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Purpose: Hypothalamic-pituitary thyrotropic activity (HPta) is crucial since TSH is the mainstay for evaluating primary hypothyroidism (hT) and replacement therapy in clinical practice. Despite TSH values, some patients experience symptoms and metabolic alterations, raising several issues about hT. The aim of the study was to investigate factors influencing the TSH values achieved after a period of hT induced in a standardized and controlled manner (TSH_time1)., Methods: Our institutional database was searched to extract records of differentiated thyroid cancer (DTC) patients undergoing a LT4 withdrawal protocol prior to radioiodine (RAI) administration. We collected clinical and biochemical parameters before LT4 discontinuation and after one month of induced hT. We performed Mann-Whitney U-test and linear regression analyses., Results: We included 102 patients, with a median age of 44 years. In univariate analyses, TSH_time1 was correlated with age (p 0.005) and the dose pro Kg/die of LT4 assumed until the discontinuation of LT4 (LT4_dose) (p 0.023). The higher the age, the lower the TSH_time1 level. The higher the LT4_dose, the higher the TSH_time1 level. After multivariate analysis, the fittest model included age, BMI, LT4_dose, and systemic inflammation response index with an adjusted R 2 of 0.4., Conclusion: The study highlights new mechanisms that influence HPta. HPta progressively reduces with age, and this must be considered when evaluating TSH values in the elderly. Furthermore, we report for the first time a rebound effect of HPta, determined by the dose pro Kg/die of LT4 taken prior to its discontinuation. Inflammation and metabolic status also affect these phenomena., (© 2024. The Author(s).)

The continuous emergence of new SARS-CoV-2 variants requires that COVID vaccines be updated to match circulating strains. We generated B/HPIV3-vectored vaccines expressing 6P-stabilized S protein of the ancestral, B.1.617.2/Delta, or B.1.1.529/Omicron variants as pediatric vaccines for intranasal immunization against HPIV3 and SARS-CoV-2 and characterized these in hamsters. Following intranasal immunization, these B/HPIV3 vectors replicated in the upper and lower respiratory tract and induced mucosal and serum anti-S IgA and IgG. B/HPIV3 expressing ancestral or B.1.617.2/Delta-derived S-6P induced serum antibodies that effectively neutralized SARS-CoV-2 of the ancestral and B.1.617.2/Delta lineages, while the cross-neutralizing potency of B.1.1.529/Omicron S-induced antibodies was lower. Despite the lower cross-neutralizing titers induced by B/HPIV3 expressing S-6P from B.1.1.529/Omicron, a single intranasal dose of all three versions of B/HPIV3 vectors was protective against matched or heterologous WA1/2020, B.1.617.2/Delta or BA.1 (B.1.1.529.1)/Omicron challenge; hamsters were protected from challenge virus replication in the lungs, while low levels of challenge virus were detectable in the upper respiratory tract of a small number of animals. Immunization also protected against lung inflammatory response after challenge, with mild inflammatory cytokine induction associated with the slightly lower level of cross-protection of WA1/2020 and B.1.617.2/Delta variants against the BA.1/Omicron variant. Serum antibodies elicited by all vaccine candidates were broadly reactive against 20 antigenic variants, but the antigenic breadth of antibodies elicited by B/HPIV3-expressed S-6P from the ancestral or B.1.617.2/Delta variant exceeded that of the S-6P B.1.1.529/Omicron expressing vector. These results will guide development of intranasal B/HPIV3 vectors with S antigens matching circulating SARS-CoV-2 variants., Competing Interests: Competing Interest U.J.B., C.L., X.L, and C.LN. are inventors on the provisional patent application number 63/180,534, entitled “Recombinant chimeric bovine/human parainfluenza virus 3 expressing SARS-CoV-2 spike protein and its use”, filed by the United States of America, Department of Health and Human Services

Purpose: MKNR3 is a paternally expressed gene whose mutations are the main cause of central precocious puberty (CPP). Protein circulating levels can be easily measured, as demonstrated in idiopathic CPP and healthy controls. No data are available for patients harboring an MKRN3 mutation. Our aim was to perform MKRN3 mutation screening and to investigate if circulating protein levels could be a screening tool to identify MKRN3 mutation in CPP patients., Methods: We enrolled 140 CPP girls and performed MKRN3 mutation analysis. Patients were stratified into two groups: idiopathic CPP (iCPP) and MKRN3 mutation-related CPP (MKRN3-CPP). Clinical characteristics were collected. Serum MKRN3 values were measured by a commercially available ELISA assay kit in MKRN3-CPP and a subgroup of 15 iCPP patients., Results: We identified 5 patients with MKRN3 mutations: one was a novel mutation (p.Gln352Arg) while the others were previously reported (p.Arg328Cys, p.Arg345Cys, p.Pro160Cysfs*14, p.Cys410Ter). There was a significant difference in circulating MKRN3 values in MKRN3-CPP compared to iCPP (p < 0.001). In MKRN3-CPP, the subject harboring Pro160Cysfs*14 presented undetectable levels. Subjects carrying the missense mutations p.Arg328Cys and p.Gln352Arg showed divergent circulating protein levels, respectively 40.56 pg/mL and undetectable. The patient with the non-sense mutation reported low but measurable MKRN3 levels (12.72 pg/mL)., Conclusions: MKRN3 defect in patients with CPP cannot be predicted by MKRN3 circulating levels, although those patients presented lower protein levels than iCPP. Due to the great inter-individual variability of the assay and the lack of reference values, no precise cut-off can be identified to suspect MKRN3 defect., (© 2023. The Author(s), under exclusive licence to Italian Society of Endocrinology (SIE).)

