PolyQ length-based molecular encoding of vocalization frequency in FOXP2.

The transcription factor FOXP2, a regulator of vocalization- and speech/language-related phenotypes, contains two long polyQ repeats (Q1 and Q2) displaying marked, still enigmatic length variation across mammals. We found that the Q1/Q2 length ratio quantitatively encodes vocalization frequency ranges, from the infrasonic to the ultrasonic, displaying striking convergent evolution patterns. Thus, species emitting ultrasonic vocalizations converge with bats in having a low ratio, whereas species vocalizing in the low-frequency/infrasonic range converge with elephants and whales, which have higher ratios. Similar, taxon-specific patterns were observed for the FOXP2-related protein FOXP1. At the molecular level, we observed that the FOXP2 polyQ tracts form coiled coils, assembling into condensates and fibrils, and drive liquid-liquid phase separation (LLPS). By integrating evolutionary and molecular analyses, we found that polyQ length variation related to vocalization frequency impacts FOXP2 structure, LLPS, and transcriptional activity, thus defining a novel form of polyQ length-based molecular encoding of vocalization frequency.

Fetal MR Imaging Anatomy of the Transverse Temporal Gyrus (Heschl Gyrus).

BACKGROUND AND PURPOSE: The human auditory system develops early in fetal life. This retrospective MR imaging study describes the in vivo prenatal anatomic development of the transverse temporal gyrus (Heschl gyrus) site of the primary auditory cortex. MATERIALS AND METHODS: Two hundred seventy-two MR imaging studies of the fetal brain (19-39 weeks' gestational age) acquired from a single institution's 1.5T scanner were retrospectively examined by 2 neuroradiologists. MR imaging with pathologic findings and extreme motion artifacts was excluded. Postnatal Heschl gyrus landmarks were used as a reference on T2-weighted ssFSE sequences in the 3 orthogonal planes. The frequency of the Heschl gyrus was reported for gestational age, hemisphere, and planes. Descriptive statistics and a McNemar test were performed. RESULTS: Two hundred thirty MR imaging studies were finally included. Fetal brains were divided by gestational age (in weeks) into 8 groups (parentheses indicate the number of observations): 19-21 (29), 22-23 (32), 24-25 (21), 26-27 (18), 28-29 (35), 30-31 (30), 32-33 (33) and >34 (32). The Heschl gyrus appeared on MR imaging between 24 and 25 weeks' gestational age (14/21 fetuses, 67%) and was visible in all fetuses after the 28th week of gestation. By its appearance (24-28 weeks' gestational age), the sagittal plane was the most sensitive in its detectability. After 28-29 weeks' gestational age, the Heschl gyrus was evident in all acquisition planes and fetuses. Results did not differ between hemispheres. CONCLUSIONS: The Heschl gyrus appears on MR imaging at 24-25 weeks' gestational age, paralleling the functional activation of the auditory system. We propose the Heschl gyrus as an early additional MR imaging marker of fetal brain development.

Pantothenate biosynthesis in Toxoplasma gondii tachyzoites is not a drug target.

Toxoplasma gondii is a pervasive apicomplexan parasite that can cause severe disease and death in immunocompromised individuals and the developing foetus. The treatment of toxoplasmosis often leads to serious side effects and novel drugs and drug targets are therefore actively sought. In 2014, Mageed and colleagues suggested that the T. gondii pantothenate synthetase, the enzyme responsible for the synthesis of the vitamin B5 (pantothenate), the precursor of the important cofactor, coenzyme A, is a good drug target. Their conclusion was based on the ability of potent inhibitors of the M. tuberculosis pantothenate synthetase to inhibit the proliferation of T. gondii tachyzoites. They also reported that the inhibitory effect of the compounds could be antagonised by supplementing the medium with pantothenate, supporting their conclusion that the compounds were acting on the intended target. Contrary to these observations, we find that compound SW314, one of the compounds used in the Mageed et al. study and previously shown to be active against M. tuberculosis pantothenate synthetase in vitro, is inactive against the T. gondii pantothenate synthetase and does not inhibit tachyzoite proliferation, despite gaining access into the parasite in situ. Furthermore, we validate the recent observation that the pantothenate synthetase gene in T. gondii can be disrupted without detrimental effect to the survival of the tachyzoite-stage parasite in the presence or absence of extracellular pantothenate. We conclude that the T. gondii pantothenate synthetase is not essential during the tachyzoite stage of the parasite and it is therefore not a target for drug discovery against T. gondii tachyzoites.

MRI-Based Radiomics Approach Predicts Tumor Recurrence in ER + /HER2 - Early Breast Cancer Patients.

