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Targeting early-stage lung cancer is vital to improve survival. Yet, the mechanisms and components of the early tumor suppressor response in lung cancer are not well understood. In this report we study the role of TLR2, a regulator of oncogene-induced senescence which is a key tumor suppressor response in premalignancy. Using human lung cancer samples and genetically engineered mouse models we show that TLR2 is active early in lung tumorigenesis where it correlates with improved survival and clinical regression.Mechanistically, TLR2 impairs early lung cancer progression via activation of cell intrinsic cell cycle arrest pathways and the proinflammatory senescence-associated secretory phenotype (SASP). The SASP regulates non-cell autonomous anti-tumor responses such as immune surveillance of premalignant cells, and we observe impaired myeloid cell recruitment to lung tumors following Tlr2 loss. Lastly, we show that administration of a TLR2 agonist reduces lung tumor growth, highlighting TLR2 as a possible therapeutic target.

Trabajo presentado en el 19th International Congress of Developmental Biology, celebrado en Guia (Portugal) del 16 al 20 de octubre de 2022., Regeneration in mammals is limited to the digit tips. The amputation of the distal tip of the terminal phalanx triggers the formation of an undifferentiated pool of progenitor cells called a blastema that will regenerate the multiple tissues required to restore the missing part. Digit tip regeneration is linked to the presence of the dorsal nail organ, as only amputations that conserve the nail matrix regenerate. However, the regenerative potential of digits without nails has not yet been tested. To evaluate nailless digits, we used dLARM1/2 mutants that develop digits with no nails due to the limb-specific loss of the dorsal limb determinant Lmx1b. We report that dLARM1/2 mutant digits are unable to regenerate, although they form a blastema. To test whether the lack of regeneration was due to the absence of dorso-ventral (DV) polarity, a concept suggested in amphibia, we evaluated Del(27) mutants that also lack DV polarity with nails on both dorsal and ventral aspects of distal phalanges due to ventral overexpression of Lmx1b. Unlike dLARM1/2 mutants, Del(27) mutant digits regenerated indicating that DV polarity is dispensable for regeneration. Interestingly, Lmx1b expression (which continues in the nail dermis postnatally) is detected uniformly in the blastema of WT, but not dLARM1/2 mutant mice. In addition, comparison of dLARM1/2 vs WT blastemal transcriptomes revealed that the mutants had defective pro regenerative processes such as vasculogenesis and extracellular matrix remodeling, while inflammatory pathways were exacerbated. Gene expressions typical of the nail dermis were also lost in dLARM1/2 digit tips, suggesting regulation by Lmx1b. Collectively, our data suggest a direct role for Lmx1b in digit tip regeneration maintaining the nail dermis and promoting a competent blastema.

Trabajo presentado en el 19th International Congress of Developmental Biology, celebrado en Guia (Portugal) del 16 al 20 de octubre de 2022., The dorso-ventral (DV) limb asymmetry is crucial for its function; however, little is known about how it is accomplished during morphogenesis. Lmx1b is the key regulator of DV patterning during limb development as it is both necessary and sufficient to specify dorsal fates, but its regulation remains poorly known. We recently demonstrated a complex autoregulatory mode of transcriptional Lmx1b regulation mediated by two limb-specific enhancers, LARM1 and LARM2. The deletion of both enhancers (dLARM1/2) reproduces the Lmx1b-null phenotype in the limb, while the removal of each enhancer produced a double ventral phenotype restricted to the posterior (dLARM1) or anterior (dLARM2) half of the limb. To investigate the regulatory logic of the Lmx1b locus in detail, we dissected the LARM1 element into its enhancer and silencer sequences and explored the functional contribution of the two Lmx1b-associated lncRNAs. The results confirm the presence of a functional repressor contained in LARM1 and underscore the positional specificity of the LARM sequences. The data suggests that Lmx1b transcription uses different regulatory elements in different positions probably reflecting the integration of the local transcriptional environment. Indeed, published ChIP-seq data showed the Gli3 and Hox13 are significantly enriched in the LARM sequences exposing their interaction with major limb regulators. Finally, considering that the double-ventral limbs of dLARM1/2 mutants fail to support locomotion, even to lift the body weight, we hypothesized that the elaboration of the DV asymmetry by modification of Lmx1b regulation, or its functional targets, had to accompany the fin to limb transition. We have identified orthologs of the LARMs in fishes and all of them show activity in the pectoral fin in zebrafish reporter essays, revealing the deep roots of Lmx1b regulation., Spanish Ministry of Science and Innovation Grant PID2020-114525GB-I00 to Marian Ros. Sofía Zdral is supported by a PhD fellowship of the University of Cantabria and Alejandro Castilla Ibeas by a PhD fellowship of the Ministry of Science and Innovation (FSE/AEI/PRE2018-083421).