Eggs and embryo manipulation is an important biotechnological challenge to enable positioning, entrapment, and selection of reproductive cells to advance into a new era of nature-like assisted reproductive technologies. Oviductin (OVGP1) is an abundant protein in the oviduct that binds reversibly to the zona pellucida, an extracellular matrix that surrounds eggs and embryos. Here, the study reports a new method coupling OVGP1 to magnetic nanoparticles (NP) forming a complex (NPOv). NPOv specifically surrounds eggs and embryos in a reversible manner. Eggs/embryos bound to NPOv can be moved or retained when subjected to a magnetic force, and interestingly only mature-competent eggs are attracted. This procedure is compatible with normal development following gametes function, in vitro fertilization, embryo development and resulting in the birth of healthy offspring. The results provide in vitro proof-of-concept that eggs and embryos can be precisely guided in the absence of physical contact by the use of magnets., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Immunization via the respiratory route is predicted to increase the effectiveness of a SARS-CoV-2 vaccine. Here, we evaluate the immunogenicity and protective efficacy of one or two doses of a live-attenuated murine pneumonia virus vector expressing SARS-CoV-2 prefusion-stabilized spike protein (MPV/S-2P), delivered intranasally/intratracheally to male rhesus macaques. A single dose of MPV/S-2P is highly immunogenic, and a second dose increases the magnitude and breadth of the mucosal and systemic anti-S antibody responses and increases levels of dimeric anti-S IgA in the airways. MPV/S-2P also induces S-specific CD4 + and CD8 + T-cells in the airways that differentiate into large populations of tissue-resident memory cells within a month after the boost. One dose induces substantial protection against SARS-CoV-2 challenge, and two doses of MPV/S-2P are fully protective against SARS-CoV-2 challenge virus replication in the airways. A prime/boost immunization with a mucosally-administered live-attenuated MPV vector could thus be highly effective in preventing SARS-CoV-2 infection and replication., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The semen of boar is characterized by ejaculation in well-differentiated fractions with specific concentration, composition, and volume. The 'sperm-rich fraction' (SRF), the most concentrated seminal fraction, is habitually collected in insemination centers to make artificial insemination (AI) doses. The absence of the other fractions in AI doses could alter the uterine reaction to AI and not trigger essential responses that could maximize fertility. Thus, there is an urge to ascertain the impact of different ejaculate fractions on the uterus after AI to optimize the semen doses. This work analyzed specific parameters related to fertility in pregnant artificially inseminated sows (n = 15) with ac-cumulative fractions of the semen of boars (n = 6): F1, composed of the sperm-rich fraction (SRF); F2, composed of F1 plus the intermediate fraction; F3, composed of F2 plus the post-SRF. Non-inseminated sows (n = 5) were included as control (C). The different types of seminal dose did not affect the number of ovulated follicles (CL; corpora lutea, p > 0.05) but did affect the embryo development (p < 0.05). The proportion of embryos in morula stages was significantly higher in AI-F1 sows (84.4%, p < 0.05). Morulas and blastocysts were balanced in AI-F2 or AI-F3 (p > 0.05). Independently of the type of seminal dose (F1, F2, or F3), we observed by immunohistochemistry that AI significantly increased uterine vascularization, although with some anatomical differences. The cranial region of the uterine horns was significantly more vascularized in AI-F1 or AI-F2 sows (26.7 ± 2.3 and 28.6 ± 2.0%, respectively), and AI-F3 showed significantly less vascularization at that point (17.8 ± 1.6%, p < 0.05). To summarize, the synergistic effect of all ejaculate fractions accelerates embryo development, at least during the preimplantation period, and increases the uterine reaction to AI in certain parts of the uterus., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Respiratory syncytial virus (RSV) is the leading viral cause of bronchiolitis and pneumonia in infants and toddlers, but there currently is no licensed pediatric vaccine. A leading vaccine candidate that has been evaluated for intranasal immunization in a recently completed phase 1/2 clinical trial is an attenuated version of RSV strain A2 called RSV/ΔNS2/Δ1313/I1314L (hereafter called ΔNS2). ΔNS2 is attenuated by deletion of the interferon antagonist NS2 gene and introduction into the L polymerase protein gene of a codon deletion (Δ1313) that confers temperature-sensitivity and is stabilized by a missense mutation (I1314L). Previously, introduction of four amino acid changes derived from a second RSV strain "line 19" (I79M, K191R, T357K, N371Y) into the F protein of strain A2 increased the stability of infectivity and the proportion of F protein in the highly immunogenic pre-fusion (pre-F) conformation. In the present study, these four "line 19" assignments were introduced into the ΔNS2 candidate, creating ΔNS2-L19F-4M. During in vitro growth in Vero cells, ΔNS2-L19F-4M had growth kinetics and peak titer similar to the ΔNS2 parent. ΔNS2-L19F-4M exhibited an enhanced proportion of pre-F protein, with a ratio of pre-F/total F that was 4.5- to 5.0-fold higher than that of the ΔNS2 parent. The stability of infectivity during incubation at 4°C, 25°C, 32°C and 37°C was greater for ΔNS2-L19F-4M; for example, after 28 days at 32°C, its titer was 100-fold greater than ΔNS2. ΔNS2-L19F-4M exhibited similar levels of replication in human airway epithelial (HAE) cells as ΔNS2. The four "line 19" F mutations were genetically stable during 10 rounds of serial passage in Vero cells. In African green monkeys, ΔNS2-L19F-4M and ΔNS2 had similar growth kinetics, peak titer, and immunogenicity. These results suggest that ΔNS2-L19F-4M is an improved live attenuated vaccine candidate whose enhanced stability may simplify its manufacture, storage and distribution, which merits further evaluation in a clinical trial in humans., Competing Interests: J.A.-S., M.K., C. Lai., M.P., S.D. and L.Z. are current or former employees of Sanofi and may hold Sanofi stocks. C. Luongo., P.L.C., and U.J.B. are inventors on patents covering part of this material, filed by the United States, Department of Health and Human Services., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Background: Respiratory syncytial virus (RSV) is the leading cause of pediatric lower respiratory illness (LRI) and a vaccine for immunization of children is needed. RSV/6120/ΔNS2/1030s is a cDNA-derived live-vaccine candidate attenuated by deletion of the interferon antagonist NS2 gene and the genetically stabilized 1030s missense polymerase mutation in the polymerase, conferring temperature sensitivity., Methods: A single intranasal dose of RSV/6120/ΔNS2/1030s was evaluated in a double-blind, placebo-controlled trial (vaccine to placebo ratio, 2:1) at 105.7 plaque-forming units (PFU) in 15 RSV-seropositive 12- to 59-month-old children, and at 105 PFU in 30 RSV-seronegative 6- to 24-month-old children., Results: RSV/6120/ΔNS2/1030s infected 100% of RSV-seronegative vaccinees and was immunogenic (geometric mean RSV plaque-reduction neutralizing antibody titer [RSV-PRNT], 1:91) and genetically stable. Mild rhinorrhea was detected more frequently in vaccinees (18/20 vaccinees vs 4/10 placebo recipients, P = .007), and LRI occurred in 1 vaccinee during a period when only vaccine virus was detected. Following the RSV season, 5 of 16 vaccinees had ≥4-fold rises in RSV-PRNT with significantly higher titers than 4 of 10 placebo recipients with rises (1:1992 vs 1:274, P = .02). Thus, RSV/6120/ΔNS2/1030s primed for substantial anamnestic neutralizing antibody responses following naturally acquired RSV infection., Conclusions: RSV/6120/ΔNS2/1030s is immunogenic and genetically stable in RSV-seronegative children, but the frequency of rhinorrhea in vaccinees exceeded that in placebo recipients., Clinical Trials Registration: NCT03387137., Competing Interests: Potential conflicts of interest . C. L., P. L. C., and U. J. B. are inventors on US patents pertaining to this vaccine candidate and its attenuating mutations. R. A. K. receives additional grant funding from Sanofi through her institution. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The diagnostic accuracy of thyroid fine-needle aspiration (FNA) can be highly influenced by the technical skills of the operator performing the procedure and by interobserver variability in microscopic interpretation. This is particularly true for the indeterminate categories. Recently, molecular testing has been proposed as an ancillary tool for monitoring the performance of different thyroid cytopathology practices. The objective of this multicenter study was to evaluate the quality of different local cytopathology practices by assessing the impact of interventional cytopathologists on FNA adequacy for molecular testing and the variations in mutation rates across different health care centers operating in the Campania region., Methods: The study included 4651 thyroid FNA samples diagnosed in different Southern Italian clinical laboratories belonging to the TIRNET (the Tiroide Network). FNA samples were collected by different proceduralists and were classified by local cytopathologists according to The Bethesda System for Reporting Thyroid Cytopathology. FNAs classified as atypia of undetermined significance, follicular neoplasm, suspicious for malignancy, and malignant were centralized for a real-time polymerase chain reaction-based, seven-gene test at the authors' institution., Results: Centers that employed interventional cytopathologists obtained fewer unsatisfactory FNA samples for molecular testing (11.3%) than centers that employed noncytopathologists (16.7%; p < .05). Furthermore, a significant variation in the mutation rate was observed in FNAs diagnosed by different local cytopathologists; indeterminate categories had the highest percentage of mutation rate variability among centers., Conclusions: Interventional cytopathologists obtained higher yields of diagnostic material for molecular testing. Finally, the current results suggest that the variability in mutation rates among different centers may highlight the low reproducibility of microscopic criteria among cytopathologists, particularly for indeterminate cases., (© 2023 The Authors. Cancer Cytopathology published by Wiley Periodicals LLC on behalf of American Cancer Society.)

Next-generation SARS-CoV-2 vaccines are needed that induce systemic and mucosal immunity. Murine pneumonia virus (MPV), a murine homolog of respiratory syncytial virus, is attenuated by host-range restriction in nonhuman primates and has a tropism for the respiratory tract. We generated MPV vectors expressing the wild-type SARS-CoV-2 spike protein (MPV/S) or its prefusion-stabilized form (MPV/S-2P). Both vectors replicated similarly in cell culture and stably expressed S. However, only S-2P was associated with MPV particles. After intranasal/intratracheal immunization of rhesus macaques, MPV/S and MPV/S-2P replicated to low levels in the airways. Despite its low-level replication, MPV/S-2P induced high levels of mucosal and serum IgG and IgA to SARS-CoV-2 S or its receptor-binding domain. Serum antibodies from MPV/S-2P-immunized animals efficiently inhibited ACE2 receptor binding to S proteins of variants of concern. Based on its attenuation and immunogenicity in macaques, MPV/S-2P will be further evaluated as a live-attenuated vaccine for intranasal immunization against SARS-CoV-2., Competing Interests: U.J.B., C.L.N., S.M., C.L., and J.K. are inventors on a provisional patent application entitled “Recombinant murine pneumonia virus expressing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein”, filed by the United States, Department of Health and Human Services.

