; investigation ; methodology ; supervision ; writing – original draft ; writing – review and editing . Wei Cai: Conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; supervision ; visualization ; writing – original draft ; writing – review and editing . Weihua Qiu: Conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; project administration ; resources ; software ; supervision ; validation ; visualization ; writing – original draft ; writing – review and editing .This research was supported by Shanghai Jiaotong University (YG2019ZDA15), and the National Natural Science Foundation of China .No conflict of interest exists in the research.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


Blackcurrant possesses various health‐endorsing attributes owing to its polyphenol profile. Recent studies have demonstrated its therapeutic potential against various health disorders. Various bioactives present in blackcurrants have different functional and pharmacological aspects including anti‐inflammatory, antioxidant, and antimicrobial properties. The most dominant and important bioactive include anthocyanins, flavonols, phenolic acids, and polyunsaturated fatty acids. Food formats derived from blackcurrants comprise pomace, juice, powder, and extracts. All these food formats have industrial, prebiotic, and pharmacological benefits. In the current article, the nutritional composition, industrial applications, and therapeutic potential are discussed in the recent literature. Moreover, novel extraction techniques for the extraction of bioactive compounds present in blackcurrants and their safety concerns have been elaborated. Blackcurrant possesses various health‐endorsing attributes owing to its polyphenol profile. Food formats derived from blackcurrants have industrial, prebiotic, and pharmacological benefits. The separation, isolation, and classification of anthocyanins and other phenolic chemicals have been the primary objective of studies so far  , organic acids, vitamins , soluble and insoluble dietary fibers, tannins, and soluble sugars , soluble and insoluble dietary fiber, tannins, soluble sugars, also organic acids and vitamins C and E  varies. MoreoveThe antioxidant properties and chemical composition of blackcurrant were analyzed. Moisture comprised 77.64%, nitrogen‐free extract comprised 17.41%, crude fiber comprised 3.08%, cured protein comprised 1.28%, crude ash comprised 0.31%, and crude lipid comprised 0.28%. The following were the basic concentrations. P (54.74 mg/100 g), Ca (26.45 mg/100 g), and K (177.36 mg/100 g) were the mineral constituents shown in Table They are manufactured in a great variety of items  because these are a high source of pectin, carbohydrates, and aromatic components . It could be a viable option for traditional methods, because of the faster extraction process and reduced solvent consumption. To enable faster extraction of essential and hydrophilic chemicals from the sample into the extraction solvent, microwave energy is consumed in this type of extraction to melt solvents in association with samples  is utilized. Mass exchange and solvent solubility were easily achieved by UAE. As a consequence, it decreases the quantity of solvent intake and extraction temperature while enhancing the extraction yield and speed , a strong‐intensity zone of ultrasound  with distilled water. The blend was kept in the ultrasound bath. For 2 h, the ultrasound of 27 kHz frequency and 6 W/cmBy utilizing 96% ethanol as a solvent, the fruit samples (10.0 g) were obtained. The extraction process took place using an ultrasonic bath, which operated at room temperature for one hour. Following purification, 5 mL of the liquid powder was utilized to evaluate extraction concentrations. A rotary evaporator was applied to evaporate the solvent through a vacuum and dried at 30°C to stable weight  and a shunt resistor (1 ohm) in combination with the container, as well as two channels, along with an oscilloscope 100 MHz, the pulse current and voltage were evaluated. These evidences were documented throughout the duration of procedure. For all the investigations, the pulse width was 100 milliseconds  as a rapid and efficient method to extract essential components, such as polyphenols, from complex living organisms containing a wide range of chemical compounds, including primary and secondary metabolites  depends on the capability of specific enzymes to decompose or destroy the cell membranes to liberate substances , pH of the solvent , % ethanol in solvent , and enzyme unit per g of sample   with 2% enzyme mix (papain: pectinase = 2) was placed in a 500 mL beaker, and then a buffer solution (pH = 5.3) was introduced at 20:1 mL/g. The polysaccharides were subsequently isolated at 40°C using an ultrasonic cell disintegrator for 45 min, and the ultrasonic power was 600 W , del‐3‐rut (28.3%–39.1%), cy‐3‐glc (4.5–11.9%), and del‐3‐glc (8.0%–20.3%)  discoveThe phenolic concentrations in the blackcurrants demonstrated maximum values of total polyphenols in fruits, 955 mg/100 g FW in total  gallocatechin benzylthioether were liberated as the free flavan‐3‐ols and free flavan‐3‐ols adducts, correspondingly, by the thiolytic breakdown of blackcurrant procyanidins. The contents of procyanidins in blackcurrant berries range from 140 mg/100 g FW for “Ben Conan” to 267 mg/100 g FW for “Ben Sarek” , galactose, malonylglucose, arabinose, and glucose  or as polymers. Blackcurrant comprises flavan‐3‐ ols catechin, epicatechin, epigallocatechin, and their galloyl variants more extensively . Hui et al.  evaluate6.32O2) and NO are neutralized along with the inhibition of the development of lipid and protein peroxidation  system is mainly responsible for the well‐known anti‐inflammation of blackcurrant anthocyanins. As a transcription factor, NF‐B modulates a variety of genes implicated in immunological process and inflammation. These included chemokines, cytokines that cause inflammation, adhesion molecules, and inactivating enzymes, like cyclooxygenases and inducible nitric oxide synthase (iNOS) Moynagh, . In seve6.5Research on blackcurrant's possible influence on cancer cells was not initiated until now. It has been proved that blackcurrant extract inhibits the progression of colon cancer, breast cancer cells HT‐29/ HCT‐166, and human promyelocytic leukemia cells MCF‐7 BCE ; writing – original draft ; writing – review and editing . Sadaf Waliat: Formal analysis ; validation ; writing – review and editing . Muhammad Afzaal: Supervision ; validation ; writing – review and editing . Farhan Saeed: Formal analysis ; supervision ; validation . Aftab Ahmad: Data curation ; validation . Ahmad Din: Formal analysis ; validation . Huda Ateeq: Investigation ; validation . Asma Asghar: Data curation ; validation . Yasir Abbas Shah: Data curation ; formal analysis ; writing – review and editing . Ahmad Rafi: Data curation ; validation . Mahbubur Rahman Khan: Data curation ; validation .The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.The authors have no relevant financial or non‐financial interests to disclose.This article does not contain any studies with human participants or animals performed by any of the authors.For this type of study, formal consent is not required.Corresponding and all the co‐authors are willing to participate in this manuscript.


