Interactions of Phenylalanine Derivatives with Human Tyrosinase: Lessons from Experimental and Theoretical tudies.

The pigmentation of the skin, modulated by different actors in melanogenesis, is mainly due to the melanins (protective pigments). In humans, these pigments' precursors are synthetized by an enzyme known as tyrosinase (TyH). The regulation of the enzyme activity by specific modulators (inhibitors or activators) can offer a means to fight hypo- and hyper-pigmentations responsible for medical, psychological and societal handicaps. Herein, we report the investigation of phenylalanine derivatives as TyH modulators. Interacting with the binuclear copper active site of the enzyme, phenylalanine derivatives combine effects induced by combination with known resorcinol inhibitors and natural substrate/intermediate (amino acid part). Computational studies including docking, molecular dynamics and free energy calculations combined with biological activity assays on isolated TyH and in human melanoma MNT-1 cells, and X-ray crystallography analyses with the TyH analogue Tyrp1, provide conclusive evidence of the interactions of phenylalanine derivatives with human tyrosinase. In particular, our findings indicate that an analogue of L-DOPA, namely (S)-3-amino-tyrosine, stands out as an amino phenol derivative with inhibitory properties against TyH.

Comorbidities and use of health services in people with diabetes mellitus according to risk levels by adjusted morbidity groups.

BACKGROUND: People with diabetes mellitus frequently have other comorbidities and involve greater use of primary and hospital care services. The aim of this study was to describe the comorbidities and use of primary and hospital care services of people with diabetes according to their risk level by adjusted morbidity groups (AMG) and to analyse the factors associated with the utilisation of these services. METHODS: Cross-sectional study. People with diabetes were identified within the population of patients with chronic conditions of an urban health care centre by the AMG stratification tool integrated into the primary health care electronic clinical record of the Community of Madrid. Sociodemographic, functional, clinical characteristics and annual health care services utilisation variables were collected. Univariate, bivariate and Poisson regression analyses were performed. RESULTS: A total of 1,063 people with diabetes were identified, representing 10.8% of patients with chronic conditions within the health centre. A total of 51.4% were female, the mean age was 70 years, 94.4% had multimorbidity. According to their risk level, 17.8% were high-risk, 40.6% were medium-risk and 41.6% were low-risk. The most prevalent comorbidities were hypertension (70%), dyslipidaemia (67%) and obesity (32.4%). Almost 50% were polymedicated. Regarding health services utilisation, 94% were users of primary care, and 59.3% were users of hospital care. Among the main factors associated with the utilisation of both primary and hospital care services were AMG risk level and complexity index. In primary care, utilisation was also associated with the need for primary caregivers, palliative care and comorbidities such as chronic heart failure and polymedication, while in hospital care, utilisation was also associated with comorbidities such as cancer, chronic obstructive pulmonary disease or depression. CONCLUSIONS: People with diabetes were older, with important needs for care, many associated comorbidities and polypharmacy that increased in parallel with the patient's risk level and complexity. The utilisation of primary and hospital care services was very high, being more frequent in primary care. Health services utilization were principally associated with functional factors related to the need of care and with clinical factors such as AMG medium and high-risk level, more complexity index, some serious comorbidities and polymedication.

Perception of abortion in first-year students of the Faculty of Medicine after the Human Embryology course.

BACKGROUND: Multiple studies have been recorded regarding the perception of abortion among students in the health area. OBJECTIVE: To know if medical-scientific training is sufficient to generate a change in the perception of first-year students of the National Autonomous University of Mexico Faculty of Medicine. MATERIAL AND METHODS: A cross-sectional survey was applied in two times, with the level of relationship between the items of each dimension being analyzed using Cramer's V test. The results were compared to analyze if there was a change in the perception of abortion after the human embryology course. RESULTS: With an initial population of 2,150 students, 393 pre-course and 394 post-course surveys were conducted. The results indicated that despite the existence of changes in the perception of Faculty of Medicine students with regard to abortion, they are not significant. CONCLUSIONS: The medical-scientific training provided by the human embryology course is not enough to provide foundations to the perception of students on issues such as abortion or conscientious objection.