The seminal plasma (SP) is the liquid component of semen that facilitates sperm transport through the female genital tract. SP modulates the activity of the ovary, oviductal environment and uterine function during the periovulatory and early pregnancy period. Extracellular vesicles (EVs) secreted in the oviduct (oEVs) and uterus (uEVs) have been shown to influence the expression of endometrial genes that regulate fertilization and early embryo development. In some species, semen is composed of well-separated fractions that vary in concentration of spermatozoa and SP composition and volume. This study aimed to investigate the impact of different accumulative fractions of the porcine ejaculate (F1, composed of the sperm-rich fraction, SRF; F2, composed of F1 plus the intermediate fraction; F3, composed of F2 plus the post-SRF) on oEVs and uEVs protein cargo. Six days after the onset of estrus, we determined the oEVs and uEVs size and protein concentration in pregnant sows by artificial insemination (AI-sows) and in non-inseminated sows as control (C-sows). We also identified the main proteins in oEVs and uEVs, in AI-F1, AI-F2, AI-F3, and C-sows. Our results indicated that although the size of EVs is similar between AI- and C-sows, the protein concentration of both oEVs and uEVs was significantly lower in AI-sows ( p < 0.05). Proteomic analysis identified 38 unique proteins in oEVs from AI-sows, mainly involved in protein stabilization, glycolytic and carbohydrate processes. The uEVs from AI-sows showed the presence of 43 unique proteins, including already-known fertility-related proteins (EZR, HSPAA901, PDS). We also demonstrated that the protein composition of oEVs and uEVs differed depending on the seminal fraction(s) inseminated (F1, F2, or F3). In conclusion, we found specific protein cargo in oEVs and uEVs according to the type of semen fraction the sow was inseminated with and whose functions these specific EVs proteins are closely associated with reproductive processes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Toledo-Guardiola, Luongo, Abril-Parreño, Soriano-Úbeda and Matás.)

Boar ejaculate is composed of sperm cells and seminal plasma (SP) and is emitted in different fractions (pre-sperm fraction; spermatic-rich fraction; intermediate fraction; post-spermatic fraction), with different composition of SP and volume, which could influence the sperm quality during seminal doses preparation, conservation, and interaction with the female reproductive tract. In artificial insemination (AI) centers, seminal doses are usually prepared with the spermatic-rich and intermediate fractions, but the inclusion of other ejaculate fractions, although controversial, is beginning to be applied. The objective was to evaluate the synergic effect of accumulative ejaculated fractions on sperm functionality during seminal doses preparation, throughout storage and after incubation with uterine fluid (UF). For this purpose, a total of 57 ejaculates were collected, and the following experimental groups were prepared (n = 19 per group): (F1) spermatic-rich fraction; (F2) F1 plus intermediate fraction; (F3) F2 plus post-spermatic fraction. Each group was stored for 5 days at ∼16 °C, and the following parameters were evaluated: sperm metabolism of pure and diluted semen (day 1), sperm quality parameters (days 1, 3, 5), thermal-resistance test (TRT) and incubation with uterine fluid (UF) (day 5). Sperm metabolic rates between accumulative ejaculate fractions from pure and diluted semen did not show differences. Also, sperm quality parameters were not affected by the ejaculate fraction during storage. However, sperm subjected to TRT showed similar results except for progressive motility, which was better in F2 and F3 than F1. When sperm were incubated with UF, the quality decreased in each group, but sperm from F2 and F3 were less affected than those from F1. In conclusion, the post-spermatic fraction can be included in seminal doses for their use in AI-centers, with functionality of sperm of different SP origins not being impaired throughout the storage, and responding better to thermal and UF stress. However, further research in AI-centers is necessary to test the sperm behaviour under presented conditions., (© 2023. Springer Nature Limited.)

In a previous study, our group detected the cholecystokinin (CCK) protein in the porcine oviduct. This fact, together with the involvement of CCK in the regulation of sperm protein tyrosine phosphorylation by the modulation of HCO 3 - for 1 h at 38.5°C. Sperm motion (total and progressive motility), kinetic parameters, viability, acrosome status, and mitochondrial activity were determined. No differences between groups (0, 25 or 50 μM of CCK) were observed when HCO 3 - for 1 h at 38.5°C. Sperm motion (total and progressive motility), kinetic parameters, viability, acrosome status, and mitochondrial activity were determined. No differences between groups (0, 25 or 50 μM of CCK) were observed when HCO 3 - was absent in the media (p > .05). However, the results showed that when the media was supplemented with 5 mmol/L HCO 3 - in 1-day seminal dose storage, the linearity index (LIN, %), straightness index (STR, %) and oscillation index (WOB, %) (sperm kinetics parameters) increased in the presence of CCK regardless the concentration (p < .05). Nevertheless, CCK in sperm from 5-day storage only increased the WOB parameter in comparison to the control (p < .05). Furthermore, the average amplitude of the lateral displacement of the sperm head (ALH, μm) and curvilinear velocity (VCL, μm/s) decreased when CCK was present, depending on its concentration and sperm aging (1-day vs. 5-days) (p < .05). In the case of the media supporting capacitation supplemented with 25 mmol/L HCO 3 - , any differences were observed except for sperm viability in the 5-day seminal doses, which increased in the 50 μM-CCK group compared to the control (p < .05). In conclusion, these data suggest an implication of CCK protein during sperm capacitation under low bicarbonate concentration increasing the sperm linear trajectory., (© 2023 The Authors. Reproduction in Domestic Animals published by Wiley-VCH GmbH.)

Objective: To evaluate (i) the prevalence and association of euthyroid sick syndrome (ESS) [decreased FT3 and/or FT4 and normal/decreased TSH] with severity indexes of type 1 diabetes mellitus (T1DM) onset such as diabetic ketoacidosis (DKA) and kidney damage [acute kidney injury (AKI) based on KDIGO criteria, acute tubular necrosis (ATN), renal tubular damage (RTD)], (ii) relationship between clinical/metabolic parameters at T1DM onset and thyroid hormones, and (iii) ESS as a prognostic indicator of delayed recovery from kidney damage., Methods: A total of 161 children with T1DM onset were included. RTD was defined by abnormal urinary beta-2-microglobulin and/or neutrophil gelatinase-associated lipocalin (NGAL) and/or tubular reabsorption of phosphate <85% and/or fractional excretion of Na>2%. ATN was defined by RTD+AKI., Results: Of 161 participants, 60 (37.3%) presented ESS. It was more prevalent in case of more severe T1DM presentation both in terms of metabolic derangement (DKA) and kidney function impairment (AKI, RTD and ATN). Only ATN, however, was associated with ESS at adjusted analysis. FT3 inversely correlated with serum triglycerides and creatinine, and urinary calcium/creatinine ratio and NGAL. Participants with euthyroidism showed earlier recovery from AKI than those with ESS. ESS spontaneously disappeared., Conclusions: ESS is associated with T1DM onset severity and spontaneously disappears. ESS delayed the recovery from AKI., Impact: This is the first longitudinal study describing in detail the relationship between clinical/metabolic factors at type 1 diabetes mellitus (T1DM) onset and thyroid hormones, with particular attention to the relationship between diabetic ketoacidosis (DKA)-related kidney function impairment and euthyroid sick syndrome (ESS). Participants with more severe T1DM onset presentation both in terms of metabolic derangement and kidney function impairment had an increased prevalence of ESS. Children with ESS had a slower recovery from acute kidney injury compared with those without ESS. ESS spontaneously disappeared in all participants., (© 2023. The Author(s).)

DICER1 syndrome is a rare genetic disorder that predisposes patients to the development of malignant and non-malignant diseases. Presently, DICER1 syndrome diagnosis still occurs late, usually following surgical operations, affecting patients' outcomes, especially for further neoplasms, which are entailed in this syndrome. For this reason, herein we present a multicenter report of DICER1 syndrome, with the prospective aim of enhancing post-surgical surveillance. A cohort of seven patients was collected among the surgical registries of Pediatric Surgery at the University of Pisa with the General and Oncologic Surgery of Federico II, University of Naples, and the Pediatric Surgery, Regina Margherita Hospital, University of Turin. In each case, the following data were analyzed: sex, age at diagnosis, age at first surgery, clinical features, familial, genetic investigations, and follow-up. A comprehensive literature review of DICER1 cases, including case reports and multicenter studies published from 1996 to June 2022, was performed. Eventually, the retrieved data from the literature were compared with the data emerging from our cohort of patients.