Oncotype Dx Recurrence Score (RS) has been validated in patients with ER + /HER2 - invasive breast carcinoma to estimate patient risk of recurrence and guide the use of adjuvant chemotherapy. We investigated the role of MRI-based radiomics features extracted from the tumor and the peritumoral tissues to predict the risk of tumor recurrence. A total of 62 patients with biopsy-proved ER + /HER2 - breast cancer who underwent pre-treatment MRI and Oncotype Dx were included. An RS > 25 was considered discriminant between low-intermediate and high risk of tumor recurrence. Two readers segmented each tumor. Radiomics features were extracted from the tumor and the peritumoral tissues. Partial least square (PLS) regression was used as the multivariate machine learning algorithm. PLS ß-weights of radiomics features included the 5% features with the largest ß-weights in magnitude (top 5%). Leave-one-out nested cross-validation (nCV) was used to achieve hyperparameter optimization and evaluate the generalizable performance of the procedure. The diagnostic performance of the radiomics model was assessed through receiver operating characteristic (ROC) analysis. A null hypothesis probability threshold of 5% was chosen (p < 0.05). The exploratory analysis for the complete dataset revealed an average absolute correlation among features of 0.51. The nCV framework delivered an AUC of 0.76 (p = 1.1∙10-3). When combining "early" and "peak" DCE images of only T or TST, a tendency toward statistical significance was obtained for TST with an AUC of 0.61 (p = 0.05). The 47 features included in the top 5% were balanced between T and TST (23 and 24, respectively). Moreover, 33/47 (70%) were texture-related, and 25/47 (53%) were derived from high-resolution images (1 mm). A radiomics-based machine learning approach shows the potential to accurately predict the recurrence risk in early ER + /HER2 - breast cancer patients.

Heptad stereotypy, S/Q layering, and remote origin of the SARS-CoV-2 fusion core.

The fusion of the SARS-CoV-2 virus with cells, a key event in the pathogenesis of Covid-19, depends on the assembly of a six-helix fusion core (FC) formed by portions of the spike protein heptad repeats (HRs) 1 and 2. Despite the critical role in regulating infectivity, its distinctive features, origin, and evolution are scarcely understood. Thus, we undertook a structure-guided positional and compositional analysis of the SARS-CoV-2 FC, in comparison with FCs of related viruses, tracing its origin and ongoing evolution. We found that clustered amino acid substitutions within HR1, distinguishing SARS-CoV-2 from SARS-CoV-1, enhance local heptad stereotypy and increase sharply the FC serine-to-glutamine (S/Q) ratio, determining a neat alternate layering of S-rich and Q-rich subdomains along the post-fusion structure. Strikingly, SARS-CoV-2 ranks among viruses with the highest FC S/Q ratio, together with highly syncytiogenic respiratory pathogens (RSV, NDV), whereas MERS-Cov, HIV, and Ebola viruses display low ratios, and this feature reflects onto S/Q segregation and H-bonding patterns. Our evolutionary analyses revealed that the SARS-CoV-2 FC occurs in other SARS-CoV-1-like Sarbecoviruses identified since 2005 in Hong Kong and adjacent regions, tracing its origin to >50 years ago with a recombination-driven spread. Finally, current mutational trends show that the FC is varying especially in the FC1 evolutionary hotspot. These findings establish a novel analytical framework illuminating the sequence/structure evolution of the SARS-CoV-2 FC, tracing its long history within Sarbecoviruses, and may help rationalize the evolution of the fusion machinery in emerging pathogens and the design of novel therapeutic fusion inhibitors.

Discovery of Novel Inhibitors of Uridine Diphosphate-N-Acetylenolpyruvylglucosamine Reductase (MurB) from Pseudomonas aeruginosa, an Opportunistic Infectious Agent Causing Death in Cystic Fibrosis Patients.

Pseudomonas aeruginosa is of major concern for cystic fibrosis patients where this infection can be fatal. With the emergence of drug-resistant strains, there is an urgent need to develop novel antibiotics against P. aeruginosa. MurB is a promising target for novel antibiotic development as it is involved in the cell wall biosynthesis. MurB has been shown to be essential in P. aeruginosa, and importantly, no MurB homologue exists in eukaryotic cells. A fragment-based drug discovery approach was used to target Pa MurB. This led to the identification of a number of fragments, which were shown to bind to MurB. One fragment, a phenylpyrazole scaffold, was shown by ITC to bind with an affinity of Kd = 2.88 mM (LE 0.23). Using a structure guided approach, different substitutions were synthesized and the initial fragment was optimized to obtain a small molecule with Kd = 3.57 µM (LE 0.35).