Controversy has persisted over the clinical benefits of low‐dose sacubitril/valsartan in patients with heart failure (HF).Low‐dose sacubitril/valsartan might also be effective and safe in HF patients.Electronic databases including PubMed, Ovid, and Cochrane Library were systematically retrieved from inception to August 5, 2021. Review manager 5.4 and Stata 15.1 were employed in this systematic review and meta‐analysis. Key efficacy outcomes of interest included HF hospitalization, all‐cause mortality, left ventricular ejection fraction (LVEF), N‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP), together with New York Heart Association (NYHA) functional class. The safety outcome was systolic blood pressure (SBP). The grading of recommendations assessment, development, and evaluation approach was conducted to evaluate the quality of evidence for each outcome.p < .0001) and the risk of all‐cause mortality . However, there were no appreciable differences in improvements of NYHA , changes of LVEF , changes of NT‐proBNP  and changes of SBP  between groups with low‐dose and high‐dose sacubitril/valsartan.A total of 1269 studies were screened and 9 real‐world studies met the inclusion criteria were included in the meta‐analysis, with 1697 participants. Compared with low‐dose sacubitril/valsartan, high‐dose sacubitril/valsartan significantly reduced the risk of HF hospitalization (odds ratio [OR]: 0.4, 95% confidence interval [CI]: 0.27–0.61, Compared with high‐dose sacubitril/valsartan, low‐dose sacubitril/valsartan was associated with increased risks of HF hospitalization and all‐cause mortality. However, no distinct between‐group differences in improvements of NYHA, changes of LVEF, changes of NT‐proBNP and changes of SBP were observed. As a result, large proportion of patients (74.76%) achieved target dose of sacubitril/valsartan in the trial.Therefore, there are still controversies on the effect of low‐dose sacubitril/valsartan versus high‐dose sacubitril/valsartan for patients with HF, which might bring some difficulties in decision‐making regarding the choice of sacubitril/valsartan doses in the clinical management of HF patients. This systematic review and meta‐analysis were conducted to evaluate the efficacy and safety of low‐dose sacubitril/valsartan for HF patients based on real‐world data, by comparing with high‐dose sacubitril/valsartan in daily practice.22.1This systematic review and meta‐analysis were presented according to the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA).2.2The baseline characteristics of studies and patients were extracted by two authors (Xiu‐Zhen Zhang and Yuan‐Min Li) independently, and the discrepancy was resolved through consensus. The following data were extracted from each included study: first author's name, year of publication, study design, location, number of participants, age, gender, ejection fraction, follow‐up duration, and mean daily dose in high‐dose group and low‐dose group. Additionally, we also tried to contact the authors if a study did not offer the necessary data. Key efficacy outcomes of interest included HF hospitalization, all‐cause mortality, left ventricular ejection fraction (LVEF), NT‐proBNP, together with NYHA functional class. The safety outcome was systolic blood pressure (SBP).2.3χ2 test and the I2 test were used to examine heterogeneity in the Review manager. Odds ratio (OR) and 95% confidence interval (CI) were calculated for the risk of HF hospitalization, the risk of all‐cause mortality, and the improvement of NYHA class. Weighted mean difference and 95% CI were calculated for the changes in LVEF, NT‐proBNP, and SBP. Sensitivity analysis was performed to seek the reason of heterogeneity by the “one‐study removed” method. In addition, the Egger's and Bgger's test, and also the visual inspection of funnel plots, were hired to assess publication bias. A two‐tailed p < .05 was considered statistically significant.Review manager 5.4 and Stata 15.1 were adopted in this systematic review and meta‐analysis. The 33.1The process of literature screening and study selection is shown in Figure The characteristics of the included studies are summarized in Table 3.2p < .0001, Figure p < .0001, Figure I2 = 0%). Improvements of NYHA were also presented in three trials. However, there were no obvious differences in the improvements of NYHA between groups with low‐dose and high‐dose sacubitril/valsartan .The quality was assessed. All the included studies had a high quality with an NOS score of ≥7 points  was observed with reduced heterogeneity  after removing the study.Apart from HF hospitalization and all‐cause mortality, changes in NYHA functional class, LVEF, and NT‐proBNP levels before and after initiation of sacubitril/valsartan were also assessed in many real‐world studies. Similar to other real‐world studies,p < .001) and 9.4%  as compared with baseline, respectively. At 12 months, 75% of the study subjects had an LVEF increase of 4.9% or more and 25% experienced an LVEF increase of 13.4% or more.I2 = 45%, pheterogeneity = 0.16), but there was also no significant difference in changes of LVEF between the two groups .It is generally recognized that improvement of LVEF during treatment for HF is strongly associated with improved survival.p = .67). Similarly, our meta‐analysis observed that no significant differences in changes of NT‐proBNP between patients with high‐dose sacubitril/valsartan and patients with low‐dose sacubitril/valsartan. Although the quality of evidence for the outcome was very low, the conclusion was still acceptable. Because quality of all the included observational studies was high and no significant heterogeneity and publication bias were found.Clinically, elevated levels of natriuretic peptides are useful for evaluating prognosis in HF, and hence a series of randomized controlled trials (RCTs)I2 = 10%, PHeterogeneity = 0.33), but difference in changes of SBP between the two groups was still no significant .With regard to the safety, real‐world studies demonstrated that hypotension occurred more frequently in patients receiving sacubitril/valsartan.4.1Some limitations should be acknowledged. First, real‐world observational studies with small sample sizes were employed in the present meta‐analysis to perform comparisons of efficacy and safety outcomes between high‐dose and low‐dose sacubitril/valsartan. This might contribute to the low quality of evidence assessed by GRADE approach for each outcome in this study, with implications for the reliability of the results of this meta‐analysis. In the future, the validity of this meta‐analysis needs to be confirmed by further RCTs with large sample sizes. Second, the high‐dose group and low‐dose group were classified according to the