The pediatric live-attenuated bovine/human parainfluenza virus type 3 (B/HPIV3)-vectored vaccine expressing the prefusion-stabilized SARS-CoV-2 spike (S) protein (B/HPIV3/S-2P) was previously evaluated in vitro and in hamsters. To improve its immunogenicity, we generated B/HPIV3/S-6P, expressing S further stabilized with 6 proline mutations (S-6P). Intranasal immunization of hamsters with B/HPIV3/S-6P reproducibly elicited significantly higher serum anti-S IgA/IgG titers than B/HPIV3/S-2P; hamster sera efficiently neutralized variants of concern (VoCs), including Omicron variants. B/HPIV3/S-2P and B/HPIV3/S-6P immunization protected hamsters against weight loss and lung inflammation following SARS-CoV-2 challenge with the vaccine-matched strain WA1/2020 or VoCs B.1.1.7/Alpha or B.1.351/Beta and induced near-sterilizing immunity. Three weeks post-challenge, B/HPIV3/S-2P- and B/HPIV3/S-6P-immunized hamsters exhibited a robust anamnestic serum antibody response with increased neutralizing potency to VoCs, including Omicron sublineages. B/HPIV3/S-6P primed for stronger anamnestic antibody responses after challenge with WA1/2020 than B/HPIV3/S-2P. B/HPIV3/S-6P will be evaluated as an intranasal vaccine to protect infants against both HPIV3 and SARS-CoV-2., Competing Interests: U.J.B., C.L., X.L, and C.LN. are inventors on the provisional patent application number 63/180,534, entitled “Recombinant chimeric bovine/human parainfluenza virus 3 expressing SARS-CoV-2 spike protein and its use”, filed by the United States of America, Department of Health and Human Services., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Anaplastic thyroid cancer (ATC) is a rare thyroid tumor that frequently originates from the dedifferentiation of a well-differentiated papillary or follicular thyroid cancer. Type 2 deiodinase (D2), responsible for the activation of the thyroid hormone thyroxine into tri-iodothyronine (T3), is expressed in normal thyroid cells and its expression is strongly downregulated in papillary thyroid cancer. In skin cancer, D2 has been associated with cancer progression, dedifferentiation, and epithelial-mesenchymal transition. Here, we show that D2 is highly expressed in anaplastic compared to papillary thyroid cancer cell lines and that D2-derived T3 is required for ATC cell proliferation. D2 inhibition is associated with G1 growth arrest and induction of cell senescence, together with reduced cell migration and invasive potential. Finally, we found that mutated p5372R(R248W), frequently found in ATC, is able to induce D2 expression in transfected papillary thyroid cancer cells. Our results show that the action of D2 is crucial for ATC proliferation and invasiveness, providing a potential new therapeutic target for the treatment of ATC.

Treatment with tyrosine kinase inhibitors (TKIs) has been associated with alterations in circulating thyroid hormone levels, possibly related to perturbations in peripheral thyroid hormone metabolism. In this study, we evaluated the effect of the multi-kinase inhibitor vandetanib on the expression of the three deiodinase selenoenzymes, responsible for the thyroid hormone activation (type 1 and type 2 deiodinases) or for its inactivation (type 3 deiodinase). Here, we show that the multi-kinase inhibitor vandetanib determines a strong cell-specific downregulation of type 2 deiodinase (D2) expression and a significant reduction in D2 enzymatic activity. This occurs in the diffused population of fibro/adipogenic progenitors, which reside in different tissues - including the muscles - and normally express D2. Given the widespread diffusion of mesenchymal cells within the body, our results may explain at least partially the alterations in thyroid hormone levels that occur in vandetanib-treated patients. Our findings represent a step forward into the understanding of the mechanisms by which TKIs induce hypothyroidism and identify a resident cell population in which such an effect takes place.

Background: The DIO2 Thr92Ala polymorphism (rs225014), which occurs in about 15-30% of Caucasian people, determines a less efficient type 2 deiodinase (D2) enzyme. The aim of this study was to determine the impact of DIO2 Thr92Ala polymorphism on the serum thyrotropin (TSH) levels in thyroidectomized patients with hypothyroidism and to evaluate whether TSH levels and aging could be related, at pituitary level, to D2 activity. Methods: This prospective study was performed on 145 thyroid cancer patients, treated with total thyroidectomy, and undergoing radioiodine treatment after 3 weeks of levothyroxine (LT4) withdrawal. A mouse model has been used to determine D2 protein and mRNA levels in pituitary during aging. Results: Genetic analysis identified DIO2 Thr92Ala polymorphism in 56% of participants: 64/145 (44%) patients were homozygous wild type (WT) (Thr/Thr), 64 (44%) heterozygous (Thr/Ala), and 17 (12%) homozygous mutant (Ala/Ala). A significant negative relationship was observed between aging and the rise in serum TSH levels during LT4 withdrawal. However, this negative correlation found in WT was reduced in heterozygous and lost in mutant homozygous patients (Thr/Thr r  = -0.45, p  = 0.0002, 95% confidence interval [CI] -0.63 to -0.23; Ala/Thr r  = -0.39, p  = 0.0012, CI -0.60 to -0.67; and Ala/Ala r  = -0.30, p  = 0.2347; CI -0.70 to 0.20). Accordingly, when we compared the TSH measured in each patient to its theoretical value predicted from age, the TSH did not reach its putative target in 47% of WT patients, in 70% of Ala/Thr, and 76% of Ala/Ala carrying patients ( p  = 0.0036). This difference was lost in individuals older than 60 years, suggesting a decline of D2 associated with aging. The hypothesis that the pituitary D2 decreases with age was confirmed by the evidence that D2 mRNA and protein levels were lower in pituitary from old versus young mice. Conclusion: An age-related decline in TSH production in response to hypothyroidism was correlated with decreased D2 levels in pituitary. The presence of DIO 2 homozygous Ala/Ala polymorphism was associated with a reduced level of TSH secretion in response to hypothyroidism, indicating a decreased pituitary sensitivity to serum thyroxine variation (Institutional Research Ethics board approval number no. 433/21).

Background: This United States-based study compared 2 candidate vaccines: RSV/ΔNS2/Δ1313/I1314L, attenuated by NS2 gene-deletion and temperature-sensitivity mutation in the polymerase gene; and RSV/276, attenuated by M2-2 deletion., Methods: RSV-seronegative children aged 6-24 months received RSV/ΔNS2/Δ1313/I1314L (106 plaque-forming units [PFU]), RSV/276 (105 PFU), or placebo intranasally. Participants were monitored for vaccine shedding, reactogenicity, and RSV serum antibodies, and followed over the subsequent RSV season., Results: Enrollment occurred September 2017 to October 2019. During 28 days postinoculation, upper respiratory illness and/or fever occurred in 64% of RSV/ΔNS2/Δ1313/I1314L, 84% of RSV/276, and 58% of placebo recipients. Symptoms were generally mild. Cough was more common in RSV/276 recipients than RSV/ΔNS2/Δ1313/I1314L (48% vs 12%; P = .012) or placebo recipients (17%; P = .084). There were no lower respiratory illness or serious adverse events. Eighty-eight and 96% of RSV/ΔNS2/Δ1313/I1314L and RSV/276 recipients were infected with vaccine (shed vaccine and/or had ≥4-fold rises in RSV antibodies). Serum RSV-neutralizing titers and anti-RSV F IgG titers increased ≥4-fold in 60% and 92% of RSV/ΔNS2/Δ1313/I1314L and RSV/276 vaccinees, respectively. Exposure to community RSV during the subsequent winter was associated with strong anamnestic RSV-antibody responses., Conclusions: Both vaccines had excellent infectivity and were well tolerated. RSV/276 induced an excess of mild cough. Both vaccines were immunogenic and primed for strong anamnestic responses., Clinical Trials Registration: NCT03227029 and NCT03422237., Competing Interests: Potential conflicts of interest. U. J. B., C. L., and P. L. C. are listed as inventors on patents related to live-attenuated RSV vaccines, including vaccines containing genetically stabilized attenuating mutations and received research support and royalties paid by Sanofi. C. K. C. and R. A. K. have served as paid consultants to Sanofi. E. J. M. participated in an advisory board meeting for Sanofi. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pediatric SARS-CoV-2 vaccines are needed that elicit immunity directly in the airways as well as systemically. Building on pediatric parainfluenza virus vaccines in clinical development, we generated a live-attenuated parainfluenza-virus-vectored vaccine candidate expressing SARS-CoV-2 prefusion-stabilized spike (S) protein (B/HPIV3/S-6P) and evaluated its immunogenicity and protective efficacy in rhesus macaques. A single intranasal/intratracheal dose of B/HPIV3/S-6P induced strong S-specific airway mucosal immunoglobulin A (IgA) and IgG responses. High levels of S-specific antibodies were also induced in serum, which efficiently neutralized SARS-CoV-2 variants of concern of alpha, beta, and delta lineages, while their ability to neutralize Omicron sub-lineages was lower. Furthermore, B/HPIV3/S-6P induced robust systemic and pulmonary S-specific CD4 + and CD8 + T cell responses, including tissue-resident memory cells in the lungs. Following challenge, SARS-CoV-2 replication was undetectable in airways and lung tissues of immunized macaques. B/HPIV3/S-6P will be evaluated clinically as pediatric intranasal SARS-CoV-2/parainfluenza virus type 3 vaccine., Competing Interests: Declaration of interests X.L., C.L., C.L.N., S.M., and U.J.B. are inventors on the provisional patent application number 63/180,534, entitled “recombinant chimeric B/HPIV3 expressing SARS-CoV-2 spike protein and its use,” filed by the United States Department of Health and Human Services., (Published by Elsevier Inc.)