Trabajo presentado en la 16th International Conference on Limb Development, regeneration, and evolution, celebrada en Cambridge (Estados Unidos) del 08 al 11 de agosto de 2022., The dorso-ventral (DV) organization of limb morphology is crucial for its function but little is known about how it is implemented during morphogenesis. Lmx1b is the key regulator of DV patterning as it is both necessary and sufficient to specify dorsal fates, but its regulation remains poorly known. Recently, we reported that Lmx1b is subject to a complex and autoregulatory mode of transcriptional regulation mediated by two limb-specific enhancers termed LARM1 and LARM2. Interestingly, these enhancers display anterior-posterior (AP) spatial modularity with LARM1 controlling Lmx1b expression in the anterior and LARM2 in the posterior half of the limb. To investigate the regulatory logic of the Lmx1b locus in higher detail, we have dissected the LARM1 enhancer and explored the functional contribution of two Lmx1b associated lncRNAs. The results confirm the presence of a functional repressor and underscore the AP positional specificity of the LARM sequences. Conjointly, the data suggests that Lmx1b transcription uses different regulatory elements in different positions probably reflecting the integration of the local transcriptional environment. Finally, since the double-ventral limbs of dLARM1/2 mutants fail to support locomotion, we hypothesized that the elaboration of the DV axis, possible driven by changes in the regulatory genome, had to accompany the fin transition towards appendages capable of lifting the body weight. In this direction, we have identified orthologs of the LARM sequences from chondrichthyans. All these sequences show activity in the pectoral fin in zebrafish reporter assays, revealing the deep roots of Lmx1b regulation.

Trabajo presentado en el 16th International Conference on Limb Development, Regeneration, and Evolution, celebrado en Cambridge (Estados Unidos) del 08 al 11 de agosto de 2022., Mammalian limb regeneration is restricted to the distal third of terminal phalanges. Amputation of distal digit tips results in blastema formation and restoration of the missing part. This regenerative response has been linked to the presence of the nail, but digits without nails have not been evaluated. Here we interrogate the regenerative ability of the double-ventral digits of dLARM1/2 mutants, which lack dorsal nails due to the limb-specific absence of Lmx1b. We report that these nail-less digits fail to regenerate, supporting the nail requirement. Because the absence of dorso-ventral (DV) polarity could also impair regeneration, as reported in amphibians, we tested the double-dorsal double-nail digits of Del(27) mutants, which lack DV polarity due to the limb-restricted loss of En1. Since Del(27) digits did regenerate, we concluded that DV polarity is not required for digit tip regeneration. We documented continued dorsal expression of Lmx1b postnatally in the wild-type (WT) nail dermis. Moreover, there is uniform Lmx1b expression in WT regeneration-competent blastemas. Transcriptomic comparison between WT and dLARM1/2 mutant blastemas revealed differential up-regulation of gene signatures functionally associated with vascularization and connective tissue remodeling in WT mice. In contrast, the dLARM1/2 non-regenerative blastemas that lack Lmx1b expression show differential up-regulation of genes associated with inflammation and inflammatory processes. Conjointly, our data implicate a direct role for Lmx1b in digit tip regeneration by maintaining the nail dermis and promoting a competent blastema.

To determine whether a newly modified capsular tension ring (CTR) is effective at preventing toric intraocular lens (TIOL) rotation and misalignment.John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA.Experimental study.Ten human cadaver eyes were used to test the ease or difficulty of TIOL rotation in the capsular bag under 3 experimental conditions: a TIOL alone, a TIOL with a standard CTR, or a TIOL with a newly modified CTR with indentations in a sinusoidal pattern. Scores for the ease of IOL rotation were compared by using the nonparametric Friedman analysis of variance test. In addition, both anterior and posterior Miyake-Apple views were filmed to observe the rotational stability of TIOLs in the capsular bag under the 3 test conditions.In the ten eyes of five patients, the rotational stability improved with a standard CTR, but further improvement was statistically observed (P.05) with the newly modified CTR under all test conditions. This was true for both IOLs used (AcrySof and TECNIS toric IOLs), with or without ophthalmic viscosurgical device, and for either clockwise or counterclockwise rotations.A newly designed CTR prototype represents a new technology for improving the rotational stability of a TIOL in the capsular bag. Under all test conditions, the prototype performed significantly better than a standard CTR. The results support the use of this new CTR design to improve the accuracy and refractive success of TIOLs.

Mammalian digit tip regeneration is linked to the presence of nail tissue, but a nail-explicit model is missing. Here, we report that nail-less double-ventral digits of ΔLARM1/2 mutants that lack limb-specific Lmx1b enhancers fail to regenerate. To separate the nail’s effect from the lack of DV polarity, we also interrogate double-dorsal double-nail digits and show that they regenerate. Thus, DV polarity is not a prerequisite for regeneration and the nail requirement is supported. Transcriptomic comparison between wild-type and non-regenerative ΔLARM1/2 mutant blastemas reveals differential up-regulation of vascularization and connective tissue functional signatures in wild-type versus upregulation of inflammation in the mutant. These results, together with the finding of uniform Lmx1b expression in the wild-type blastema and in the dorsal dermis underneath the nail, indicate that, in addition of the nail’s effect, a direct role for Lmx1b in driving the progression of digit tip regeneration is likely.

