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In recent years, the loop-mediated isothermal amplification (LAMP) technique, designed for microbial pathogen detection, has acquired fundamental importance in the biomedical field, providing rapid and precise responses. However, it still has some drawbacks, mainly due to the need for a thermostatic block, necessary to reach 63 °C, which is the BstI DNA polymerase working temperature. Here, we report the identification and characterization of the DNA polymerase I Large Fragment from Deinococcus radiodurans (DraLF-PolI) that functions at room temperature and is resistant to various environmental stress conditions. We demonstrated that DraLF-PolI displays efficient catalytic activity over a wide range of temperatures and pH, maintains its activity even after storage under various stress conditions, including desiccation, and retains its strand-displacement activity required for isothermal amplification technology. All of these characteristics make DraLF-PolI an excellent candidate for a cutting-edge room-temperature LAMP that promises to be very useful for the rapid and simple detection of pathogens at the point of care.

Early life exposure to Endocrine Disruptor Chemicals (EDCs), such as the organophosphate pesticide Chlorpyrifos (CPF), affects the thyroid activity and dependent process, including the glucose metabolism. The damage of thyroid hormones (THs) as a mechanism of action of CPF is underestimated because the studies rarely consider that TH levels and signaling are customized peripherally. Here, we investigated the impairment of metabolism/signaling of THs and lipid/glucose metabolism in the livers of 6-month-old mice, developmentally and lifelong exposed to 0.1, 1, and 10 mg/kg/die CPF (F1) and their offspring similarly exposed (F2), analyzing the levels of transcripts of the enzymes involved in the metabolism of T3 ( Dio1 ), lipids ( Fasn , Acc1 ), and glucose ( G6pase , Pck1 ). Both processes were altered only in F2 males, affected by hypothyroidism and by a systemic hyperglycemia linked to the activation of gluconeogenesis in mice exposed to 1 and 10 mg/kg/die CPF. Interestingly, we observed an increase in active FOXO1 protein due to a decrease in AKT phosphorylation, despite insulin signaling activation. Experiments in vitro revealed that chronic exposure to CPF affected glucose metabolism via the direct modulation of FOXO1 activity and T3 levels in hepatic cells. In conclusion, we described different sex and intergenerational effects of CPF exposure on the hepatic homeostasis of THs, their signaling, and, finally, glucose metabolism. The data points to FOXO1-T3-glucose signaling as a target of CPF in liver.

Although the imbalance of circulating levels of Thyroid Hormones (THs) affects female fertility in vertebrates, its involvement in the promotion of Premature Ovarian Aging (POA) is debated. Therefore, altered synthesis of THs in both thyroid and ovary can be a trait of POA. We investigated the relationship between abnormal TH signaling, dysthyroidism, and POA in evolutionary distant vertebrates: from zebrafish to humans. Ovarian T3 signaling/metabolism was evaluated by measuring T3 levels, T3 responsive transcript, and protein levels along with transcripts governing T3 availability (deiodinases) and signaling (TH receptors) in distinct models of POA depending on genetic background and environmental exposures (e.g., diets, pesticides). Expression levels of well-known ( Amh , Gdf9 , and Inhibins) and novel ( miR143/145 and Gas5 ) biomarkers of POA were assessed. Ovarian dysthyroidism was slightly influenced by genetics since very few differences were found between C57BL/6J and FVB/NJ females. However, diets exacerbated it in a strain-dependent manner. Similar findings were observed in zebrafish and mouse models of POA induced by developmental and long-life exposure to low-dose chlorpyrifos (CPF). Lastly, the T3 decrease in follicular fluids from women affected by diminished ovarian reserve, as well as of the transcripts modulating T3 signaling/availability in the cumulus cells, confirmed ovarian dysthyroidism as a common and evolutionary conserved trait of POA.