Each year, tens of thousands of people worldwide die of end-stage organ failure due to the limited availability of organs for use in transplantation. To meet this clinical demand, one of the last frontiers of regenerative medicine is the generation of humanized organs in pigs from pluripotent stem cells (PSCs) via blastocyst complementation. For this, organ-disabled pig models are needed. As endothelial cells (ECs) play a critical role in xenotransplantation rejection in every organ, we aimed to produce hematoendothelial-disabled pig embryos targeting the master transcription factor ETV2 via CRISPR-Cas9-mediated genome modification. In this study, we designed five different guide RNAs (gRNAs) against the DNA-binding domain of the porcine ETV2 gene, which were tested on porcine fibroblasts in vitro. Four out of five guides showed cleavage capacity and, subsequently, these four guides were microinjected individually as ribonucleoprotein complexes (RNPs) into one-cell-stage porcine embryos. Next, we combined the two gRNAs that showed the highest targeting efficiency and microinjected them at higher concentrations. Under these conditions, we significantly improved the rate of biallelic mutation. Hence, here, we describe an efficient one-step method for the generation of hematoendothelial-disabled pig embryos via CRISPR-Cas9 microinjection in zygotes. This model could be used in experimentation related to the in vivo generation of humanized organs.

Current vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are administered parenterally and appear to be more protective in the lower versus the upper respiratory tract. Vaccines are needed that directly stimulate immunity in the respiratory tract, as well as systemic immunity. We used avian paramyxovirus type 3 (APMV3) as an intranasal vaccine vector to express the SARS-CoV-2 spike (S) protein. A lack of pre-existing immunity in humans and attenuation by host-range restriction make APMV3 a vector of interest. The SARS-CoV-2 S protein was stabilized in its prefusion conformation by six proline substitutions (S-6P) rather than the two that are used in most vaccine candidates, providing increased stability. APMV3 expressing S-6P (APMV3/S-6P) replicated to high titers in embryonated chicken eggs and was genetically stable, whereas APMV3 expressing non-stabilized S or S-2P were unstable. In hamsters, a single intranasal dose of APMV3/S-6P induced strong serum IgG and IgA responses to the S protein and its receptor-binding domain, and strong serum neutralizing antibody responses to SARS-CoV-2 isolate WA1/2020 (lineage A). Sera from APMV3/S-6P-immunized hamsters also efficiently neutralized Alpha and Beta variants of concern. Immunized hamsters challenged with WA1/2020 did not exhibit the weight loss and lung inflammation observed in empty-vector-immunized controls; SARS-CoV-2 replication in the upper and lower respiratory tract of immunized animals was low or undetectable compared to the substantial replication in controls. Thus, a single intranasal dose of APMV3/S-6P was highly immunogenic and protective against SARS-CoV-2 challenge, suggesting that APMV3/S-6P is suitable for clinical development., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

This study aimed to determine uterine blood flow indices by transabdominal Doppler ultrasound in sows (n = 18) under different conditions: (i) sows after estrus detection (day 0, D0); (ii) sows 2 h after artificial insemination (AI), performed 24 h after detection of estrus (day 1, D1); (iii) sows in early diestrus (day 5, D5). Moreover, three different types of seminal doses were used for AI depending on the ejaculate fraction included (F1: doses containing only the rich fraction of the ejaculate; F2: F1 + the transition fraction between rich and poor fractions; F3: F2 and poor fraction). The statistical analysis revealed significant differences in some indices regarding the period of analysis (D0, D1, and D5). Diastolic velocity and mean velocity showed lower values at D5 in comparison with D0 and D1 (p < 0.01). On the other hand, the pulsatility index and the relationship systolic velocity/diastolic velocity indicated higher values at D5 in comparison with D0 and D1 (p < 0.01). No differences were observed regarding the type of seminal dose used in any of the time points analyzed (p > 0.05). Neither insemination per se nor the type of ejaculate fraction used immediately modified the uterine vascularity, but some indices are affected by the stage of the estrus cycle (estrus vs. early diestrus).

Adaptive thermogenesis in small mammals and infants takes place in brown adipose tissue (BAT). Heat is produced via uncoupling protein 1 (UCP1)-mediated uncoupling between oxidation of energy substrates and adenosine 5'-triphosphate synthesis. Thyroid hormone (TH) signaling plays a role in this process. The deiodinases activate thyroxine (T4) to 3,5,3'-triiodothyronine (T3) (D2) or inactivate T4 and T3 to 3,3,5'-triiodothyronine and T2 (D3), respectively. Using a mouse model with selective inactivation of Dio3 in BAT (flox-Dio3 × UCP1-cre = BAT-D3KO), we now show that knocking out D3 resulted in premature exposure of developing brown adipocytes (embryonic days 16.5-18.5) to T3 signaling, leading to an earlier expression of key BAT genes, including Cidea, Cox8b, Dio2, Ucp1, and Pgc1α. Adult BAT-D3KO mice exhibited increased expression of 1591 genes as assessed by RNA sequencing, including 19 gene sets related to mitochondria, 8 related to fat, and 8 related to glucose homeostasis. The expression of 243 genes was changed by more than 1.5-fold, 36 of which play a role in metabolic/thermogenic processes. BAT-D3KO mice weigh less and exhibit smaller white adipocyte area, but maintain normal energy expenditure at room temperature (22 °C) and in the cold (4 °C). They also defend their core temperature more effectively and do not lose as much body weight when exposed to cold. We conclude that the coordinated actions of Dio3 in the embryonic BAT define the timing and intensity of T3 signaling during brown adipogenesis. Enhanced T3 signaling during BAT embryogenesis (Dio3 inactivation) results in selective life-long modifications in the BAT transcriptome., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Increased incidence of central precocious puberty (CPP) after coronavirus infectious disease-19 lockdown has been reported. Our study aims in investigating changes in CPP rates and in sleep patterns in CPP and healthy controls., Methods: CPP were retrospectively evaluated from April 2020 to April 2021. Parents of girls diagnosed with CPP during lockdown and of matched healthy controls filled out a questionnaire about sleep disturbances (SDSC questionnaire) and sleep schedules., Results: Thirty-five CPP and 37 controls completed the survey. Incidence of new CPP cases significantly increased in 2020-2021 compared to 2017-2020 (5:100 vs 2:100, p = 0.02). Sleep disturbance rates did not differ between CPP and healthy controls before lockdown. During lockdown, CPP reported higher rates of sleep disturbs for total score (p = 0.005), excessive somnolence (p = 0.049), sleep breathing disorders (p = 0.049), and sleep-wake transition disorders (p = 0.005). Moreover, CPP group more frequently shifted toward later bedtime (p = 0.03) during lockdown compared to controls. Hours of sleep and smartphone exposure around bedtime did not differ between groups., Conclusions: Our study confirms the observation of increased incidence of CPP after lockdown measures. Additionally, CPP showed higher rates of sleep disturbances and later bedtime compared to controls. The causality link between sleep disturbances and CPP should be further investigated to gain knowledge in this association., (© 2022. The Author(s).)

Background: Rearranged during transfection ( RET ) gene fusions are detected in 10-20% of thyroid cancer patients. Recently, RET fusion-positive metastatic thyroid cancers have attracted much attention owing to the FDA approval of two highly selective anti-RET tyrosine kinase inhibitors, namely, selpercatinib, and pralsetinib., Areas Covered: This review summarizes the available evidence on the biological and predictive role of RET gene fusions in thyroid carcinoma patients and the latest screening assays currently used to detect these genomic alterations in histological and cytological specimens., Expert Opinion: Management of advanced thyroid carcinoma has significantly evolved over the last decade thanks to the approval of three multikinase inhibitors, i.e. sorafenib, lenvatinib, cabozantinib, and of two selective RET-tyrosine inhibitors, i.e. selpercatinib and pralsetinib. In this setting, the detection of RET-fusions in advanced thyroid cancer specimens through the use of next-generation sequencing has become a commonly used strategy in clinical practice to select the best treatment options.

Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 μM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible.

Boar ejaculate is released in several well-characterized fractions, differing in terms of sperm concentration, seminal plasma volume, and composition. However, the inclusion of the last part of the ejaculate for artificial insemination (AI) purposes is still under debate due to its controversial effects. Thus, there is a need to study the potential synergistic impact of the different ejaculate fractions. We aimed to evaluate the effect of accumulative ejaculate fractions on sperm conservation, AI performance, and offspring health. Ejaculates ( n = 51) were collected and distributed as follows: F1: sperm-rich fraction; F2: sperm-rich + intermediate fractions; F3: sperm-rich + intermediate + poor fractions. Each group was diluted in a commercial extender, packaged in seminal doses (2000 × 10 6 sperm/60 mL), and stored at ~16 °C. On day 3 of conservation, sperm were analyzed and used for AI ( n = 174). High sperm quality was observed after storage without a significant difference between the groups ( p > 0.05). Moreover, no differences were obtained for AI performance (pregnancy and farrowing rates, and litter size; p > 0.05) and offspring health (growth and blood analysis; p > 0.05). Conclusively, the presence of all ejaculate fractions within the seminal doses does not impair the reproductive performance, reporting important economic savings according to the economic model included here.

Anaplastic thyroid cancer (ATC) is a rare but aggressive thyroid cancer, responsible for about 50% of all thyroid cancer-related deaths. During the last two decades, the development of a multimodal personalized approach resulted in an increased survival. Here, we present an unusual case of a 54-year old woman with a paucicellular metastatic ATC, a rare variant of ATC, who was treated with a combination of surgery, radiation therapy and cytotoxic chemotherapy. More than two years later, when the disease was rapidly growing, a combination of lenvatinib and pembrolizumab induced a partial tumor response of lung metastasis that persisted over 18 months. Paucicellular ATC may initially show a less aggressive behavior compared to other histological ATC variants. However, over the time, its clinical course can rapidly progress like common ATC. The combination of lenvatinib and pembrolizumab was effective as a salvage therapy for a long period of time.

Single-dose vaccines with the ability to restrict SARS-CoV-2 replication in the respiratory tract are needed for all age groups, aiding efforts toward control of COVID-19. We developed a live intranasal vector vaccine for infants and children against COVID-19 based on replication-competent chimeric bovine/human parainfluenza virus type 3 (B/HPIV3) that express the native (S) or prefusion-stabilized (S-2P) SARS-CoV-2 S spike protein, the major protective and neutralization antigen of SARS-CoV-2. B/HPIV3/S and B/HPIV3/S-2P replicated as efficiently as B/HPIV3 in vitro and stably expressed SARS-CoV-2 S. Prefusion stabilization increased S expression by B/HPIV3 in vitro. In hamsters, a single intranasal dose of B/HPIV3/S-2P induced significantly higher titers compared to B/HPIV3/S of serum SARS-CoV-2-neutralizing antibodies (12-fold higher), serum IgA and IgG to SARS-CoV-2 S protein (5-fold and 13-fold), and IgG to the receptor binding domain (10-fold). Antibodies exhibited broad neutralizing activity against SARS-CoV-2 of lineages A, B.1.1.7, and B.1.351. Four weeks after immunization, hamsters were challenged intranasally with 10 4.5 50% tissue-culture infectious-dose (TCID 50 ) of SARS-CoV-2. In B/HPIV3 empty vector-immunized hamsters, SARS-CoV-2 replicated to mean titers of 10 6.6 TCID 50 /g in lungs and 10 7 TCID 50 /g in nasal tissues and induced moderate weight loss. In B/HPIV3/S-immunized hamsters, SARS-CoV-2 challenge virus was reduced 20-fold in nasal tissues and undetectable in lungs. In B/HPIV3/S-2P-immunized hamsters, infectious challenge virus was undetectable in nasal tissues and lungs; B/HPIV3/S and B/HPIV3/S-2P completely protected against weight loss after SARS-CoV-2 challenge. B/HPIV3/S-2P is a promising vaccine candidate to protect infants and young children against HPIV3 and SARS-CoV-2., Competing Interests: Competing interest statement: X.L, C.L., C.L.N., S.M., and U.J.B. are inventors on the provisional patent application number 63/180,534, entitled “Recombinant chimeric bovine/human parainfluenza virus 3 expressing SARS-CoV-2 spike protein and its use,” filed by the United States, Department of Health and Human Services., (Copyright © 2021 the Author(s). Published by PNAS.)

The Italian Cancer Registry Association has estimated that for the five-year period 2016-2020, in line with the previous five years, approximately 7000 neoplasms have been diagnosed among children and 4000 among adolescents. Leukemias, brain tumors and lymphomas together account for more than two-thirds of all pediatric cancers. Fortunately, the five-years survival rate has progressively improved reaching 80% thanks to the continuing improvement of therapeutic protocols but most of these cancer survivors will have at least one chronic health condition by 40 years of age. Long-term complications concern various organs and systems and have a multifactorial etiopathogenesis. Obesity, diabetes, and metabolic syndrome represent chronic diseases that affect life expectancy. Cardiovascular risk partly linked to therapies and genetic susceptibility and partly linked to the presence of obesity, diabetes and metabolic syndrome predispose childhood cancer survivors to heart failure, coronary artery disease, valvular disease, arrhythmia. Hence the cardio- metabolic risk of childhood cancer survivors can have a significant impact on their lives, families, and on society at-large. Therefore, it is very important to know the risk factors that predispose to the development of cardio-metabolic pathologies in childhood cancer survivors, the possible primary and secondary prevention strategies, the methods of surveillance and the therapeutic approaches.

Skeletal muscle atrophy is a condition associated with various physiological and pathophysiological conditions, such as denervation, cachexia, and fasting. It is characterized by an altered protein turnover in which the rate of protein degradation exceeds the rate of protein synthesis, leading to substantial muscle mass loss and weakness. Muscle protein breakdown reflects the activation of multiple proteolytic mechanisms, including lysosomal degradation, apoptosis, and ubiquitin-proteasome. Thyroid hormone (TH) plays a key role in these conditions. Indeed, skeletal muscle is among the principal TH target tissue, where TH regulates proliferation, metabolism, differentiation, homeostasis, and growth. In physiological conditions, TH stimulates both protein synthesis and degradation, and an alteration in TH levels is often responsible for a specific myopathy. Intracellular TH concentrations are modulated in skeletal muscle by a family of enzymes named deiodinases; in particular, in muscle, deiodinases type 2 (D2) and type 3 (D3) are both present. D2 activates the prohormone T4 into the active form triiodothyronine (T3), whereas D3 inactivates both T4 and T3 by the removal of an inner ring iodine. Here we will review the present knowledge of TH action in skeletal muscle atrophy, in particular, on the molecular mechanisms presiding over the control of intracellular T3 concentration in wasting muscle conditions. Finally, we will discuss the possibility of exploiting the modulation of deiodinases as a possible therapeutic approach to treat muscle atrophy.

Neck ultrasound (nUS) is the cornerstone of clinical management of thyroid nodules in pediatric patients, as well as adults. The current study was carried out to explore and compare the diagnostic performance of the main US-based risk stratification systems (RSSs) (i.e., the American College of Radiology (ACR), European (EU), Korean (K) TI-RADSs and ATA US RSS criteria) for detecting malignant thyroid lesions in pediatric patients. We conducted a retrospective analysis of consecutive children and adolescents who received a diagnosis of thyroid nodule. We included subjects with age <19 years having thyroid nodules with benign cytology/histology or final histological diagnosis. We excluded subjects with (a) a previous malignancy, (b) a history of radiation exposure, (c) cancer genetic susceptibility syndromes, (d) lymph nodes suspicious for metastases of thyroid cancer at nUS, (e) a family history of thyroid cancer, or (f) cytologically indeterminate nodules without histology and nodules with inadequate cytology. We included 41 nodules in 36 patients with median age 15 years (11-17 years). Of the 41 thyroid nodules, 29 (70.7%) were benign and 12 (29.3%) were malignant. For both ACR TI-RADS and EU-TIRADS, we found a sensitivity of 41.7%. Instead, for both K-TIRADS and ATA US RSS, we found a sensitivity of 50%. The missed malignancy rate for ACR-TIRADS and EU-TIRADS was 58.3%, while that for K-TIRADS and ATA US RSS was 50%. The unnecessary FNA prevalence for ACR TI-RADS and EU-TIRADS was 58.3%, while that for K-TIRADS and ATA US RSS was 76%. Our findings suggest that the four US-based RSSs (i.e., ACR-TIRADS, EU-TIRADS, K-TIRADS, and ATA US RSS) have suboptimal performance in managing pediatric patients with thyroid nodules, with one-half of cancers without indication for FNA according to their recommendations.