Trabajo presentado en The Company of Biologists Workshop Molecular mechanisms of developmental and regenerative biology (EMBO), celebrado en modalidad virtual del 26 al 29 de abril de 2022., The regeneration of the digit tip is an exceptional model of multi-tissue regeneration in mammals, with remarkable potential to advance regenerative medicine. Digit tip regeneration positively correlates with the presence of the nail organ, most likely because of the environment of the nail stem cells, but the underlying mechanisms are still unclear. The fact that the nail is a dorsal structure links regeneration with dorso-ventral (DV) polarity. Here, to evaluate the nail function more directly, and to disentangle it from DV positional information, we tested the regenerative ability of double-ventral digits and double-dorsal digits resulting from the limb-specific loss of Lmx1b and of En1 expression, respectively. Our results indicate that DV polarity is not required for digit tip regeneration but that an excessive inflammatory response prevents regeneration in the absence of Lmx1b. The possible causes of the exacerbated inflammation in the absence of Lmx1b are discussed.

Targeting early-stage lung cancer is vital to improve survival. However, the mechanisms and components of the early tumor suppressor response in lung cancer are not well understood. In this report, we study the role of Toll-like receptor 2 (TLR2), a regulator of oncogene-induced senescence, which is a key tumor suppressor response in premalignancy. Using human lung cancer samples and genetically engineered mouse models, we show that TLR2 is active early in lung tumorigenesis, where it correlates with improved survival and clinical regression. Mechanistically, TLR2 impairs early lung cancer progression via activation of cell intrinsic cell cycle arrest pathways and the proinflammatory senescence-associated secretory phenotype (SASP). The SASP regulates non-cell autonomous anti-tumor responses, such as immune surveillance of premalignant cells, and we observe impaired myeloid cell recruitment to lung tumors after Tlr2 loss. Last, we show that administration of a TLR2 agonist reduces lung tumor growth, highlighting TLR2 as a possible therapeutic target., F.R.M. is funded by a Wellcome Trust clinical research fellowship through the Edinburgh Clinical Academic Track (ECAT) program (203913/Z/16/Z), a Wellcome Trust-ISSF3 award (IS3-R1.07 20/21), and a Wellcome Trust iTPA award (209710/Z/17/Z). J.C.A. core lab funding was received from Cancer Research UK (C47559/A16243, Training and Career Development Board – Career Development Fellowship), the University of Edinburgh (Chancellor’s Fellowship), and the Ministry of Science and Innovation of the Government of Spain (Proyecto PID2020-117860GB-100 financiado por MCIN/AEI/10.13039/501100011033). S.W. is supported by a Cancer Research UK senior fellowship (A29576). J.C. is supported by a Wellcome Trust clinical lectureship through the ECAT program (203913/Z/16/Z). M.M. is supported by a CRUK Edinburgh Centre Award (C157/A25140). S.V. and J.F.P. are funded by National Institute on Aging (NIA) grants (R01AG 68048-1 and UG3CA 268103-1).

MicroRNAs (miRNAs), key regulators of gene expression at the post-transcriptional level, are grossly misregulated in some human cancers, including non-small-cell lung carcinoma (NSCLC). The aberrant expression of specific miRNAs results in the abnormal regulation of key components of signalling pathways in tumour cells. MiRNA levels and the activity of the gene targets, including oncogenes and tumour suppressors, produce feedback that changes miRNA expression levels and indicates the cell’s genetic activity. In this study, we measured the expression of five circulating miRNAs (miR-195, miR-504, miR-122, miR-10b and miR-21) and evaluated their association with EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) mutation status in 66 NSCLC patients. Moreover, we examined the discriminative power of circulating miRNAs for EGFR mutant‐positive and -negative NSCLC patients using two different data normalisation approaches. We extracted total RNA from the plasma of 66 non-squamous NSCLC patients (31 of whom had tumours with EGFR mutations) and measured circulating miRNA levels using quantitative reverse transcription polymerase chain reaction (RT-qPCR). The miRNA expression levels were normalised using two endogenous controls: miR-191 and miR-16. We found significant associations between the expression of circulating miR-504 and EGFR-activating mutations in NSCLC patients regardless of the normalisation approach used (p = 0.0072 and 0.0236 for miR-16 and miR-191 normalisation, respectively). The greatest discriminative power of circulating miR-504 was observed in patients with EGFR exon 19 deletions versus wild-type EGFR normalised to miR-191 (area under the curve (AUC) = 0.81, p

Pathophysiological conditions can modify the skeletal chemical concentration. This study analyzes the elemental composition in two anatomical regions from dry femoral bone using a portable X-Ray Fluorescence (pXRF) and evaluates its impact in the bone mineral density (BMD). The left femora of 97 female skeletons (21–95 years old individuals) from the Coimbra Identified Skeletal Collection were studied. Diagenetic biases were discarded at the outset and BMD was determined with Dual-energy X-ray absorptiometry. Chemical measurements were performed at the midpoint of the femoral neck and at the midshaft using a pXRF device, and comparisons were made considering the age and the BMD values. Only elements with a Technical Measurement Error ≤ 5% were selected: P, S, Ca, Fe, Zn, As, Sr, Pb and the Ca/P ratio. Statistically significant differences were found between regions, with higher concentrations of P, Ca, Zn and S at the midshaft, and the Ca/P ratio at the femoral neck. The concentration of P is higher in individuals, This study was supported by the ERASMUS+ internship [SZ] performed in the Research Centre for Anthropology and Health (CIAS: PEstOE/SADG/UI0283/2020) at the University of Coimbra and the FCT-Fellowship SFRH/BD/115691/2016 [AMC]. FCT-Fellowship SFRH/BD/115691/2016 [AMC], Erasmus + Program [SZ].