Background: Immune-checkpoint inhibitors (ICIs) have increased and improved the treatment options for patients with non-oncogene-addicted advanced stage non-small cell lung cancer (NSCLC). However, the role of ICIs in oncogene-addicted advanced stage NSCLC patients is still debated. In this study, in an attempt to fill in the informational gap on the effect of ICIs on other driver mutations, we set out to provide a molecular landscape of clinically relevant oncogenic drivers in programmed death-ligand 1 (PD-L1) positive NSCLC patients., Methods: We retrospectively reviewed data on 167 advanced stage NSCLC PD-L1 positive patients (≥1%) who were referred to our clinic for molecular evaluation of five driver oncogenes, namely, EGFR , KRAS , BRAF , ALK and ROS1 ., Results: Interestingly, n = 93 (55.7%) patients showed at least one genomic alteration within the tested genes. Furthermore, analyzing a subset of patients with PD-L1 tumor proportion score (TPS) ≥ 50% and concomitant gene alterations ( n = 8), we found that n = 3 (37.5%) of these patients feature clinical benefit with ICIs administration, despite the presence of a concomitant KRAS gene alteration., Conclusions: In this study, we provide a molecular landscape of clinically relevant biomarkers in NSCLC PD-L1 positive patients, along with data evidencing the clinical benefit of ICIs in patient NSCLC PD-L1 positive alterations.

Generation of the 3' overhang is a critical step during homologous recombination (HR) and replication fork rescue processes. This event is usually performed by a series of DNA nucleases and/or helicases. The nuclease NurA and the ATPase HerA, together with the highly conserved MRE11/RAD50 proteins, play an important role in generating 3' single-stranded DNA during archaeal HR. Little is known, however, about HerA-NurA function and activation of this fundamental and complicated DNA repair process. Herein, we analyze the functional relationship among NurA, HerA and the single-strand binding protein SSB from Saccharolubus solfataricus . We demonstrate that SSB clearly inhibits NurA endonuclease activity and its exonuclease activities also when in combination with HerA. Moreover, we show that SSB binding to DNA is greatly stimulated by the presence of either NurA or NurA/HerA. In addition, if on the one hand NurA binding is not influenced, on the other hand, HerA binding is reduced when SSB is present in the reaction. In accordance with what has been observed, we have shown that HerA helicase activity is not stimulated by SSB. These data suggest that, in archaea, the DNA end resection process is governed by the strictly combined action of NurA, HerA and SSB.

Muscle invasive bladder cancer (MIBC) is a widespread malignancy with a worse prognosis often related to a late diagnosis. For early-stage MIBC pts, a multidisciplinary approach is mandatory to evaluate the timing of neoadjuvant chemotherapy (NAC) and surgery. The current standard therapy is platinum-based NAC (MVAC-methotrexate, vinblastine, doxorubicin, and cisplatin or Platinum-Gemcitabine regimens) followed by radical cystectomy (RC) with lymphadenectomy. However, preliminary data from Vesper trial highlighted that dose-dense NAC MVAC is endowed with a good pathological response but shows low tolerability. In the last few years, translational-based research approaches have identified several candidate biomarkers of NAC esponsiveness, such as ERCC2, ERBB2, or DNA damage response (DDR) gene alterations. Moreover, the recent consensus MIBC molecular classification identified six molecular subtypes, characterized by different sensitivity to chemo- or targeted or immunotherapy, that could open a novel procedure for patient selection and also for neoadjuvant therapies. The Italian PURE-01 phase II Trial extended data on efficacy and resistance to Immune Checkpoint Inhibitors (ICIs) in this setting. In this review, we summarize the most relevant literature data supporting NAC use in MIBC, focusing on novel therapeutic strategies such as immunotherapy, considering the better patient stratification and selection emerging from novel molecular classification.

All organisms have evolved many DNA repair pathways to counteract the different types of DNA damages. The detection of DNA damage leads to distinct cellular responses that bring about cell cycle arrest and the induction of DNA repair mechanisms. In particular, DNA double-strand breaks (DSBs) are extremely toxic for cell survival, that is why cells use specific mechanisms of DNA repair in order to maintain genome stability. The choice among the repair pathways is mainly linked to the cell cycle phases. Indeed, if it occurs in an inappropriate cellular context, it may cause genome rearrangements, giving rise to many types of human diseases, from developmental disorders to cancer. Here, we analyze the most recent remarks about the main pathways of DSB repair with the focus on homologous recombination. A thorough knowledge in DNA repair mechanisms is pivotal for identifying the most accurate treatments in human diseases.