ANTECEDENTES: Actualmente se han registrado diversos estudios sobre la percepción del aborto en el alumnado del área de la salud. OBJETIVO: Conocer si la formación médico-científica puede generar un cambio en la percepción del alumnado que cursa el primer año en la Facultad de Medicina de la Universidad Nacional Autónoma de México. MATERIAL Y MÉTODOS: Se aplicó una encuesta transversal en dos tiempos y se analizó el nivel de relación de los ítems de cada dimensión mediante coeficiente V de Crámer. Los resultados se compararon para identificar si existió un cambio de percepción acerca del aborto después del curso de embriología humana. RESULTADOS: Con una población inicial de 2150 alumnos, se realizaron 393 encuestas previas al curso y 394 encuestas posteriores. Los resultados indicaron que a pesar de existir cambios en la percepción de los alumnos de la Facultad de Medicina sobre el aborto, no fueron significativos. CONCLUSIONES: La formación médico-científica que proporciona el curso de embriología humana no es suficiente para fundamentar la percepción del alumnado en temas como el aborto o la objeción de conciencia.

Assembly of The Mitochondrial Complex†I Assembly Complex Suggests a Regulatory Role for Deflavination.

Fatty acid ß-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are mitochondrial redox processes that generate ATP. The biogenesis of the respiratory Complex I, a 1 MDa multiprotein complex that is responsible for initiating OXPHOS, is mediated by assembly factors including the mitochondrial complex I assembly (MCIA) complex. However, the organisation and the role of the MCIA complex are still unclear. Here we show that ECSIT functions as the bridging node of the MCIA core complex. Furthermore, cryo-electron microscopy together with biochemical and biophysical experiments reveal that the C-terminal domain of ECSIT directly binds to the vestigial dehydrogenase domain of the FAO enzyme ACAD9 and induces its deflavination, switching ACAD9 from its role in FAO to an MCIA factor. These findings provide the structural basis for the MCIA complex architecture and suggest a unique molecular mechanism for coordinating the regulation of the FAO and OXPHOS pathways to ensure an efficient energy production.

Phenylthiourea Binding to Human Tyrosinase-Related Protein 1.

Tyrosinase-related protein 1 (TYRP1) is one of the three human melanogenic enzymes involved in the biosynthesis of melanin, a pigment responsible for the color of the skin, hair, and eyes. It shares high sequence identity with tyrosinase, but has two zinc ions in its active site rather than two copper ions as in tyrosinase. Typical tyrosinase inhibitors do not directly coordinate to the zinc ions of TYRP1. Here, we show, from an X-ray crystal structure determination, that phenylthiourea, a highly potent tyrosinase inhibitor, does neither coordinate the active site zinc ions, but binds differently from other structurally characterized TYRP1-inhibitor complexes. Its aromatic ring is directed outwards from the active site, apparently as a result from the absence of polar oxygen substituents that can take the position of water molecules bound in the active site. The compound binds via hydrophobic interactions, thereby blocking substrate access to the active site.

Indications and results of extended total laryngectomy with en-bloc resection of overlying cervical skin.

PURPOSE: Extended total laryngectomy with en-bloc resection of overlying cervical skin (ETL) is indicated in cases with infiltration of the pre-laryngeal soft tissues. The present study analyses the surgical indications and the results of ETL in our hospital. METHODS: Retrospective review of 38 patients treated with an ETL during the period 1988-2016. RESULTS: The indications for ETL were the initial treatment of tumors with extralaryngeal extension (n = 16), salvage treatment after failure of partial surgery or radiotherapy (n = 17), and total laryngectomy in patients with infection or fibrosis of the skin over the larynx (n = 5). The surgical defect was reconstructed with local flaps in 3 cases, with pectoralis major flaps in 34 cases, and with an internal mammary artery perforator flap in one case. The rate of pharyngocutaneous fistula in the postoperative period was 16%. 5-year cancer-specific survival for patients treated with an ETL was 67.1%. The patients with positive margins and those operated as a salvage treatment after failure of previous treatments showed worse survival. CONCLUSIONS: ETL offers acceptable oncological results for patients with tumors with extralaryngeal extension. Myocutaneous or myofascial pectoralis major flaps allow for adequate reconstruction of the surgical defect with a low rate of complications.

Pharyngocutaneous fistula after total laryngectomy: multivariate analysis of risk factors and a severity-based classification proposal.

PURPOSE: The aim of the study is to determine the predisposing factors for pharyngocutaneous fistula (PCF) in patients undergoing total laryngectomy (TL) or extended TL and, secondarily, to propose a new severity-based classification system. METHODS: This is a retrospective study of 400 patients who underwent TL or extended TL. Major fistula was defined as a fistula (1) persisting for ≥ 4 weeks, (2) requiring surgical treatment, or (3) associated with perioperative mortality. RESULTS: PCF formation occurred in 93 patients (23.3%) and major fistula in 72 (18.0%). Extended surgery with partial or total pharyngectomy, previous treatment with radiotherapy, and postoperative hemoglobin levels < 99 g/L were associated with a significantly higher risk of developing major fistula. CONCLUSIONS: We propose a new PCF classification system according to clinical severity. Predictors of major fistula were the type of surgery, previous radiotherapy, and low (< 99 g/L) postoperative hemoglobin levels. We consider the use of onlay flaps in irradiated patients who require partial pharyngectomy.