Background: Nephrotic syndrome (NS) is associated with kidney podocyte injury and may occur as part of thyroid autoimmunity such as Graves' disease. Therefore, the present study was designed to ascertain if and how podocytes respond to and regulate the input of biologically active thyroid hormone (TH), 3,5,3'-triiodothyronine (T3); and also to decipher the pathophysiological role of type 3 deiodinase (D3), a membrane-bound selenoenzyme that inactivates TH, in kidney disease., Methods: To study D3 function in healthy and injured (PAN, puromycin aminonucleoside and LPS, Lipopolysaccharide-mediated) podocytes, immunofluorescence, qPCR and podocyte-specific D3 knockout mouse were used. Surface plasmon resonance (SPR), co-immunoprecipitation and Proximity Ligation Assay (PLA) were used for the interaction studies., Findings: Healthy podocytes expressed D3 as the predominant deiodinase isoform. Upon podocyte injury, levels of Dio3 transcript and D3 protein were dramatically reduced both in vitro and in the LPS mouse model of podocyte damage. D3 was no longer directed to the cell membrane, it accumulated in the Golgi and nucleus instead. Further, depleting D3 from the mouse podocytes resulted in foot process effacement and proteinuria. Treatment of mouse podocytes with T3 phenocopied the absence of D3 and elicited activation of αvβ3 integrin signaling, which led to podocyte injury. We also confirmed presence of an active thyroid stimulating hormone receptor (TSH-R) on mouse podocytes, engagement and activation of which resulted in podocyte injury., Interpretation: The study provided a mechanistic insight into how D3-αvβ3 integrin interaction can minimize T3-dependent integrin activation, illustrating how D3 could act as a renoprotective thyrostat in podocytes. Further, injury caused by binding of TSH-R with TSH-R antibody, as found in patients with Graves' disease, explained a plausible link between thyroid disorder and NS., Funding: This work was supported by American Thyroid Association (ATA-2018-050.R1)., Competing Interests: Declaration of Competing Interest Jochen Reiser has patents on novel strategies for kidney therapeutics and stands to gain royalties from their commercialization. He is the co-founder of Walden Biosciences (Cambridge, MA, USA), a biotechnology company in which he has financial interest, including stock. Antonio Bianco is a consultant for Synthonics, Allergan, Abbvie, and BLA Technology. Other authors have nothing to disclose and there are no competing or conflicting interests., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Purpose: Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that enhance the immune response against cancer cells. ICIs are generally well tolerated, although endocrine immune-related adverse events (irAEs) are common. We investigated the risk factors for thyroid irAEs in patients treated with ICIs. Moreover, we evaluated the clinical outcome of subjects who became hypothyroid compared to euthyroid patients., Patients and Methods: We retrospectively analyzed a series of 195 consecutively subjects treated with ICIs for metastatic tumors at the University of Naples "Federico II" between January 2014 and March 2020. Only subjects tested for thyroid function before and during the treatment with ICIs were included., Results: In the 96 patients treated with ICIs who were included [66 males, median age: 62 years (27-87)], thyroid irAEs occurred in 36 (37.5%), 16 (16.7%) a transient thyrotoxicosis, and 20 (20.8%) an hypothyroidism (in nine subjects hypothyroidism was preceded by a transient thyrotoxicosis). Only baseline TSH levels above 1.67 mIU/L and positive anti-thyroid antibodies (Ab-T) were associated with a higher risk of hypothyroidism. Patients with hypothyroidism during ICI treatment showed an improved 2-year PFS (HR = 0.82 CI 0.47-1.43; p = 0.0132) and OS (HR = 0.38 CI 95% 0.17-0.80; p = 0.011) compared to euthyroid patients., Conclusions: Baseline TSH levels above 1.67 mIU/L and presence of Ab-T are risk factors for the development of thyroid irAEs. Patients affected by thyroid irAEs showed a longer survival than patients who remained euthyroid., (© 2021. The Author(s).)

Purpose: The efficacy of lenvatinib for advanced and progressive radioactive iodine refractory differentiated thyroid cancer is well established. Herein, we retrospectively evaluated the long-term safety and efficacy of lenvatinib in 23 patients treated at a single Institution., Methods: Clinical data of all patients treated for a differentiated thyroid cancer with lenvatinib from April 2015 to September 2020 were retrospectively analyzed., Results: A total of 23 patients were included. In all, 21 patients received lenvatinib as first-line systemic therapy. Median age at initiation of lenvatinib treatment was 68 (44-90) years. Median duration of the study from initiation of lenvatinib to study end was 23 (2-65) months. The indication for lenvatinib treatment was documented progression of distant metastases in 20 patients and of locally advanced disease in the other 3 and median duration of lenvatinib therapy was 15 (2-64) months. Best treatment responses were: partial response in 6 patients, stable disease in 14, progressive disease in 1, and not evaluable in 2. Median progression-free survival was 25 months (95% CI: 12-40) and median overall survival was 46 months (95% CI: 28-65). Three patients had to discontinue lenvatinib treatment due to serious adverse events and no drug-related death was observed. Ten patients continued lenvatinib for more than 24 months and the only newly registered adverse event after this period of time was one case of G2 proteinuria. Six patients continued lenvatinib treatment beyond documented tumor progression due to oligoprogression or slowly progressive disease (median time 18.5 months, 8-42 months). A total of 14 patients were alive at the end of the study: 11 showed partial response/stable disease on lenvatinib, including 3 who had a stable disease after local ablative therapy for oligoprogressive metastases; 3 had to change treatment, including 2 for lenvatinib-related serious adverse events and 1 for progressive disease., Conclusions: Long-term lenvatinib treatment is safe and some patients may experience persistent long-term control of the disease. Late treatment-related AEs rarely occurred. Oligoprogressive and slowly progressive disease can be managed without treatment withdrawal as long as there are some clinical benefits.

Proteins play an important role in many reproductive functions such as sperm maturation, sperm transit in the female genital tract or sperm-oocyte interaction. However, in general, little information concerning reproductive features is available in the case of aquatic animals. The present study aims to characterize the proteome of both spermatozoa and seminal plasma of bottlenose dolphins ( Tursiops truncatus ) as a model organism for cetaceans. Ejaculate samples were obtained from two trained dolphins housed in an aquarium. Spermatozoa and seminal plasma were analyzed by means of proteomic analyses using an LC-MS/MS, and a list with the gene symbols corresponding to each protein was submitted to the DAVID database. Of the 419 proteins identified in spermatozoa and 303 in seminal plasma, 111 proteins were shared by both. Furthermore, 70 proteins were identified as involved in reproductive processes, 39 in spermatozoa, and 31 in seminal plasma. The five most abundant proteins were also identified in these samples: AKAP3, ODF2, TUBB, GSTM3, ROPN1 for spermatozoa and CST11, LTF, ALB, HSP90B1, PIGR for seminal plasma. In conclusion, this study provides the first characterization of the proteome in cetacean sperm and seminal plasma, opening the way to future research into new biomarkers, the analysis of conservation capacity or possible additional applications in the field of assisted reproductive technologies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fuentes-Albero, González-Brusi, Cots, Luongo, Abril-Sánchez, Ros-Santaella, Pintus, Ruiz-Díaz, Barros-García, Sánchez-Calabuig, García-Párraga, Avilés, Izquierdo Rico and García-Vázquez.)