Thyroid hormone levels are usually genetically determined. Thyrocytes produce a unique set of enzymes that are dedicated to thyroid hormone synthesis. While thyroid transcriptional regulation is well-characterized, post-transcriptional mechanisms have been less investigated. Here, we describe the involvement of ZFP36L2, a protein that stimulates degradation of target mRNAs, in thyroid development and function, by in vivo and in vitro gene targeting in thyrocytes. Thyroid-specific Zfp36l2 -/- females were hypothyroid, with reduced levels of circulating free Thyroxine (cfT4) and Triiodothyronine (cfT3). Their hypothyroidism was due to dyshormonogenesis, already evident one week after weaning, while thyroid development appeared normal. We observed decreases in several thyroid-specific transcripts and proteins, such as Nis and its transcriptional regulators ( Pax8 and Nkx2 .1), and increased apoptosis in Zfp36l2 -/- thyroids. Nis , Pax8, and Nkx2 .1 mRNAs were also reduced in Zfp36l2 knock-out thyrocytes in vitro (L2KO), in which we confirmed the increased apoptosis. Finally, in L2KO cells, we showed an altered response to TSH stimulation regarding both thyroid-specific gene expression and cell proliferation and survival. This result was supported by increases in P21/WAF1 and p-P38MAPK levels. Mechanistically, we confirmed Notch1 as a target of ZFP36L2 in the thyroid since its levels were increased in both in vitro and in vivo models. In both models, the levels of Id4 mRNA, a potential inhibitor of Pax8 activity, were increased. Overall, the data indicate that the regulation of mRNA stability by ZFP36L2 is a mechanism that controls the function and survival of thyrocytes.

Thyroid hormones (THs) regulate many biological processes in vertebrates, including reproduction. Testicular somatic and germ cells are equipped with the arrays of enzymes (deiodinases), transporters, and receptors necessary to locally maintain the optimal level of THs and their signalling, needed for their functions and spermatogenesis. Pesticides, as chlorpyrifos (CPF) and ethylene thiourea (ETU), impair the function of thyroid and testis, affecting male fertility. However, their ability to disarrange testicular T3 (t-T3) metabolism and signalling is poorly considered. Here, a multi-species analysis involving zebrafish and mouse suggests the damage of t-T3 metabolism and signalling as a mechanism of gonadic toxicity of low-doses CPF and ETU. Indeed, the developmental exposure to both compounds reduces Dio2 transcript in both models, as well as in ex-vivo cultures of murine seminiferous tubules, and it is linked to alteration of steroidogenesis and germ cell differentiation. A major impact on spermatogonia was confirmed molecularly by the expression of their markers and morphologically evidenced in zebrafish. The results reveal that in the adopted models, exposure to both pesticides alters the t-T3 metabolism and signalling, affecting the reproductive capability. Our data, together with previous reports suggest zebrafish as an evaluable model in assessing the action of compounds impairing locally T3 signalling.

Neurodevelopmental exposure to psychoactive compounds in cannabis, specifically THC, is associated with a variety of long-term psychopathological outcomes. This increased risk includes a higher prevalence of schizophrenia, mood and anxiety disorders, and cognitive impairments. Clinical and pre-clinical research continues to identify a wide array of underlying neuropathophysiological sequelae and mechanisms that may underlie THC-related psychiatric risk vulnerability, particularly following adolescent cannabis exposure. A common theme among these studies is the ability of developmental THC exposure to induce long-term adaptations in the mesocorticolimbic system which resemble pathological endophenotypes associated with these disorders. This narrative review will summarize recent clinical and pre-clinical evidence that has elucidated these THC-induced developmental risk factors and examine how specific pharmacotherapeutic interventions may serve to reverse or perhaps prevent these cannabis-related risk outcomes.