Structure and Function of Human Tyrosinase and Tyrosinase-Related Proteins.

Melanin is the main pigment responsible for the color of human skin, hair and eye. Its biosynthesis requires three melanogenic enzymes, tyrosinase (TYR), and the tyrosinase-related proteins TYRP1 and TYRP2. The difficulty of isolating pure and homogeneous proteins from endogenous sources has hampered their study, and resulted in many contradictory findings regarding their physiological functions. In this review, we summarize recent advances on the structure and function of TYR and TYRPs by virtue of the crystal structure of human TYRP1, which is the first available structure of a mammalian melanogenic enzyme. This structure, combined with tyrosinase structures from other lower eukaryotes and mutagenesis studies of key active site residues, sheds light on the mechanism of TYR and TYRPs. Furthermore, a TYRP1-based homology model of TYR provides a high-quality platform to map and analyze albinism-related mutations, as well as the design of specific antimelanogenic compounds. Finally, we provide perspectives for future structure/function studies of TYR and TYRPs.

N-Methyl Inversion and Accurate Equilibrium Structures in Alkaloids: Pseudopelletierine.

A rotational spectroscopy investigation has resolved the conformational equilibrium and structural properties of the alkaloid pseudopelletierine. Two different conformers, which originate from inversion of the N-methyl group from an axial to an equatorial position, have been unambiguously identified in the gas phase, and nine independent isotopologues have been recorded by Fourier-transform microwave spectroscopy in a jet expansion. Both conformers share a chair-chair configuration of the two bridged six-membered rings. The conformational equilibrium is displaced towards the axial form, with a relative population in the supersonic jet of Naxial /Nequatorial ≈2/1. An accurate equilibrium structure has been determined by using the semiexperimental mixed-estimation method and alternatively computed by quantum-chemical methods up to the coupled-cluster level of theory. A comparison with the N-methyl inversion equilibria in related tropanes is also presented.

Prognostic significance of extracapsular spread in isolated neck recurrences in head and neck squamous cell carcinoma patients.

Few studies have analyzed the appearance of extracapsular spread (ECS) in salvage neck dissections carried out after regional recurrence of the disease. The aim of our study was to evaluate the frequency of ECS in patients with an isolated regional recurrence treated with a salvage neck dissection, and to assess the influence of ECS on prognosis. We conducted a retrospective study of 123 patients treated with a salvage neck dissection. Eighty-two patients (66.7 %) had nodes with ECS. Five-year salvage-specific survival for patients without ECS was 77.2 %, whereas for patients with ECS it was 32.0 % (P = 0.0001). According to the results of a multivariate analysis, the presence of ECS in the salvage neck dissection was the only variable significantly related to the salvage-specific survival. Sixty-six percent of the patients with nodes with ECS had adjuvant treatment with radiotherapy or chemoradiotherapy. Five-year salvage-specific survival for patients with ECS who had not received adjuvant treatment (n = 26) was 15.2 %, whereas for patients treated with adjuvant radiotherapy (n = 39) or chemotherapy (n = 17), 5-year salvage-specific survival was 36.4 and 47.1 %, respectively. Patients with ECS could benefit from adjuvant treatment with radiotherapy or chemoradiotherapy.

Relationship between response to induction chemotherapy and disease control in patients with advanced laryngeal carcinoma included in an organ preservation protocol.

The aim of the study is to analyze the relationship between the degree of response to induction chemotherapy and the disease control in patients with locally advanced laryngeal carcinomas candidates to total laryngectomy. This retrospective study includes 389 patients with T3-T4 laryngeal tumors candidates to total laryngectomy, diagnosed between 1985 and 2013, treated with induction chemotherapy in an organ preservation protocol. Five-year local recurrence-free survival for patients receiving conservative treatment with radiotherapy after a complete response to induction chemotherapy was 75.4%; for patients with partial response greater than 50%, it was 62.0%; and for patients with the absence of response, it was 32.7%. There were significant differences in local recurrence-free survival and laryngeal dysfunction-free survival according to the response to induction chemotherapy (P = 0.0001) at the expense of patients with absence of response to induction chemotherapy. Patients with partial response greater than 50% treated with radiotherapy had a tendency to have worse local recurrence-free survival and laryngeal dysfunction-free survival than patients with complete response, but the differences did not reach statistical significance. Patients with the absence of response after induction chemotherapy had significant differences in disease-specific survival according to the second treatment: for patients treated with surgery it was 70.2%, whereas for patients treated with radiotherapy, it was 28.2% (P = 0.0001). In patients with the absence of response to induction chemotherapy, conservative treatment with radiotherapy implies a significant decrease in survival.