Trabajo presentado en el EMBO Workshop: Enhanceropathies: understanding enhancer function to understand human disease, celebrado en Santander (España) del 06 al 09 de octubre de 2021., Lmx1b is the key regulator of dorso-ventral (DV) pattern in the limb, as it is both necessary and sufficient to specify dorsal fates. Accordingly, targeted disruption of Lmx1b results in double ventral limbs. In humans, mutations in the LMX1B gene are responsible for the autosomal dominant Nail-Patella Syndrome (OMIM #161200), characterized by incomplete limb dorsalization. Here, we report on two enhancers located upstream of the gene, LARM1 and LARM2, that regulate Lmx1b specifically in the limb. These sequences contain several canonical Lmx1b binding sites that mediate a positive loop of Lmx1b autoregulation. Deletion of these enhancers together results in mice with double ventral limbs, like these of Lmx1b-nul mice. Remarkably, deletion of each individual enhancer produced a double ventral phenotype clearly restricted to the posterior half of the limb in LARM1 mutants and to the anterior in LARM2 mutants, indicating differential activity of these enhancers along the anterior-posterior axis. These results reflect an unexpected complex, modular and autoregulatory mode of Lmx1b transcriptional regulation during limb development. We also report on several NPS patient families with normal LMX1B coding sequence but variations in the LARM region that exhibit limb restricted phenotypes, stressing the role of regulatory modules in disease pathogenesis.

Pathophysiological conditions can modify the skeletal chemical concentration. This study analyzes the elemental composition in two anatomical regions from dry femoral bone using a portable X-Ray Fluorescence (pXRF) and evaluates its impact in the bone mineral density (BMD). The left femora of 97 female skeletons (21-95 years old individuals) from the Coimbra Identified Skeletal Collection were studied. Diagenetic biases were discarded at the outset and BMD was determined with Dual-energy X-ray absorptiometry. Chemical measurements were performed at the midpoint of the femoral neck and at the midshaft using a pXRF device, and comparisons were made considering the age and the BMD values. Only elements with a Technical Measurement Error ≤ 5% were selected: P, S, Ca, Fe, Zn, As, Sr, Pb and the Ca/P ratio. Statistically significant differences were found between regions, with higher concentrations of P, Ca, Zn and S at the midshaft, and the Ca/P ratio at the femoral neck. The concentration of P is higher in individuals 50 years, while S and Ca/P ratio increase in individuals ≥ 50 years. The decrease of P with age can be simultaneously related to the decline of its concentration in osteoporosis. Decreased BMD is also associated with higher levels of S and Pb. Osteoporosis enhances the absorption of osteolytic elements in specific locations. This fast and non-destructive technique has proved effective for the comprehension of chemical changes related to bone mass loss. This study highlights the potential of identified skeletal collections to improve the knowledge about bone fragility.

Objective To improve the differential diagnosis of osteopoikilosis in past populations using a clinical case as an example of this rare condition. Materials A patient referred to our Genetic Service with suspected Buschke Ollendorff Syndrome after finding a connective nevus. Methods Radiological images from different body regions were accompanied by a genetic study using next-generation sequencing. Results Small circular-to-ellipsoid sclerotic lesions were found in the epiphysis and metaphysis of long bones, as well as in the pelvis. These lesions were bilaterally distributed and with well-defined margins, compatible with the characteristics of Buschke Ollendorff Syndrome, bone manifestation osteopoikilosis. A heterozygous mutation on LEMD3 (NM_001167614:c.1918 + 1G > C) was identified by next-generation sequencing. Based on this confirmed case, we have discussed the most probable causes of similar bone lesions found in the archaeological record. Conclusion It has been demonstrated how a current case of a rare disease can provide useful tools to improve the differential diagnosis of this disease in ancient skeletons. Significance This work underlines the great need for multidisciplinary platforms that integrates clinical research into paleopathology in order to successfully address the study of rare diseases from the past. Limitations Since OPK is only detected by X-rays, suspected cases of this bone lesion will only be identified when radiographs are taken for other purposes. Suggestions for further research Retrospective and large-scale studies of radiographs from other research in past populations.

Significance In this study, we report a unique and necessary function for the HoxC gene cluster in the development of some ectodermal organs, as illustrated both by the hair and nail phenotype displayed by mice lacking the Hoxc13 function and by the congenital anonychia (absence of nails) in full HoxC cluster mutants. We show that Hoxc genes are activated in a colinear manner in the embryonic limb ectoderm and are subsequently transcribed in developing nails and hairs. We identify two mammalian-specific enhancers located upstream of the HoxC cluster, which display an exclusive ectodermal specificity. Individual or combined enhancer deletions suggest that they act in combination to raise the transcription level of several Hoxc genes during hair and nail development., Vertebrate Hox genes are critical for the establishment of structures during the development of the main body axis. Subsequently, they play important roles either in organizing secondary axial structures such as the appendages, or during homeostasis in postnatal stages and adulthood. Here, we set up to analyze their elusive function in the ectodermal compartment, using the mouse limb bud as a model. We report that the HoxC gene cluster was co-opted to be transcribed in the distal limb ectoderm, where it is activated following the rule of temporal colinearity. These ectodermal cells subsequently produce various keratinized organs such as nails or claws. Accordingly, deletion of the HoxC cluster led to mice lacking nails (anonychia), a condition stronger than the previously reported loss of function of Hoxc13, which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identified two mammalian-specific ectodermal enhancers located upstream of the HoxC gene cluster, which together regulate Hoxc gene expression in the hair and nail ectodermal organs. Deletion of these regulatory elements alone or in combination revealed a strong quantitative component in the regulation of Hoxc genes in the ectoderm, suggesting that these two enhancers may have evolved along with the mammalian taxon to provide the level of HOXC proteins necessary for the full development of hair and nail.