average daily dose of sacubitril/valsartan in the meta‐analysis. Therefore, the efficacy and safety of sacubitril/valsartan at the routine dosages  could not be assessed. Third, several efficacy  and safety outcomes  could not be evaluated between high‐dose and low‐dose sacubitril/valsartan due to the small number of included studies and limited data, which needs to be further investigated. Fourth, due to patients with chronic kidney disease (CKD) mixed in all studies that had been included in the meta‐analysis, we could not achieve independent data to perform a subgroup analysis to explore if the effects of low‐dose sacubitril/valsartan were different in patients with CKD. Last, all patients of those studies that had been included in the meta‐analysis had LVEF of 40% or less. Therefore, we did not have sufficient data to allow for meaningful subgroup to explore if the efficacy and safety of low‐dose sacubitril/valsartan were different at different level of ejection fraction.5Compared with high‐dose sacubitril/valsartan, low‐dose sacubitril/valsartan was associated with increased risks of HF hospitalization and all‐cause mortality. However, no obvious differences in improvements of NYHA, changes of LVEF, changes of NT‐proBNP, and changes of SBP were observed in both study groups. These data showed that the clinical benefits of low dose of sacubitril/valsartan were not as good as those of high dose of sacubitril/valsartan. Additionally, since all the included studies were observational studies, level of evidence for every analysis assessed by GRADE approach was low and conclusions of the meta‐analysis should be applied with caution. Further RCTs with large sample sizes should be required to confirm the efficacy and safety of sacubitril/valsartan at different dose levels.Wen‐Wen Chen: Study design and manuscript. Juan Jiang, Jie Gao, Xiu‐Zhen Zhang, and Yuan‐Min Li: Data collection, data analysis, and validation. Yan‐Lin Liu and He‐Qin Dang: Scientific revision of the manuscript.The authors declare no conflict of interest.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


Based on the eggshell analysis result, SEM images and FTIR spectra demonstrated that the components were firmly integrated into the blended coatings as well as had more intertwined than their pure ones, which was also reflected in the evaluation results of their internal quality parameters. In addition, the results showed that by enhancing the ratio of polyvinyl alcohol from 25 to 75 wt%, the blended coating barrier efficiency was relatively improved (p < .05). Meanwhile, the lowest percentage of weight loss (0.57 ± 0.08%), pH value of albumin (8.30 ± 0.04), the highest values of Haugh unit (61.00 ± 0.07), yolk index (0.37 ± 0.02) were observed in eggs coated with CH/PVA 25:75 wt%. But there was no difference in 50 or 70 wt% PVA significantly. Therefore, the CH/PVA blended coatings containing around 50–75 wt% PVA, as egg biodegradable packaging, can be used to extend the shelf life for 2–3 weeks at ambient temperature.Using biopolymers in the form of edible coating as egg eco‐friendly packaging is a progressive approach. The blending of biopolymers is one of the procedures for overcoming mechanical weakness and benefiting from their maximum synergistic effect. Aiming to determine the relative ratios of chitosan (CH 4 w/v%) and polyvinyl alcohol (PVA 5 w/v%) in the composition of blended coatings, an experiment was conducted with six treatments ( r = 3) including different ratios of CH/PVA  wt% in a period of 4 weeks of egg storage at ambient temperature, emphasizing eggshell barrier properties. The CH/PVA blended coatings containing around 50–75 wt% PVA, as egg biodegradable packaging, can be used to extend the shelf life for 2–3 weeks at ambient temperature.Aiming to determine the relative ratios of chitosan (CH 4 w/v%) and polyvinyl alcohol (PVA 5 w/v%) in the composition of blended coatings, an experiment was conducted with six treatments ( Besides, the obtained results were matched to confirm with scanning electron microscopy (SEM) images and Fourier transform infrared (FTIR) spectroscopy.22.1Chitosan (CH) white‐colored powder brought from shrimp shells was purchased from the Nano Novin Polymer Co. with a high molecular weight of 310–375 KDa and an 85% degree of deacetylated. Polyvinyl alcohol (PVA), fully hydrolyzed (average Mw: 85–146 KDa) in the form of a white powder was delivered by the Sigma‐Aldrich Co. Glacial acetic acid and glycerol (99%) were procurement from Merck. All chemicals used were of analytical grade; in addition, the solutions were freshly prepared in all of the experiments.Fresh, feces‐free, without surface cracks, white shell eggs (from 28‐week‐old Hy‐line White hens) with an average weight of 55 ± 5 g were obtained from a commercial layer farm at Mazandaran, Iran.2.2According to the method proposed by Olewnik‐Kruszkowska et al. , in brieAll of the prepared coating solutions were used to coat the eggs with the immersing procedure explained by Rachtanapun et al. . Before 2.3h − 1.7w0.37 + 7.6), where h is the albumen height (mm), and w is the weight of the egg (g)  and a digital caliper , respectively. All eggs were weighed at the initial and as well as end of each weekly storage interval, with a digital balance . Haugh unit (HU) values were calculated as 100 log  Haugh, . The yol) Haugh, . The ave) Haugh, . The pH 2.4−1.A scanning electron microscope  was used to evaluate the eggshell morphology at the initial and the end of storage. The Fourier transform infrared–attenuated total reflectance (FTIR‐ATR) spectra of the outer surface of eggshells were recorded using an FTIR spectrophotometer (Thermo Fisher Scientific) with attenuated total reflectance (ATR) accessories in the frequency range of 500–4000 cm2.5p < .05) were evaluated using Duncan's multiple range tests. Five replicates for each treatment were used for the evaluations.A two‐way analysis of variance (ANOVA) using SPSS software version 20.0 was used to determine differences in the means of data. Statistically significant differences found (33.1p < .05). However, the WL of CH/PVA 25:75‐coated eggs was approximately 16% less than that of CH/PVA 50:50 (0.57% vs. 0.66%) at the end of the storage.The escape of gases causes many adverse internal physical and chemical changes, which ultimately lead to a deterioration in the quality of the eggs. Therefore, determining the WL values is one of the effective indicators in assessing the interior quality of eggs . The CH/PVA blended coating delayed egg weight loss by sealing eggshell pores with the highly water‐resistant coating material, which decreased the evaporation rate of the internal content of the eggs. The prevention of egg weight loss using highly water‐resistant