Particle therapy in which deep seated tumours are treated using 12 C ions (Carbon Ions RadioTherapy or CIRT) exploits the high conformity in the dose release, the high relative biological effectiveness and low oxygen enhancement ratio of such projectiles. The advantages of CIRT are driving a rapid increase in the number of centres that are trying to implement such technique. To fully profit from the ballistic precision achievable in delivering the dose to the target volume an online range verification system would be needed, but currently missing. The 12 C ions beams range could only be monitored by looking at the secondary radiation emitted by the primary beam interaction with the patient tissues and no technical solution capable of the needed precision has been adopted in the clinical centres yet. The detection of charged secondary fragments, mainly protons, emitted by the patient is a promising approach, and is currently being explored in clinical trials at CNAO. Charged particles are easy to detect and can be back-tracked to the emission point with high efficiency in an almost background-free environment. These fragments are the product of projectiles fragmentation, and are hence mainly produced along the beam path inside the patient. This experimental signature can be used to monitor the beam position in the plane orthogonal to its flight direction, providing an online feedback to the beam transverse position monitor chambers used in the clinical centres. This information could be used to cross-check, validate and calibrate, whenever needed, the information provided by the ion chambers already implemented in most clinical centres as beam control detectors. In this paper we study the feasibility of such strategy in the clinical routine, analysing the data collected during the clinical trial performed at the CNAO facility on patients treated using 12 C ions and monitored using the Dose Profiler (DP) detector developed within the INSIDE project. On the basis of the data collected monitoring three patients, the technique potential and limitations will be discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Toppi, Baroni, Battistoni, Bisogni, Cerello, Ciocca, De Maria, De Simoni, Donetti, Dong, Embriaco, Ferrero, Fiorina, Fischetti, Franciosini, Kraan, Luongo, Malekzadeh, Magi, Mancini-Terracciano, Marafini, Mattei, Mazzoni, Mirabelli, Mirandola, Morrocchi, Muraro, Patera, Pennazio, Schiavi, Sciubba, Solfaroli-Camillocci, Sportelli, Tampellini, Traini, Valle, Vischioni, Vitolo and Sarti.)

Recent evidence supports involvement of the acute phase protein haptoglobin in numerous events during mammalian reproduction. The present study represents an in-depth investigation of haptoglobin expression and secretion in the porcine oviduct and uterus, and assesses its effect on porcine in vitro embryo production. A systematic study was made of sows in different oestrous stages: late follicular, early luteal and late luteal stages. Relative haptoglobin mRNA abundance was quantified by RT-qPCR. In addition, expression of the protein was analysed by immunohistochemistry and the results were complemented by Western-blot and proteomic analyses of the oviductal and uterine fluids. In vitro porcine fertilization and embryo culture were carried out in the presence of haptoglobin. The results indicate that haptoglobin mRNA expression in the porcine oviduct and uterus is most abundant during the late luteal stage of the oestrous cycle. By means of Western blot and proteomic analyses haptoglobin presence was demonstrated in the oviduct epithelium and in the oviductal and uterine fluids in different stages of the oestrous cycle. The addition of haptoglobin during gamete co-incubation had no effect on sperm penetration, monospermy or efficiency rates; however, compared with the control group, blastocyst development was significantly improved when haptoglobin was present (haptoglobin: 64.50% vs. control: 37.83%; p < 0.05). In conclusion, the presence of haptoglobin in the oviduct and uterus of sows at different stages of the oestrous cycle suggests that it plays an important role in the reproduction process. The addition of haptoglobin during in vitro embryo production improved the blastocyst rates.

Bottlenose dolphin ( Tursiops truncatus ) males follow many reproductive strategies to ensure their paternity. However, little is known about the sperm traits, including morphometric features, that contribute to their reproductive success. Our aim was to study dolphin sperm morphometry (a total of 13 parameters) in two adult males to evaluate (i) presumptive sperm subpopulations, (ii) the correlation of sperm morphometry with testosterone levels and (iii) the effect of refrigerated storage on the sperm morphometry. Sperm populations were classified into four principal components (PCs) based on morphometry (>94% of cumulative variance). The PCs clustered into two different sperm subpopulations, which differed between males. Furthermore, the levels of serum testosterone were positively correlated with the length of the midpiece but negatively correlated with head width and the principal piece, flagellum and total sperm lengths. Most of the sperm morphometric parameters changed during the storage period (day 1 vs. day 7), but only the principal piece length was affected by the storage temperature (5 °C vs. 15 °C). This is the first study to identify dolphin sperm subpopulations based on morphometry and the influence of serum testosterone and refrigeration on sperm morphometry.

Adult neural stem cell (NSC) generation in vertebrate brains requires thyroid hormones (THs). How THs enter the NSC population is unknown, although TH availability determines proliferation and neuronal versus glial progenitor determination in murine subventricular zone (SVZ) NSCs. Mice display neurological signs of the severely disabling human disease, Allan-Herndon-Dudley syndrome, if they lack both MCT8 and OATP1C1 transporters, or MCT8 and deiodinase type 2. We analyzed the distribution of MCT8 and OATP1C1 in adult mouse SVZ. Both are strongly expressed in NSCs and at a lower level in neuronal cell precursors but not in oligodendrocyte progenitors. Next, we analyzed Mct8/Oatp1c1 double-knockout mice, where brain uptake of THs is strongly reduced. NSC proliferation and determination to neuronal fates were severely affected, but not SVZ-oligodendroglial progenitor generation. This work highlights how tight control of TH availability determines NSC function and glial-neuron cell-fate choice in adult brains., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The type 2 deiodinase (DIO2) converts thyroxine to 3,3',5-triiodothyronine (T3), modulating intracellular T3. An increase in DIO2 within muscle stem cells during skeletal muscle regeneration leads to T3-dependent potentiation of differentiation. The muscle stem cell niche comprises numerous cell types, which coordinate the regeneration process. For example, muscle stem cells provide secretory signals stimulating endothelial cell-mediated vascular repair, and, in turn, endothelial cells promote muscle stem differentiation. We hypothesized that Dio2 loss in muscle stem cells directly impairs muscle stem cell-endothelial cell communication, leading to downstream disruption of endothelial cell function. Methods: We assessed the production of proangiogenic factors in differentiated C2C12 cells and in a C2C12 cell line without Dio2 (D2KO C2C12) by real-time quantitative-polymerase chain reaction and enzyme-linked immunosorbent assay. Conditioned medium (CM) was collected daily in parallel to evaluate its effects on human umbilical vein endothelial cell (HUVEC) proliferation, migration and chemotaxis, and vascular network formation. The effects of T3-treatment on vascular endothelial growth factor ( Vegfa ) mRNA expression in C2C12 cells and mouse muscle were assessed. Chromatin immunoprecipitation (ChIP) identified thyroid hormone receptor (TR) binding to the Vegfa gene. Using mice with a targeted disruption of Dio2 (D2KO mice), we determined endothelial cell number by immunohistochemistry/flow cytometry and evaluated related gene expression in both uninjured and injured skeletal muscle. Results: In differentiated D2KO C2C12 cells, Vegfa expression was 46% of wildtype (WT) C2C12 cells, while secreted VEGF was 45%. D2KO C2C12 CM exhibited significantly less proangiogenic effects on HUVECs. In vitro and in vivo T3 treatment of C2C12 cells and WT mice, and ChIP using antibodies against TRα, indicated that Vegfa is a direct genomic T3 target. In uninjured D2KO soleus muscle, Vegfa expression was decreased by 28% compared with WT mice, while endothelial cell numbers were decreased by 48%. Seven days after skeletal muscle injury, D2KO mice had 36% fewer endothelial cells, coinciding with an 83% decrease in Vegfa expression in fluorescence-activated cell sorting purified muscle stem cells. Conclusion: Dio2 loss in the muscle stem cell impairs muscle stem cell-endothelial cell crosstalk via changes in the T3-responsive gene Vegfa , leading to downstream impairment of endothelial cell function both in vitro and in vivo .

The high dose conformity and healthy tissue sparing achievable in Particle Therapy when using C ions calls for safety factors in treatment planning, to prevent the tumor under-dosage related to the possible occurrence of inter-fractional morphological changes during a treatment. This limitation could be overcome by a range monitor, still missing in clinical routine, capable of providing on-line feedback. The Dose Profiler (DP) is a detector developed within the INnovative Solution for In-beam Dosimetry in hadronthErapy (INSIDE) collaboration for the monitoring of carbon ion treatments at the CNAO facility (Centro Nazionale di Adroterapia Oncologica) exploiting the detection of charged secondary fragments that escape from the patient. The DP capability to detect inter-fractional changes is demonstrated by comparing the obtained fragment emission maps in different fractions of the treatments enrolled in the first ever clinical trial of such a monitoring system, performed at CNAO. The case of a CNAO patient that underwent a significant morphological change is presented in detail, focusing on the implications that can be drawn for the achievable inter-fractional monitoring DP sensitivity in real clinical conditions. The results have been cross-checked against a simulation study.