Resumen del trabajo presentado en el 17th Spanish Society for Developmental Biology Meeting Virtual Meeting, celebrado en modalidad virtual del 18 al 20 de noviembre de 2020., Vertebrate Hox genes are critical developmental regulators in the establishment of the basic body plan of bilaterian animals and, subsequently, in the organization of secondary axial structures such asthe appendages. At postnatal stages and in adulthood, Hox genes also play important roles in homeostasis and disease. Here we concentrate in the study of the function of the HoxC cluster in the ectoderm, using the mouse limb bud as a model. We report that Hoxc genes are activated in a colinear manner in the embryonic limb ectoderm and are subsequently transcribed in developing nails and hairs. Accordingly, mice lacking the complete HoxC cluster display anonychia (absence of the nail organ), a condition stronger than the misshaped nails previously reported in the loss of function of Hoxc13, which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identify two mammalian-specific enhancers located upstream of the HoxC cluster that show ectodermal restricted activity in mouse transient transgenic reporter assays. The individual and combined deletions of these two enhancers indicate that they act together to control the transcription level of Hoxc gene necessary for correct hair and nail development.

VertebrateHoxgenes are key players in the establishment of structures during the development of the main body axis. Subsequently, they play important roles either in organizing secondary axial structures such as the appendages, or during homeostasis in postnatal stages and adulthood. Here we set up to analyze their elusive function in the ectodermal compartment, using the mouse limb bud as a model. We report that theHoxCgene cluster was globally co-opted to be transcribed in the distal limb ectoderm, where it is activated following the rule of temporal colinearity. These ectodermal cells subsequently produce various keratinized organs such as nails or claws. Accordingly, deletion of theHoxCcluster led to mice lacking nails (anonychia) and also hairs (alopecia), a condition stronger than the previously reported loss of function ofHoxc13, which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identified two ectodermal, mammalian-specific enhancers located upstream of theHoxCgene cluster, which act synergistically to regulateHoxcgene expression in the hair and nail ectodermal organs. Deletion of these regulatory elements alone or in combination revealed a strong quantitative component in the regulation ofHoxcgenes in the ectoderm, suggesting that these two enhancers may have evolved along with mammals to provide the level of HOXC proteins necessary for the full development of hairs and nails.Significance StatementIn this study, we report a unique and necessary function for theHoxCgene cluster in the development of some ectodermal organs, as illustrated both by the hair and nail phenotype displayed by mice lacking theHoxc13function and by the congenital anonychia (absence of nails) in fullHoxCcluster mutants. We show thatHoxcgenes are activated in a colinear manner in the embryonic limb ectoderm and are subsequently transcribed in developing nails and hairs. We identify two mammalian-specific enhancers located upstream of theHoxCcluster with and exclusive ectodermal specificity. Individual or combined enhancer deletions suggest that they act in combination to raise the transcription level of severalHoxcgenes during hairs and nails development.

© 2020 the Author(s)., Vertebrate Hox genes are critical for the establishment of structures during the development of the main body axis. Subsequently, they play important roles either in organizing secondary axial structures such as the appendages, or during homeostasis in postnatal stages and adulthood. Here, we set up to analyze their elusive function in the ectodermal compartment, using the mouse limb bud as a model. We report that the HoxC gene cluster was co-opted to be transcribed in the distal limb ectoderm, where it is activated following the rule of temporal colinearity. These ectodermal cells subsequently produce various keratinized organs such as nails or claws. Accordingly, deletion of the HoxC cluster led to mice lacking nails (anonychia), a condition stronger than the previously reported loss of function of Hoxc13, which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identified two mammalian-specific ectodermal enhancers located upstream of the HoxC gene cluster, which together regulate Hoxc gene expression in the hair and nail ectodermal organs. Deletion of these regulatory elements alone or in combination revealed a strong quantitative component in the regulation of Hoxc genes in the ectoderm, suggesting that these two enhancers may have evolved along with the mammalian taxon to provide the level of HOXC proteins necessary for the full development of hair and nail., This work was supported by funds from the Ecole Polytechnique Fédérale (Lausanne), the University of Geneva and the Swiss National Research Fund (310030B_138662), and the European Research Council grants RegulHox (588029) to D.D., and by the Spanish Ministry of Science and Innovation (Grant BFU2017-88265-P) to M.A.R.