Structure of Human Tyrosinase Related Protein†1 Reveals a Binuclear Zinc Active Site Important for Melanogenesis.

Tyrosinase-related protein 1 (TYRP1) is one of three tyrosinase-like glycoenzymes in human melanocytes that are key to the production of melanin, the compound responsible for the pigmentation of skin, eye, and hair. Difficulties with producing these enzymes in pure form have hampered the understanding of their activity and the effect of mutations that cause albinism and pigmentation disorders. Herein we show that the typical tyrosinase-like subdomain of TYRP1 contains two zinc ions in the active site instead of copper ions as found in tyrosinases, which explains why TYRP1 does not exhibit tyrosinase redox activity. In addition, the structures reveal for the first time that the Cys-rich subdomain, which is unique to vertebrate melanogenic proteins, has an epidermal growth factor-like fold and is tightly associated with the tyrosinase subdomain. Our structures suggest that most albinism-related mutations of TYRP1 affect its stability or activity.

Scopine Isolated in the Gas Phase.

The rotational spectrum of the tropane alkaloid scopine is detected by Fourier transform microwave spectroscopy in a pulsed supersonic jet. A nonconventional method for bringing the molecules intact into the gas phase is used in which scopine syrup is mixed with glycine powder and the solid mixture is vaporized with an ultrafast UV laser beam. Laser vaporization prevents the easy isomerization to scopoline previously observed with conventional heating methods. A single conformer is unambiguously observed in the supersonic jet and corresponds to the energetically most stable species according to quantum chemical calculations. Rotational and centrifugal distortion constants are accurately determined. The spectrum shows fine and hyperfine structure due to the hindered rotation of the methyl group and the presence of a quadrupolar nucleus (14 N), respectively. This additional information allows the angle of N-methyl inversion between the N-CH3 bond and the bicyclic C-N-C plane to be determined (131.8-137.8°), as well as the internal rotation barrier of the methyl group (6.235(1) kJ mol-1 ).

Fuzziness and noise in nucleosomal architecture.

Nucleosome organization plays a key role in the regulation of gene expression. However, despite the striking advances in the accuracy of nucleosome maps, there are still severe discrepancies on individual nucleosome positioning and how this influences gene regulation. The variability among nucleosome maps, which precludes the fine analysis of nucleosome positioning, might emerge from diverse sources. We have carefully inspected the extrinsic factors that may induce diversity by the comparison of microccocal nuclease (MNase)-Seq derived nucleosome maps generated under distinct conditions. Furthermore, we have also explored the variation originated from intrinsic nucleosome dynamics by generating additional maps derived from cell cycle synchronized and asynchronous yeast cultures. Taken together, our study has enabled us to measure the effect of noise in nucleosome occupancy and positioning and provides insights into the underlying determinants. Furthermore, we present a systematic approach that may guide the standardization of MNase-Seq experiments in order to generate reproducible genome-wide nucleosome patterns.

Management of recurrent head and neck cancer: variables related to salvage surgery.

After a local and/or regional recurrence of head and neck squamous cell carcinoma (HNSCC) not all patients are candidates to salvage treatment. The objective of this study was to identify the variables related to performance of salvage surgery with curative intent in these patients. We performed a retrospective study of 1088 HNSCC patients with a local and/or regional recurrence. According to a multivariate analysis, the variables related to performance of salvage surgery were the Karnofsky index, the location and extension of the primary tumor, the initial treatment, the disease-free interval between treatment of the initial tumor and diagnosis of the recurrence, and the year the recurrence was diagnosed. Considering salvage surgery as the dependent variable, the results of a recursive partitioning analysis defined four categories of patients in function of the category of local and regional extension of the initial tumor, the location of the primary tumor, the initial treatment and the disease-free interval between treatment of the initial tumor and diagnosis of the recurrence.

Internal dynamics in halogen-bonded adducts: a rotational study of chlorotrifluoromethane-formaldehyde.