coating materials has also been previously reported by Rachtanapun et al.  at the end of the first week to 41.07 (grade B), and 28.28 (grade C) at the end of 14 and 28 days of the storage period, respectively. The disruption of the ovalbumin structure, which is attributed to the increasing albumin pH  and water vapor permeability (WVP) of coating materials are of considerable importance in food preservation. Low OP exhibits great oxygen barrier abilities . The pH of albumen enhances because of the hydrolysis of carbonic acid and subsequently, carbon dioxide escapes via the pores of the eggshell  are attributed to C=N pyridine ring vibrations. This confirmed the interaction between the PVA and CH. The peak intensity increased with enhanced PVA contents, because of hydrogen bonding between the hydroxyl groups of PVA and the hydroxyl, or amine group of CH in the blended coatings ; data curation ; methodology ; project administration ; software ; validation ; visualization ; writing – original draft . Pezhman Riazi Kermani: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; project administration ; resources ; software ; supervision ; validation ; visualization ; writing – original draft ; writing – review and editing . Maryam Sarvarian: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; project administration ; writing – review and editing . Chinaza Godswill Awuchi: Data curation ; formal analysis ; methodology ; project administration ; validation ; visualization ; writing – review and editing .The authors declare that they have no conflict of interest.The study does not involve any human or animal testing.


It seems natural that motor responses unfold smoothly and that we are able to easily concatenate different components of movements to achieve goal-directed actions. Theoretical frameworks suggest that different motor features have to be bound to each other to achieve a coherent action. Yet, the nature of the “glue”  between elements constituting a motor sequence and enabling a smooth unfolding of motor acts is not well understood. We examined in how far motor feature bindings are affected by reward magnitude or the effects of an unsigned surprise signal. We show that the consistency of action file binding strength is modulated by unsigned surprise, but not by reward magnitude. On a conceptual and theoretical level, the results provide links between frameworks, which have until now not been brought into connection. In particular, theoretical accounts stating that only the unexpectedness (surprisingness) is essential for action control are connected to meta-control accounts of human action control. Different features defining an action (e.g. which hand/finger is to be moved) have to be “bound” to each other to achieve a coherent movement/response1. Even though recent years have seen progress in the understanding of the neurophysiological underpinnings of these motor feature binding processes10, the nature of the “glue” (i.e. bindings) between elements constituting a motor sequence and enabling a smooth unfolding of motor acts is not well understood.It seems natural that motor responses unfold smoothly and that we are able to easily concatenate different components of movements to achieve goal-directed actions. For that, different features constituting a motor response or sequence need to become integrated1, this is astonishing because reward and reinforcement learning related processes are central to build and (re −)shape associations11. An in-depth investigation of how reward and reinforcement learning principles shape bindings between different features of motor responses, will provide insights into the nature of bindings. Likewise, this will inform how theoretical frameworks detailing how smooth motor planning and responding proceeds, can be connected to theories framing effects of reward. The effects of reward have been conceptualized in reinforcement learning accounts. The latter mostly refer to the processing of reward prediction errors (RPEs)12, which are signed quantities describing the difference between expected and received rewards14. Anticipation of reward fosters behavioral performance and the size of a potential reward scales with dopamine system activity carrying reward information16. Sometimes, however, behavior is not affected by the reward magnitude or the valence of a predicted event. Instead, the mere un-expectedness of non-occurrences of predicted events, no matter whether these are “good” or “bad” events, has an effect17. Such surprises provide a signal similar to an unsigned  prediction error in reinforcement learning12, leading to the notion that violations of predictions (i.e. the surprisingness) and not necessarily the valence of rewards shape behavior. Thus, the question arises whether bindings between features determining motor sequences are affected by the magnitude and valence of rewards and/or by the surprisingness of predicted events, independent of the valence?Especially considering that "bindings" have been conceptualized as associations between action features19. Therefore, an overlap between a to-be-performed and a planned response impairs performance, which is reflected by increased reaction times (RTs) and higher error rates20. The degree of performance impairment depends on the strength of binding22. The employed experimental approach therefore uses an “ABBA design”, which requires that a planned response (A) is executed only after another response (B) is planned and performed immediately. Performance of this immediate response (B) is better when there is no feature overlap between the A and the B motor response, compared to when features overlapped between both responses20. The strength of bindings between features constituting response A determines how much response performance on B declines in the feature overlap, compared to the non-overlap condition. We used an orthogonal manipulation of reward magnitude and surprisingness of a specific reward magnitude (i.e. unsigned reward prediction error23) across different experimental groups (see methods section). Participants were rewarded upon fast responses on stimulus B. If reward magnitude modulates bindings between action features, the modulation of the action feature binding effect should be higher with higher rewards and not modulated by the surprisingness of the reward magnitude. If, however, it is the un-expectedness/non-occurrences of predicted events that is most important, binding effects should be modulated in an identical way no matter whether the magnitude of reward is higher or lower than expected. Considering behavioral performance, we not only examine mean changes in binding strengths, but also in how far the consistency in the strength of action feature