Introduction: Molecular diagnosis of non-small-cell lung cancer (NSCLC) is commonly based on qPCR which provides sufficient sensitivity and specificity but has low sample throughput and allows parallel analysis of few amplicons only. NGS offers higher throughput with comparable diagnostic parameters. We evaluated the diagnostic utility of NGS for EGFR mutation detection in NSCLC as a part of intra-lab method validation in our center. Methods: DNA was isolated from tumor specimens (FFPET n=16, cytological smear n=4) collected from 20 advanced NSCLC patients (ADC n=17, NOS n=3) with known EGFR mutation status established using Cobas qPCR test (Roche) and Sanger sequencing. DNA libraries were sequenced on MiSeq instrument and analyzed using Variant Interpreter software (Illumina). Results: The results from Cobas/Sanger and NGS were consistent in 100% though one sample showed very low allelic frequency (AF) of L858R variant (2.44%) in NGS. Various EGFR variants were detected with mean AF of 27.32% (range 8.25-92.82%): exon (ex) 19 deletions: n=8; ex18, 20 and 21 substitutions: n=8, ex20 insertion: n=1; wild-type: n=2. A rare EGFR variant (p.(Ile706Thr) was detected by NGS while not reported by Cobas. One FFPET sample showed sequence artifacts in NGS analysis due to the cytosine deamination effect. In 16 samples, biologically meaningful mutations were found in other genes (AKT, ERBB2, FOXL2, GNA11, KIT, PIK3CA, TP53). Conclusions: NGS allows for specific detection and precise identification of multiple EGFR variants, ex19 deletion and rare mutations in particular. However, the quality of FFPET material still poses a challenge as it determines the effectiveness of NGS analysis. The study is ongoing.

Alpha-1 antitrypsin (A1AT) deficiency is one of the most common genetic disorders in Caucasian population. There is a link between granulomatosis with polyangiitis (GPA) and most frequent variants of SERPINA1 gene encoding severe alpha-1 antitripsin deficiency. However, the potential effect of Pi*Z, Pi*S as well as other SERPINA1 variants on clinical course of vasculitis are not well understood. The aim of the study was to analyze the potential effect of A1AT protein phenotype representing the SERPINA1 gene variants on the clinical course of GPA. The study group consisted of 64 subjects with GPA, stratified according to the disease severity: patients in active phase (group I, n = 12), patients during remission on treatment (group II, n = 40) or untreated (group III, n = 12). Normal Pi*MM SERPINA1 genotype was detected by means of real-time polymerase chain reaction (PCR) or direct sequencing in 59 patients, Pi*MZ genotype in 2, and Pi*IM, Pi*MS or Pi*SZ in 1 patient respectively. The patients with abnormal Pi*Z, Pi*S, or Pi*I allele constituted 17% in group I, 5% in group II, and 8% in group III. The serum content of A1AT and high sensitivity C-reactive protein (hsCRP) assessed by nephelometry did not differ between the groups. Interestingly, the mean serum antiPR3-antibodies level detected by Elisa method was significantly greater in the GPA patients with Pi*Z, Pi*S, or Pi*I SERPINA1 variants than in the Pi*MM homozygotes. In summary, heterozygous Pi*MZ, Pi*MS, and Pi*SZ genotype was detected in 7.8% of total group of GPA patients, and in 10.5% of those with lung lesions. The abnormal alleles of Pi*S and Pi*Z may affect the clinical course of the disease.

Alpha-1 antitrypsin (A1AT) deficiency is one of the most common genetic disorders in Caucasian population. There is a link between granulomatosis with polyangiitis (GPA) and most frequent variants of SERPINA1 gene encoding severe alpha-1 antitripsin deficiency. However, the potential effect of Pi*Z, Pi*S as well as other SERPINA1 variants on clinical course of vasculitis are not well understood. The aim of the study was to analyze the potential effect of A1AT protein phenotype representing the SERPINA1 gene variants on the clinical course of GPA. The study group consisted of 64 subjects with GPA, stratified according to the disease severity: patients in active phase (group I, n = 12), patients during remission on treatment (group II, n = 40) or untreated (group III, n = 12). Normal Pi*MM SERPINA1 genotype was detected by means of real-time polymerase chain reaction (PCR) or direct sequencing in 59 patients, Pi*MZ genotype in 2, and Pi*IM, Pi*MS or Pi*SZ in 1 patient respectively. The patients with abnormal Pi*Z, Pi*S, or Pi*I allele constituted 17% in group I, 5% in group II, and 8% in group III. The serum content of A1AT and high sensitivity C-reactive protein (hsCRP) assessed by nephelometry did not differ between the groups. Interestingly, the mean serum antiPR3-antibodies level detected by Elisa method was significantly greater in the GPA patients with Pi*Z, Pi*S, or Pi*I SERPINA1 variants than in the Pi*MM homozygotes. In summary, heterozygous Pi*MZ, Pi*MS, and Pi*SZ genotype was detected in 7.8% of total group of GPA patients, and in 10.5% of those with lung lesions. The abnormal alleles of Pi*S and Pi*Z may affect the clinical course of the disease.