Forkhead box E1 ( FOXE1 ) is a lineage-restricted transcription factor involved in thyroid cancer susceptibility. Cancer-associated polymorphisms map in regulatory regions, thus affecting the extent of gene expression. We have recently shown that genetic reduction of FOXE1 dosage modifies multiple thyroid cancer phenotypes. To identify relevant effectors playing roles in thyroid cancer development, here we analyse FOXE1 -induced transcriptional alterations in thyroid cells that do not express endogenous FOXE1 . Expression of FOXE1 elicits cell migration, while transcriptome analysis reveals that several immune cells-related categories are highly enriched in differentially expressed genes, including several upregulated chemokines involved in macrophage recruitment. Accordingly, FOXE1 -expressing cells induce chemotaxis of co-cultured monocytes. We then asked if FOXE1 was able to regulate macrophage infiltration in thyroid cancers in vivo by using a mouse model of cancer, either wild type or with only one functional FOXE1 allele. Expression of the same set of chemokines directly correlates with FOXE1 dosage, and pro-tumourigenic M2 macrophage infiltration is decreased in tumours with reduced FOXE1 . These data establish a novel link between FOXE1 and macrophages recruitment in the thyroid cancer microenvironment, highlighting an unsuspected function of this gene in the crosstalk between neoplastic and immune cells that shape tumour development and progression.

Squamous cell carcinoma of the head and neck (SCCHN) is a complex group of malignancies, posing several challenges to treating physicians. Most patients are diagnosed with a locally advanced disease and treated with strategies integrating surgery, chemotherapy, and radiotherapy. About 50% of these patients will experience a recurrence of disease. Recurrent/metastatic SCCHN have poor prognosis with a median survival of about 12 months despite treatments. In the last years, the strategy to manage recurrent/metastatic SCCHN has profoundly evolved. Salvage treatments (surgery or re-irradiation) are commonly employed in patients suffering from locoregional recurrences and their role has gained more and more importance in the last years. Re-irradiation, using some particularly fractionating schedules, has the dual task of reducing the tumor mass and eliciting an immune response against cancer (abscopal effect). In this review, we will analyze the main systemic and/or locoregional strategies aimed at facing the recurrent/metastatic disease, underlining the enormous importance of the multidisciplinary approach in these types of patients.

The transcription factor Forkhead box E1 ( FOXE1 ) is a key player in thyroid development and function and has been identified by genome-wide association studies as a susceptibility gene for papillary thyroid cancer. Several cancer-associated polymorphisms fall into gene regulatory regions and are likely to affect FOXE1 expression levels. However, the possibility that changes in FOXE1 expression modulate thyroid cancer development has not been investigated. Here, we describe the effects of FOXE1 gene dosage reduction on cancer phenotype in vivo. Mice heterozygous for FOXE1 null allele ( FOXE1 +/- ) were crossed with a BRAF V600E -inducible cancer model to develop thyroid cancer in either a FOXE1 +/+ or FOXE1 +/- genetic background. In FOXE1 +/+ mice, cancer histological features are quite similar to that of human high-grade papillary thyroid carcinomas, while cancers developed with reduced FOXE1 gene dosage maintain morphological features resembling less malignant thyroid cancers, showing reduced proliferation index and increased apoptosis as well. Such cancers, however, appear severely undifferentiated, indicating that FOXE1 levels affect thyroid differentiation during neoplastic transformation. These results show that FOXE1 dosage exerts pleiotropic effects on thyroid cancer phenotype by affecting histology and regulating key markers of tumor differentiation and progression, thus suggesting the possibility that FOXE1 could behave as lineage-specific oncogene in follicular cell-derived thyroid cancer.