bindings are modulated. This is relevant since enhanced dopamine system signaling, as induced by rewards and surprising events14, decreases the intra-individual variability in behavior and neurophysiological correlates thereof27. It is, therefore, well-conceivable that effects of reward magnitude and/or surprise do not only modulate the degree of binding strength between action features. Instead, it may be the variability in the strength of binding that is decreased by effects of reward magnitude and/or surprise.A consequence of bindings between features constituting a motor response is that once features for specific responses are activated, they are hardly available for the planning of other motor responses partly depending on the same features until the initiated motor response has been fully executedN = 39, 18 female, Mage = 25.7, SDage = 5.9; second sample: N = 39, 23 female, Mage = 25.9, SDage = 3.8; third sample: N = 34, 19 female, Mage = 27.7, SDage = 8.1; fourth sample: N = 16, 9 female, Mage = 26.9, SDage = 4.6). Sample sizes corresponded to previous studies suggesting at least middle effect sizes for these kind of bindings28; thus we can be assured that given α = 0.05, 1-β was > 0.80. Based on self-reports, none of the participants had a history of psychiatric or neurological disorders and did not receive centrally acting medication. All participants had normal or corrected-to-normal vision and stated to be right-handed.Four samples of participants were examined . The study was approved by the Ethics Committee of the TU Dresden and was conducted according to their guidelines, the tenets of the Declaration of Helsinki, and all relevant local regulations.20, also known as R-R task, which is shown in Fig. To investigate motor feature integration (binding), we chose an established paradigm28. The participants were seated at a distance of 60 cm in front of a 17-inch CRT display. For every trial, participants had to perform task A and task B in an interleaved ABBA sequence. Task B was embedded in task A. That is, participants were instructed to withhold the execution of a response to stimulus A until after presentation of and immediate response to stimulus B. Therefore, the effect of an already formed action plan (of task A) on the efficiency to plan and execute another action (of task B) can be observed. Every trial started with a fixation cross, which was displayed for 50 ms. This was followed by Stimulus A that was presented for 2000 ms. Stimulus A consisted of a left- or right-pointing arrowhead and a “ ~ ” either above or below the arrowhead. After stimulus A, another fixation cross was displayed again for 50 ms. Next, Stimulus B displayed either a “&” or a “#” for 200 ms. Following every trial, participants received a feedback about respective received or non-received points, which was displayed for 500 ms.This task was already used to in the context of EEG recordingsThe response to stimulus A consisted of a sequence of three consecutive parts: first, a button press for the direction of the arrowhead (left or right center button), second, a button press for the position of the “ ~ ” (above or below the first button press), last, a button press again for the arrowhead direction . This response chain needed to be withhold until Response B was executed. To Stimulus B, participants had to respond immediately with either a left button press for “&”, or a right button press for “#”. After responding to stimulus B, a question mark indicates to now carry out the previously planned response to stimulus A. Since the sides of the correct button press for responses to task A and task B could be the same or different, this resulted in two conditions for the trials: feature overlap (same side of response for task A and task B) and no feature overlap (sides switched between responses for task A and task B). In the following, we will use the term “Immediate Response” for the response to task B, and the term “Planned Responses” for the three consecutive responses to task A. Even though participants were instructed to respond as fast as possible, there was no time limit for responding to either task A or B. To exclude reaction time outliers, only trials with an Immediate Response faster than 1500 ms were included in the analyses. To quantify response time for the Planned Responses, the accumulated time between the onset of the Immediate Response and the third button press of the response sequence is used. One experimental session consisted of 12 practice trials, and 256 experimental trials. Feature overlap and no feature overlap conditions occurred equally often. Every participant had at least 200 valid trials  with at least 100 of these trials in the feature overlap condition.28.For an orthogonal manipulation of reward magnitude and reward expectation (surprise) on action feature integration (binding), the R-R task was expanded. Participants were instructed to earn as many points as possible to increase their bonus for participating in the study. These points could be collected in every trial, based on the accuracy of the responses. Only trials with a correct Immediate Response were rewarded. However, if the Planned Responses were also correct, participants earned double points in that trial. This reward scheme was designed to ensure that participants try to maximize their performance throughout the task. After a general instruction of the task and the required responses, participants were further introduced to the group specific reward conditions. E.g., for the standard-low surprise-high participants the explanation was done as following: ‘If you responded correctly to the second symbol, a # or a &, you will earn 10 points. If you also responded correctly to the first symbol, those points are doubled, so you earn 20 points. No points are earned, if the response to the second symbol was incorrect, regardless of the response to the first symbol.’ Trials with an incorrect Immediate Response were neither rewarded nor analyzed, since correct Immediate Response RTs are necessary to study the binding effect in the R-R task28 and newly recruited subjects.To examine the effect of reward magnitude, both samples of reward manipulation participants were exposed to different overall reward conditions: standard low—surprise high group could earn 10 or 20 points per trial (low reward condition), and standard high—surprise low group could earn 50 or 100 points per trial (high reward condition). For comparison, a third sample in which participants performed the R-R task without receiving rewards (i.e. no reward group), was formed from a previous study of our work group29. To build schema-discrepancy, first, a schema was established in so-called standard trials, which made up 224 out of the 256 trials in the experiment. In standard trials, stimuli were displayed in white font and corrected responses were rewarded as previously described  and by being less probable (i.e. state prediction error)30. Further in this paper, we will refer to this combination of those two types, simply as “surprise”. To distinguish between two directions of reward magnitude change, the terms “positive surprise” (i.e. raise of reward magnitude) and “negative surprise” (i.e. decrease of reward magnitude) will be used. This is to avoid misconception with the terms positive or negative prediction errors30, since our design does not separate the reward prediction error from the state prediction error. The color-coding of trials was established to also ensure participants identified the presented trial condition immediately. Thus, surprise effects were evoked with the start of the trial. This design allowed us to independently modulate effects of reward magnitude and surprise.To investigate surprise effects in the reward manipulation groups, we aimed to evoke schema-discrepancy among those participantssee Fig. A. The resee Fig. B, and byTo distinguish surprise from general environmental conditions , we conducted an additional, fourth sample of participants, which were rewarded for correct responses and encountered the same color change in the same frequency, but without any change of reward magnitude. Thus, with color being the only difference between trials for this sample, we can ensure that surprise effects found in our experimental groups are actually based on surprise rather than stimulus features.28, therefore, analyses were run on Stimulus B responses. Mean accuracy (percentage of correct responses) and means of RT (for correct responses) were calculated for each participant, each feature overlap condition , and each reward condition. To quantify response variability, standard deviations (SDs) of RTs were calculated and investigated using the same experimental factors. Since performance of the Planned Response does not reflect action file binding effects, analyzing those parameters in detail was not the main focus of our analyses. All three responses to Stimulus A belong to one response chain and are therefore reported as done in previous papers regarding the R-R task: as accumulated RT and average accuracy of all three responses28. Action file binding was calculated as a difference of the feature overlap minus the no feature overlap conditions, separately for accuracy, RT and SDRT of the Immediate Response. Higher values indicate stronger binding effects. To investigate effects of reward magnitude (low reward vs high reward) and of surprise , repeated measures ANOVAs with Bonferroni-adjusted post-hoc analysis were used on accuracy, RT, and SDRT data of the reward manipulation samples. Differences between the binding effects of those reward manipulation samples were analyzed in independent t-tests. Additionally, to distinguish between reward magnitude effects and pure reward effects, binding data of the third sample, i.e. no reward group were compared separately to standard low group and to standard high group also through several independent t-tests. The reported p values of all t-tests are FDR-corrected. The Bayes factor as BF10 is reported to quantify the evidence for the null hypothesis. In the Bayesian t-tests, the default Cauchy prior was used with the scale of 0.707. For interpretation of the reported Bayes factors, we used and suggest to refer to the categorical evaluation according to Jeffreys31. A BF10 of 1 would be read as no evidence for H1, up to 3 as anecdotal, up to 10 as moderate, up to 30 as strong, up to 100 as very strong, and above 100 as extreme evidence for the H1. In contrast, values beneath 1 and up to 1/3 would be read as anecdotal, up to 1/10 as moderate, up to 1/30 as strong, up to 1/100 as very strong, and smaller than 1/100 as extreme evidence for the H0.Statistical analyses of the behavioral data were performed by using SPSS. Performances of the Immediate Response reflect action file bindingRT) of the Immediate Response. Main and interaction effects of the following factors were analyzed: Feature overlap condition (levels: feature overlap vs. no feature overlap), overall reward magnitude (levels: low vs. high), and surprise . In these first ANOVA models, we did not consider the group factor  because this factor is co-linear with the factors reward magnitude and surprise. To compare binding effects between both reward manipulation groups, we calculated an independent t-test for binding in accuracy, RT, and SDRT later on (see below). Figure RT) across three different groups  in standard trials  and rewarded high  in surprise trials . Another sample was rewarded high (50 to 100 points) in standard trials and rewarded low (10 to 20 points) in surprise trials. This way, conditions and points for both groups were exactly mirror-reversed to one another. For these groups, we first calculated repeated measures ANOVAs on accuracy, response speed (RT), and variability of response speed  = 0.11, p = 0.746, η2 = 0.00, BF10 = 0.129), overall reward magnitude  = 1.47, p = 0.229, η2 = 0.02, BF10 = 0.257), and surprise  = 2.06, p = 0.155, η2 = 0.03, BF10 = 0.454). Thus, the accuracy parameter showed no binding effect, and remained the same in all overall reward magnitude and surprise manipulations.The accuracy of the Immediate Response was unaffected by feature overlap  = 8.72, p = 0.004, η2 = 0.10, BF10 > 100) with slower response times for the feature overlap (540.31 ms ± 18.58) than for the no feature overlap trials . Additionally, the RT data showed significant main effects of overall reward magnitude  = 26.81, p < 0.001, η2 = 0.03, BF10 > 100), and surprise  = 6.35, p = 0.014, η2 = 0.08, BF10 = 0.614). That is, responses were slower when expecting a low reward (543.56 ms ± 18.88) than a high reward , and slower in standard trials (537.53 ms ± 18.25) than in surprise trials . Therefore, a high magnitude of reward and a surprise improves response speed. Regarding binding, neither overall reward magnitude  = 0.36, p = 0.550, η2 = 0.01, BF10 = 0.143), nor surprise  = 2.49, p = 0.118, η2 = 0.03, BF10 = 0.377) showed significant interaction with feature overlap.Regarding the response speed in trials with correct Immediate Response, a significant effect of feature overlap was found  = 10.67, p = 0.002, η2 = 0.12, BF10 = 1.115) was found for the variability of response speed (i.e. SDRT). That is, participants showed a higher variability of response speed in the feature overlap condition (214.20 ms ± 6.67), compared to the no feature overlap condition . The SDRT was not affected by overall reward magnitude  = 2.57, p = 0.113, η2 = 0.03, BF10 = 0.159), but