Epidermal growth factor receptor (EGFR) mutation assessment is essential for targeted therapy of non-small cell lung cancer (NSCLC) as predictive biomarker of the response to EGFR tyrosine kinase inhibitors (TKI). There is a number of well-known mutations in EGFR gene. Still infrequent or complex aberrations are also observed. p.Leu747Pro is one of rare mutations which might be misdiagnosed by commercial real-time PCR kits for EGFR mutation analysis. It is an inhibiting mutation. Importantly, it might be confused with an activating deletion in exon 19 of EGFR gene. The aim of the study was to assess the occurrence of p.Leu747Pro in a group of 1841 NSCLC patients referred for EGFR mutation analysis between 2015 and 2017. EGFR mutation analysis was performed with CE-IVD real-time PCR kit. The frequency of EGFR gene mutations was 8,4% (n=155), including deletions in exon 19 (n=84; 4,6%), p.Leu858Arg (n=56; 3%), insertions in exon 20 (n=9), p.Glu719X (n=1), p.Glu719X and p.Ser768Ile (n=5). All samples with a deletion in exon 19 were reanalysed by validated PNA-LNA PCR clamp to confirm the test result. In case of a discrepancy, Sanger sequencing was performed. Upon reanalysis, in 4 (4,8%) cases initially diagnosed as a deletion in exon 19, p.Leu747Pro substitution was decisively detected instead and confirmed with direct sequencing. All four NSCLC subjects (3 female, 1 male, average age 61) were diagnosed with adenocarcinoma. Clinical follow-up of patients harbouring c.Leu747Pro mutation is ongoing. This is the first report presenting p.Leu747Pro in Caucasian NSCLC patients. It has been previously described only in Asian population as a primary resistance mutation to EGFR TKI.

Background: Alpha-1 antitrypsin (AAT) is encoded by the highly polymorphic SERPINA1 gene. Mutations in this gene cause AAT deficiency (AATD) - a risk factor for a number of chronic lung diseases. The causative link between AATD and non-small cell lung cancer (NSCLC) is still controversial due to insufficient or inconsistent research data. Objective: We evaluated the frequency of the major pathogenic SERPINA1 gene alleles among Polish NSCLC patients. Methods: blood samples were collected from 468 NSCLC patients (ADC, SQC, LCC, NOS, stages I-IV). The serum AAT concentration was measured by nephelometry and the phenotype was analyzed by isoelectric focusing in polyacrylamide gel. The SERPINA1 gene variants were identified by DNA sequencing. The frequency of SERPINA1 gene alleles was estimated by means of Hardy-Weinberg equilibrium. Results: 446 out of 468 (95.2%) NSCLC patients presented normal Pi*MM phenotype. AATD alleles were found in 22 patients. 10 (2.1%) carried Pi*S allele, 10 (2.1%) - Pi*Z allele, and 3 (0.6%) – Pi*F allele. Accordingly, the frequency of major AATD alleles was: Pi*S 10.7/1000 (95%CI: 4.1-17.3) and Pi*Z 10.7/1000 (95%CI: 4.1-17.3). The mean serum AAT concentration in NSCLC patients was 180 mg/dL in Pi*MM individuals and 148 mg/dL in AATD allele carriers. Conclusions: The frequency of Pi*S and Pi*Z alleles in NSCLC patients differ noticeably from the general Polish population (Pi*Z 13.7/1000 and Pi*S 7.6/1000): the frequency of Pi*Z allele is lower whereas the frequency of Pi*S is higher. The NSCLC patients who carry AATD allele do not present reduced AAT concentration in blood as compared to the reference values from population studies. The study is on-going.

SERPINA1 gene encoding the alpha-1 antitrypsin (A1AT) protein is highly polymorphic. Apart from the most prevalent PI*S and PI*Z deficiency alleles, other so-called rare variants also predispose individuals to severe chronic respiratory disorders (CRDs). Our aim was to assess the frequency of common and rare SERPINA1 mutations in a group of 1749 Polish patients with CRDs referred for A1AT deficiency diagnosticsbetween 2013-2016. A1AT concentration was assessed by nephelometry, phenotype by isoelectric focusing followed by PCR genotyping and/or direct sequencing. Further on, samples from 100 consecutive patients with an A1AT concentration 85.5% samples carried the normal PI*MM genotype, whereas in 14.5%, at least one A1AT deficiency variant was detected. PI*S (1.9%) and PI*Z (11.9%) alleles were identified yielding frequencies of 0.011 and 0.069, respectively. Rare A1AT variants were detected in 11 patients: PI*F (n=5) and PI*I (n=5). Also PI*M6 Passau and c.573delG indicating heterozygous Null variant were revealed in 1 patient. Retrospective analysis of SERPINA1 gene by direct sequencing in 100 patients revealed rare A1AT alleles: PI*M2 Obernburg (n=1), c.922G>T (n=1), PI*P Lowell (n=1) and PI*M wurzburg (n=1). No PI*M malton allele was detected. The prevalence of the PI*F and PI*I alleles in Polish CRD patients seems higher than in others European studies. PI*Mmalton, the most prevalent rare variant in Southern Europe, was not detected. Common PI*Z A1AT deficiency allele is considerably more common in CRDs subjects than in general Polish population.