The rotational spectra of two isotopologues of the 1:1 complex between chlorotrifluoromethane and formaldehyde have been recorded and analyzed by using Fourier-transform microwave spectroscopy. Only one rotamer was detected, with the two constituent molecules held together through a Cl⋅⋅⋅O halogen bond (R(Cl⋅⋅⋅O) = 3.048 Å). The dimer displays two simultaneous large-amplitude intramolecular motions. The internal rotation of formaldehyde around its symmetry axis (V2 = 28(5) cm(-1)) splits all the rotational transitions into two component lines with a relative intensity ratio of 1:3. On the other hand, the almost free internal rotation (V3 ≈ 2.5 cm(-1)) of the CF3 symmetric top increases the "rigid" value of the rotational constant A by almost one order of magnitude. In addition, all the transitions display a hyperfine structure due to the (35)Cl (or (37)Cl) nucleus quadrupole effects.

Chiral recognition and atropisomerism in the sevoflurane dimer.

We have examined the stereoselectivity of molecular recognition between two molecules of the anesthetic sevoflurane using broadband rotational spectroscopy. The transient axial chirality of sevoflurane is revealed upon the formation of the dimer, as two different diastereoisomers made of either homo- or heterochiral species are detected in a supersonic jet expansion. The conformational assignment was confirmed by the observation of eighteen different isotopologues in natural abundance (all possible (13)C's and two (18)O species of the homochiral form). The two clusters are formed in practically equal proportions (1.1 : 1), probably due to their similar hydrogen bonding topologies. In both clusters the complex is stabilized by a primary C-H···O hydrogen bond, assisted by weak C-HF interactions. This intermolecular binding regime is characterized by a mixture of electrostatic and dispersive interactions, midway between classical hydrogen bonds and van der Waals clusters.

Cooperative binding of two acetylation marks on a histone tail by a single bromodomain.

A key step in many chromatin-related processes is the recognition of histone post-translational modifications by effector modules such as bromodomains and chromo-like domains of the Royal family. Whereas effector-mediated recognition of single post-translational modifications is well characterized, how the cell achieves combinatorial readout of histones bearing multiple modifications is poorly understood. One mechanism involves multivalent binding by linked effector modules. For example, the tandem bromodomains of human TATA-binding protein-associated factor-1 (TAF1) bind better to a diacetylated histone H4 tail than to monoacetylated tails, a cooperative effect attributed to each bromodomain engaging one acetyl-lysine mark. Here we report a distinct mechanism of combinatorial readout for the mouse TAF1 homologue Brdt, a testis-specific member of the BET protein family. Brdt associates with hyperacetylated histone H4 (ref. 7) and is implicated in the marked chromatin remodelling that follows histone hyperacetylation during spermiogenesis, the stage of spermatogenesis in which post-meiotic germ cells mature into fully differentiated sperm. Notably, we find that a single bromodomain (BD1) of Brdt is responsible for selectively recognizing histone H4 tails bearing two or more acetylation marks. The crystal structure of BD1 bound to a diacetylated H4 tail shows how two acetyl-lysine residues cooperate to interact with one binding pocket. Structure-based mutagenesis that reduces the selectivity of BD1 towards diacetylated tails destabilizes the association of Brdt with acetylated chromatin in vivo. Structural analysis suggests that other chromatin-associated proteins may be capable of a similar mode of ligand recognition, including yeast Bdf1, human TAF1 and human CBP/p300 (also known as CREBBP and EP300, respectively). Our findings describe a new mechanism for the combinatorial readout of histone modifications in which a single effector module engages two marks on a histone tail as a composite binding epitope.

Unravelling the hidden DNA structural/physical code provides novel insights on promoter location.

Although protein recognition of DNA motifs in promoter regions has been traditionally considered as a critical regulatory element in transcription, the location of promoters, and in particular transcription start sites (TSSs), still remains a challenge. Here we perform a comprehensive analysis of putative core promoter sequences relative to non-annotated predicted TSSs along the human genome, which were defined by distinct DNA physical properties implemented in our ProStar computational algorithm. A representative sampling of predicted regions was subjected to extensive experimental validation and analyses. Interestingly, the vast majority proved to be transcriptionally active despite the lack of specific sequence motifs, indicating that physical signaling is indeed able to detect promoter activity beyond conventional TSS prediction methods. Furthermore, highly active regions displayed typical chromatin features associated to promoters of housekeeping genes. Our results enable to redefine the promoter signatures and analyze the diversity, evolutionary conservation and dynamic regulation of human core promoters at large-scale. Moreover, the present study strongly supports the hypothesis of an ancient regulatory mechanism encoded by the intrinsic physical properties of the DNA that may contribute to the complexity of transcription regulation in the human genome.