showed significant effects of surprise  = 971.96, p < 0.001, η2 = 0.93, BF10 > 100): In standard trials, the variability of response speed was more than twice as high (289.76 ms ± 9.28) as in surprise trials . Furthermore, a significant interaction of surprise and feature overlap was found  = 10.86, p = 0.001, η2 = 0.13, BF10 = 4.422). The Bonferroni adjusted post-hoc test showed significant differences between feature overlap conditions only in standard trials  and not in surprise trials .Similarly to the RT data, a significant main effect of feature overlap , we performed a RM-ANOVA on the fourth sample with the factors feature overlap and trial color . For RT, a significant effect of feature overlap was found  = 1.556, p = 0.039, η2 = 0.04, BF10 = 1.391), with higher response times for the feature overlap (543.91 ms ± 17.19) than for the no feature overlap trials . In contrast to surprise, trial color had no significant effect on response times  = 1.374, p = 0.259, η2 = 0.017, BF10 = 0.320). For SDRT, again, a significant main effect of feature overlap  = 1.303, p = 0.048, η2 = 0.001, BF10 = 0.407) was found. That is, participants showed a higher variability of response speed in the feature overlap condition (215.40 ms ± 6.37), compared to the no feature overlap condition . The SDRT showed a significant main effect of trial color  = 145.5, p < 0.001, η2 = 0.884, BF10 > 100) equal to the main effect of surprise: In white trials , the variability of response speed was twice as high (289.71 ms ± 22.66) as in green trials . However, no significant interaction of trial color and feature overlap was found  = 0.061, p = 0.808, η2 = 0.001, BF10 = 0.419). In conclusion, the found interaction with action file binding in standard low surprise high group and standard high surprise low group is actually based on surprise and not on color differences of trials.To verify that these found surprise effects in RT and SDRT, the RT post-hoc analysis showed both, a significant difference between feature overlap conditions in standard trials , and in surprise trials .Based on these results, we also conducted a Bonferroni adjusted post-hoc test for the non-significant interaction between feature overlap and surprise on RT. In contrast to the SDRT) and no feature overlap RT (i.e. no feature overlap SDRT) presumably reflects the variability of action file binding strength. Consequently, the data suggest that surprise decreases variability of action file binding strength, but not the mean magnitude of binding. Reward magnitude had no effect on variability or the mean magnitude of binding.The difference between feature overlap RT and no feature overlap RT represents action file binding strength. Thus, the difference between variability of feature overlap RT  = 0.682, p = 0.497, d = 0.154, BF10 = 0.287). The same was the case for the RT (t(76) = 0.396, p = 0.693, d = 0.09, BF10 = 0.251) and the SDRT (t(76) = 0.396, p = 0.903, d = 0.028, BF10 = 0.236). Standard low trials and standard high trials did also not differ in accuracy (t(76) = 0.552, p = 0.583, d = 0.125, BF10 = 0.268), RT (t(76) = 0.208, p = 0.836, d = 0.047, BF10 = 0.239), or SDRT (t(76) = 0.218, p = 0.828, d =  − 0.049, BF10 = 0.240). Finally, no significant differences between surprise high trials and surprise low trials occurred in accuracy (t(58.124) = 0.916, p = 0.362, d = 0.207, BF10 = 0.338), RT (t(76) = 0.717, p = 0.476, d = 0.162, BF10 = 0.294), or SDRT (t(76) = 0.591, p = 0.556, d = 0.134, BF10 = 0.273). Thus, the factor group did not affect binding effects. As mentioned before, group membership reflects the combination of overall reward magnitude and surprise: standard low—surprise high group experienced surprise as positive , while standard high—surprise low group experienced surprise as negative . Since no significant differences in the measured binding effects was found between the groups, this indicates that surprise influences binding strength regardless of its direction .To compare binding effects between both reward manipulation groups, we calculated an independent t-test for the binding effect  in accuracy, RT, and SDTo distinguish between reward magnitude effects and surprise effects on action file binding, we performed t-tests comparing a third control sample  and each reward manipulation group. The control group performed the original R-R task without reward magnitude and surprise manipulations. We compared the binding effects (i.e. difference feature overlap minus no feature overlap) in all trials of no reward group to binding effects of the reward manipulation groups in general  and in all four trial conditions .t(71) = 2.519, p = 0.028 d = 0.591, BF10 = 0.778). Similarly, accuracy binding in standard low trials were significantly higher (0.44 ± 1.50) in comparison to no reward group (− 0.36 ± 1.15), (t(71) = 2.538, p = 0.028, d = 0.596, BF10 = 1.473). In our repeated measures ANOVA of the reward manipulation groups, we found that binding in SDRT is vastly higher in both kinds of standard trials than in both kinds of surprise trials. Therefore, the comparison between binding in both surprise trials of the reward manipulation groups and the no reward group was particularly interesting. Indeed, the SDRT binding in surprise high trials (3.94 ± 15.02) and in surprise low trials (1.48 ± 21.20) was significantly lower than in all trials of no reward group (12.24 ± 18.99), (t(71) =  − 2.084, p = 0.041, d =  − 0.489, BF10 = 2.283; t(71) =  − 2.270, p = 0.035, d =  − 0.533, BF10 = 2.957). None of the other comparisons between no reward group and the reward manipulation groups were significant . Thus, surprise, regardless of it being positive  or negative , decreases variability of action file binding strength.Comparing binding effects, only the accuracy binding of standard low—surprise high group was significantly higher (0.38 ± 1.36) than binding of no reward group (− 0.36 ± 1.15),  in surprise effects.The goal of the current study was to provide further insights into the nature of bindings between different motor features constituting complex motor sequences. To this end we examined in how far motor feature bindings are affected by reward magnitude or the effects of an unsigned surprise signal. As an experimental approach, we used an established paradigm inspired by the theory of event coding framework33. Taken together, surprise might not directly influence the volatility of action file binding but might do so via attention allocation34.We found that the consistency of action file binding strength is modulated by surprise, but not by reward magnitude. Surprise decreases variability of action file binding strength. Importantly, the quality of the surprise was not essential for this effect since RT variability