The SERPINA1 gene encoding the alpha-1 antitrypsin (A1AT) protein is highly polymorphic. It is known that, apart from the most prevalent PI*S and PI*Z A1AT deficiency variants, other so-called rare variants also predispose individuals to severe chronic respiratory disorders such as emphysema and chronic obstructive pulmonary disease. Our aim was to assess the frequencies of common and rare SERPINA1 mutations in a group of 1033 Polish patients referred for A1AT deficiency diagnostics due to chronic respiratory disorders in the period of January 2014-September 2015. All blood samples were analyzed according to the routine diagnostic protocol, including A1AT serum concentration assessment by nephelometry and immune isoelectric focusing, followed by PCR genotyping and direct sequencing when necessary. A total of 890 out of the 1033 samples (86 %) carried the normal PI*MM genotype, whereas, in 143 samples (14 %), at least one A1AT deficiency variant was detected. In 132 subjects, PI*S (2.1 %) and PI*Z (10.8 %) common deficiency alleles were identified, yielding frequencies of 0.011 and 0.062, respectively. Rare SERPINA1 variants were detected in nine patients: PI*F (c.739CT) (n = 5) and PI*I (c.187CT) (n = 4). Samples from the patients with an A1AT serum concentration below 120 mg/dl and presenting a PI*MM-like phenotypic pattern were retrospectively analyzed by direct sequencing for rare SERPINA1 mutations, revealing a PI*M2Obernburg (c.514GT) mutation in one patient and a non-pathogenic mutation (c.922GT) in another. We conclude that the deficiency PI*Z A1AT allele is considerably more common in patients with chronic respiratory disorders than in the general Polish population. The prevalence of the PI*F allele seems higher than in other European studies.

The SERPINA1 gene encoding the alpha-1 antitrypsin (A1AT) protein is highly polymorphic. It is known that, apart from the most prevalent PI*S and PI*Z A1AT deficiency variants, other so-called rare variants also predispose individuals to severe chronic respiratory disorders such as emphysema and chronic obstructive pulmonary disease. Our aim was to assess the frequencies of common and rare SERPINA1 mutations in a group of 1033 Polish patients referred for A1AT deficiency diagnostics due to chronic respiratory disorders in the period of January 2014–September 2015. All blood samples were analyzed according to the routine diagnostic protocol, including A1AT serum concentration assessment by nephelometry and immune isoelectric focusing, followed by PCR genotyping and direct sequencing when necessary. A total of 890 out of the 1033 samples (86 %) carried the normal PI*MM genotype, whereas, in 143 samples (14 %), at least one A1AT deficiency variant was detected. In 132 subjects, PI*S (2.1 %) and PI*Z (10.8 %) common deficiency alleles were identified, yielding frequencies of 0.011 and 0.062, respectively. Rare SERPINA1 variants were detected in nine patients: PI*F (c.739C>T) (n = 5) and PI*I (c.187C>T) (n = 4). Samples from the patients with an A1AT serum concentration below 120 mg/dl and presenting a PI*MM-like phenotypic pattern were retrospectively analyzed by direct sequencing for rare SERPINA1 mutations, revealing a PI*M2Obernburg (c.514G>T) mutation in one patient and a non-pathogenic mutation (c.922G>T) in another. We conclude that the deficiency PI*Z A1AT allele is considerably more common in patients with chronic respiratory disorders than in the general Polish population. The prevalence of the PI*F allele seems higher than in other European studies.

Summary Background : Chronic obstructive pulmonary disease (COPD) is accompanied by both airway and systemic inflammation and by oxidative stress. This study aimed to characterise the relationship between oxidative stress and inflammatory components in induced sputum and blood. Material & methods : We studied blood and sputum samples from stable COPD patients (mean FEV 1 60.5±7.5% predicted) at baseline (no treatment) and after 10 weeks treatment with either inhaled steroid, fluticasone propionate (FP) (1000μg/d) or 10 weeks treatment with N -acetylcysteine (600mg/d) (NAC). We assessed the inflammatory markers (IL-8, ECP, sICAM-1, NE) in sputum and serum and we compared them with blood markers of oxidative stress (SOD, GPx, TEAC, albumin, vitamin E and A). Results : At baseline blood sICAM-1 correlated with IL-8 levels ( P 0.01 , r = 0.62 ) and negatively with GPx ( P 0.01 , r = - 0.63 ) and with TEAC ( P 0.05 , r = - 0.53 ). TEAC correlated positively with GPx ( P 0.01 , r = 0.70 ). Correlation between sICAM and IL-8 disappeared after NAC treatment. The correlation between sICAM and GPx disappeared after FP treatment. The correlation between TEAC and GPx was maintained after both NAC and FP. Conclusions : The relationship between markers of inflammation, adhesion and antioxidant capacity is significantly modulated by treatment with N -acetylcysteine or inhaled corticosteroids.

Physical exercises proved to be the single best way of improving bad mood (Thayer, 2001). In the present study we replicated this result within a framework of three-dimensional model of mood (Matthews et al., 1990) taking into account a possible moderating role of personality traits. Participants (young female adults; N = 100) filled Eysencks’ EPQ-R, Zuckerman’s Sensation Seeking Scale and a Polish adaptation of UMACL checklist (Gorynska, 2005) in which they reported their momentary moods, directly before and immediately after a 1.5 h long fitness training. Conducted analyses revealed a robust effect of exercise on mood, with Cohen’s d ranging between 1.5 for Tension and 2.1 for Energy. We also found evidence for the moderating role of personality: The mood elevation was related to higher Sensation Seeking and Psychoticism. Therefore, we confirmed that exercise has significant positive effects on mood, however some personality traits may modify the magnitude of this influence.