This work describes a methodology for the definition of indoor air quality monitoring plans in schools and above all to improve the knowledge and evaluation of the indoor concentration levels of some chemical pollutants. The aim is to guide interventions to improve the health of students and exposed staff connected with the activities carried out there. The proposed methodology is based on the simultaneous study of chemical (indoor/outdoor PM 2.5 , NO 2 , CO 2 ) and physical (temperature, humidity) parameters by means of automatic analyzers coupled with gaseous compounds (benzene, toluene, ethylbenzene, xylenes, formaldehyde and NO 2 ) sampled by denuders. The important novelty is that all the data were collected daily in two different situations, i.e., during school activities and no-school activities, allowing us to evaluate the exposure of each student or person. The different behaviors of all the measured pollutants during the two different situations are reported and commented on. Finally, a statistical approach will show how the investigated compounds are distributed around the two components of combustion processes and photochemical reactions.

l-carnosine is an attractive therapeutic agent for acute ischemic stroke based on its robust preclinical cerebroprotective properties and wide therapeutic time window. However, large doses are needed for efficacy because carnosine is rapidly degraded in serum by carnosinases. The need for large doses could be particularly problematic when translating to human studies, as humans have much higher levels of serum carnosinases. We hypothesized that d-carnosine, which is not a substrate for carnosinases, may have a better pharmacological profile and may be more efficacious at lower doses than l-carnosine. To test our hypothesis, we explored the comparative pharmacokinetics and neuroprotective properties of d- and L-carnosine in acute ischaemic stroke in mice. We initially investigated the pharmacokinetics of d- and L-carnosine in serum and brain after intravenous (IV) injection in mice. We then investigated the comparative efficacy of d- and l-carnosine in a mouse model of transient focal cerebral ischemia followed by in vitro testing against excitotoxicity and free radical generation using primary neuronal cultures. The pharmacokinetics of d- and l-carnosine were similar in serum and brain after IV injection in mice. Both d- and l-carnosine exhibited similar efficacy against mouse focal cerebral ischemia. In vitro studies in neurons showed protection against excitotoxicity and the accumulation of free radicals. d- and l-carnosine exhibit similar pharmacokinetics and have similar efficacy against experimental stroke in mice. Since humans have far higher levels of carnosinases, d-carnosine may have more favorable pharmacokinetics in future human studies.

Endoderm-derived organs as liver and pancreas are potential targets for regenerative therapies, and thus, there is great interest in understanding the pathways that regulate the induction and specification of this germ layer. Currently, the knowledge of molecular mechanisms that guide the in vivo endoderm specification is restricted by the lack of early endoderm specific markers. Nephrocan ( Nepn ) is a gene whose expression characterizes the early stages of murine endoderm specification (E7.5-11.5) and encodes a secreted N-glycosylated protein. In the present study, we report the identification of a new transcript variant that is generated through alternative splicing. The new variant was found to have differential and tissue specific expression in the adult mouse. In order to better understand Nepn role during endoderm specification, we generated Nepn knock-out (KO) mice. Nepn -/- mice were born at Mendelian ratios and displayed no evident phenotype compared to WT mice. In addition, we produced nullizygous mouse embryonic stem cell (mESC) line lacking Nepn by applying (CRISPR)/CRISPR-associated systems 9 (Cas9) and employed a differentiation protocol toward endoderm lineage. Our in vitro results revealed that Nepn loss affects the endoderm differentiation impairing the expression of posterior foregut-associated markers.

Pancreatic organogenesis is a multistep process that requires the cooperation of several signaling pathways. In this context, the role of pancreatic mesenchyme is important to define the epithelium development; nevertheless, the precise space-temporal signaling activation still needs to be clarified. This study reports a dissection of the pancreatic embryogenesis, highlighting the molecular network surrounding the epithelium-mesenchyme interaction. To investigate this crosstalk, pancreatic epithelium and surrounding mesenchyme, at embryonic day 10.5, were collected through laser capture microdissection (LCM) and characterized based on their global gene expression. We performed a bioinformatic analysis to hypothesize crosstalk interactions, validating the most promising genes and verifying the precise localization of their expression in the compartments, by RNA in situ hybridization (ISH). Our analyses pointed out also the c-Met gene, a very well-known factor involved in stimulating motility, morphogenesis, and organ regeneration. We also highlighted the potential crosstalk between Versican (Vcan) and Syndecan4 (Sdc4) since these genes are involved in pancreatic tissue repair, strengthening the concept that the same signaling pathways required during pancreatic embryogenesis are also involved in tissue repair. This finding leads to novel strategies for obtaining functional pancreatic stem cells for cell replacement therapies.