modulations did not differ depending on whether the surprise was positive (increase of reward magnitude) or negative (decrease of reward magnitude). For reward magnitude and surprise modulation groups, both qualities of surprise were indicated by the stimuli color at the beginning of trials . We were able to rule out the possibility that the mere change of color had caused this effect, since the sample which did not experience any change of reward magnitude (i.e. no positive or negative surprise) when trial colors changed, did not show a decreased variability of action file binding strength. Opposed to the consistency of action file binding strength across trials, the average magnitude of action file binding effects was not modulated by reward or surprise manipulations. A more fine-grained interpretation is that surprise signals recruit an attentional orienting response reducing time-on-task effects such as attentional lapses or mind-wandering, and thereby reducing variability in processing leading in turn to less variability in action file binding effects. In other words, the surprise signals may indicate to participants that the upcoming trial is "special", thereby allocating more task-directed attention and as a consequence more consistent behavior emerges as in the other trials which are likely subject to occasional attentional lapses. Such effects of decreasing behavioral variability via attention allocation is consistent with other findings reported in the literature35. With such high accuracy scores as observed in this paradigm, it is possible that a significant main effect of feature overlap (i.e. action file binding) on accuracy is masked. Nevertheless, there is no indication that action file binding strength in accuracy would be affected by surprise, since action file binding strength in response speed was not modulated by surprise either. A modification of the classical R-R paradigm with an increased degree of difficulty, causing lower accuracy scores, could further secure our conclusion on the role of unsigned surprise on motor feature integration.The used established R-R paradigm has no time limit for Planned and Immediate Response, which can encourage to sacrifice faster reaction time in favor of a higher hit rate (i.e. speed-accuracy trade-off)12. One framework, the so-called predicted response outcome (PRO) model, suggests that medial prefrontal brain structures  predict the likely outcome of action un-related to the valence of that outcome. This theory’s aim is to clarify how we make predictions about potential outcomes before carrying out an action, as well as how we evaluate afterwards the degree in which our predictions match the actual outcomes36. According to the PRO model, only the unexpectedness (surprisingness) is essential for action control. The current findings on the modulability of action file bindings are in line with that model; however, it was not average binding strength that was altered, but the consistency in binding effects. This outcome is not directly predicted by the PRO model of unsigned surprise signaling, but can nevertheless be explained by the PRO model. Regarding this, it is important to consider that the PRO model is built on assumptions of the reinforcement learning theory and temporal difference learning model in particular12. Viewed from a neurobiological perspective, computational principles of the models are related to the functioning of the dopaminergic system, making it likely that also the dopaminergic system is triggered by surprise signals38. Crucially, the dopaminergic system is not only implicated in the processing of rewards and surprise signals. Rather, the dopaminergic system does also modulates the consistency of  processes underlying cognitive functions27. The likely reason is that increased dopaminergic activity reflects the recruitment of attention as much as it decreases neuronal noise39, which leads to more distinct and stable cortical representations resulting in decreases and variability in cognitive performance27. These effects have also been referred to as gain control, which describes a general working principle in neural networks found at sensory, cognitive42 and motor levels44. Increasing gain control can be viewed as sharpening the responsivity of the neural network and as increasing the signal–noise-ratio (SNR) of information processing. Byres and Serences45 note that mechanisms aimed to increase top-down (cognitive) control and processes subserving learning-relevant mechanisms (such as surprise processing) operate by increasing the SNR in neural circuits46. This increase in the SNR is then likely leading to a higher consistency in binding and motor feature integration effects as observed in the current study.Several theoretical frameworks have been put forward to conceptualize the effects of unsigned surprise signals47, because this theory is mostly ground in cognitive psychology. Nevertheless, propositions of neural gain control are commensurable with the theoretical framing of TEC. This becomes particularly clear when considering the so-called “meta-control state model (MSM)”48, closely connected to TEC. The dopaminergic system is known to affect metacontrol processes51. In recent years, TEC has been connected to so-called metacontrol frameworks54 describing that response selection (and cognitive control processes) are not static. According to the metacontrol hypothesis49 there is evidence suggesting that cognitive control is not a unitary function but an emerging property of the interaction of systems promoting cognitive persistence including focusing on one goal and on relevant information, and systems promoting cognitive flexibility that is needed, for instance, for switching to other plans, opening up for other opportunities, and considering a broader range of possibilities. Following the MSM, a person can be biased towards persistence or towards flexibility, when two or more goal-directed representations compete for selection49. Recent findings from our research group showed that a bias towards cognitive persistence reduces binding effects55. This is the case because more top-down control sustains an effective focus on one goal and on relevant information. Thus, not goal-compatible information is discarded reducing time-consuming binding reconfiguration processes. The current results suggest that this is also associated with an increase in the temporal consistency of binding reconfiguration processes taking place. The results obtained are thus compatible with several theoretical conceptions, for which the current results also suggest that these can be integrated.However, considerations related to principles of neural gain control have not been much around in the Theory of Event Coding