Gastric cancer (GC) is a leading cause of cancer-related deaths in the world. Molecular heterogeneity is a major determinant for the clinical outcomes and an exhaustive tumor classification is currently missing. Histologically normal tissue adjacent to the tumor (NAT) is commonly used as a control in cancer studies, nevertheless a recently published paper described the unique characteristics of the NAT in several tumor types. Little is known about the global gene expression profile of gastric NAT (gNAT) which could be an effective tool for a more realistic definition of GC molecular signature. Here, we integrated data of 512 samples from the Genotype-Tissue Expression project (GETx) and The Cancer Genome Atlas (TCGA) to analyze the transcriptome of healthy gastric tissues, gNAT, and GC samples. We validated TCGA-GETx data mining through inHouse gNAT and GC expression dataset. Differential gene expression together with pathway enrichment analyses, indeed, led to different results when using the gNAT or the healthy tissue as control. Based on our analyses, gNAT showed a peculiar gene signature and biological features, like the estrogen receptor pathways activation, suggesting a molecular behavior partially different from both healthy and GC tissues. Therefore, using gNAT as healthy control tissue in the characterization of tumor associated biological processes and pathways could lead to suboptimal results., Competing Interests: The authors declare no conflict of interest.

Epidemiological and experimental studies emphasize the link between environmental chemicals exposure and thyroid cancer. However, this association is strongly debated and the mechanisms of action of environmental thyroid carcinogens still need to be identified. The analysis of in vitro transcriptomic data developed to investigate the effects of chlorpyrifos on immortalized thyrocytes highlighted the impaired expression of genes involved in endodermal carcinogenesis. This endodermal carcinogenic gene-network (ECGN, including Zfp36l2 , Dmbt1 , Ddit4 ), was validated in cellular and mouse models of thyroid carcinogenesis, characterized by the constitutive activation of the mitogen-activated protein kinase (MAPK) pathway and in immortalized thyrocytes exposed to tetrachlorodibenzo- p -dioxin (TCDD) and chlorpyrifos (CPF). The mRNA levels of Zfp36l2 , Dmbt1 and Ddit4 were increased in models characterized by MAPK activation or following TCDD exposure, whereas they were inhibited by CPF exposure. Overall, the ECGN transcripts identify a novel gene-regulatory network associated with thyroid carcinogenesis promoted by genetic mutation or by environmental carcinogens. The latter have opposite effects on the modulation of the ECGN transcripts according to their mechanisms of action in promoting carcinogenesis. Therefore, the analyses of ECGN might be helpful in discriminating compounds that promote cellular survival associated or not to proliferation of thyrocytes.

[This corrects the article on p. 3792 in vol. 7.].

Fourteen volatile organic compounds (VOCs)-twelve hydrocarbons and two organochlorine compounds-were monitored both outdoors and indoors for three years at one site in Rome. Results showed that 118 out of 168 indoor seasonal mean values were higher than the corresponding outdoor concentrations. The most relevant source of outdoor hydrocarbons was automotive exhaust emissions. Due to the enforcement of various measures to protect health and the environment, outdoor levels of monoaromatic hydrocarbons decreased about ten fold over 15 years, and aliphatic hydrocarbons also decreased. With the decrease in these outdoor concentrations, indoor air sources are likely to be more relevant for indoor air exposures. Winter outdoor values for monoaromatic hydrocarbons were generally markedly higher than the summer ones. The gradual replacement of the current fleet of circulating cars with new cars complying with EURO 5 standards, further reducing hydrocarbon emissions, may possibly lead to an increase in the observed indoor/outdoor ratios. It is indeed more difficult to remove indoor sources, some of which are still unknown.