Synchronous unilateral male breast cancer presenting with ipslateral gynecomastia: A triple assessment approach / Cáncer de mama masculino unilateral sincrónico con presencia de ginecomastia ipsilateral: un enfoque de triple evaluación

Male breast cancer is rare. It accounts for less than 1% of all cancers in men and less than 1% of all breast cancers. We present the case of a 73-years-old male patient with the coexistence of ipsilateral gynecomastia and two synchronous unilateral primary malignant tumors: invasive papillary carcinoma and invasive ductal carcinoma. Our case emphasizes the rare incidence of ipsilateral synchronous unilateral primary malignant tumors and gynecomastia in male breast. Since the triple radiological assessment (clinical, mammography or ultrasonography, and core biopsy) represents a mainstay of the clinical management, radiologists should be aware of the typical findings on clinical examination, mammography and ultrasound. (AU)

El cáncer de mama masculino es infrecuente, y representa menos del 1% de todos los cánceres en varones, y menos del 1% de todos los cánceres de mama. Presentamos el caso de un paciente de 73 años de edad, con coexistencia de ginecomastia ipsilateral y 2 tumores malignos primarios unilaterales sincrónicos: carcinoma papilar invasivo y carcinoma ductal invasivo. Nuestro caso resalta la rara incidencia de los tumores malignos primarios unilaterales sincrónicos e ipsilaterales y ginecomastia en la mama masculina. Como la triple evaluación radiológica (clínica, mamografía o ecografía y biopsia central) es la base del tratamiento clínico, los radiólogos deben tener en cuenta los hallazgos típicos en el examen clínico, la mamografía y la ecografía. (AU)

A G-quadruplex-binding compound shows potent activity in human gemcitabine-resistant pancreatic cancer cells.

Gemcitabine is a drug of choice in the treatment of human pancreatic cancer. Chemo-resistance to this drug is common and has been attributed to a variety of distinct mechanisms, involving > 100 genes. A recently developed small-molecule G-quadruplex ligand, the trisubstituted naphthalene diimide compound CM03, has previously been shown to have equivalent potency to gemcitabine in the pancreatic cancer cell line MIA PaCa-2. We report here on cell lines of increased resistance to gemcitabine that have been generated from this line, with the most resistant having 1,000-fold reduced sensitivity to gemcitabine. These resistant lines retain nM sensitivity to CM03. The molecular basis for the retention of potency by this G-quadruplex ligand has been examined using whole transcriptome data analysis with RNA-seq. This has revealed that the pattern of pathways down regulated by CM03 in the parental MIA PaCa-2 cell line is largely unaffected in the gemcitabine-resistant line. The analysis has also shown that the expression patterns of numerous genes involved in gemcitabine sensitivity are down regulated in the resistant line upon CM03 treatment. These results are supportive of the concept that G-quadruplex small molecules such as CM03 have potential for clinical use in the treatment of gemcitabine-resistant human pancreatic cancer.

Substituted Naphthalenediimide Compounds Bind Selectively to Two Human Quadruplex Structures with Parallel Topology.

Interactions are reported of three representative naphthalenediimide derivatives with three quadruplex targets, from the promoter region of the telomerase (hTERT) gene, a human telomeric DNA quadruplex, and a telomeric RNA quadruplex (TERRA). Thermal melting studies showed that these compounds strongly stabilize the quadruplexes, with weak stabilization of a duplex DNA. Binding studies by surface plasmon resonance and fluorescence spectroscopy found that the compounds bind to the quadruplexes with nanomolar equilibrium dissociation constants. Plausible topologies for the quadruplex complexes were deduced from CD spectra, which together with the surface plasmon resonance data indicate that the quadruplexes with parallel quadruplex folds are preferred by two compounds, which was confirmed by qualitative molecular modeling.

Novel polyamine-based Histone deacetylases-Lysine demethylase 1 dual binding inhibitors.

Epigenetic modulators Histone deacetylases (HDACs) and Lysine demethylase (LSD1) are validated targets for anticancer therapy. Both HDAC1/2 and LSD1 are found in association with the repressor protein CoREST in a transcriptional co-repressor complex, which is responsible for gene silencing. Combined modulation of both targets results in a synergistic antiproliferative activity. In the present investigation, we report about the design and synthesis of a series of polyamine-based HDACs-LSD1 dual binding inhibitors obtained by coupling Vorinostat and Tranylcypromine. Compound 4 emerged as the most promising of the synthesized series, showing good inhibitory activity towards HDAC1 and LSD1 either in vitro and in cell-based assay (Ki = 42.52 ±â€¯8.94 nM and IC50 = 3.85 µM, respectively). Furthermore, at 70.0 µM compound 4 induced a more pronounced cytotoxic effect than Vorinostat (68.6% vs 56.6% of dead cells) in MCF7 cancer cell line.

Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule.

Human pancreatic ductal adenocarcinoma (PDAC) involves the dysregulation of multiple signaling pathways. A novel approach to the treatment of PDAC is described, involving the targeting of cancer genes in PDAC pathways having over-representation of G-quadruplexes, using the trisubstituted naphthalene diimide quadruplex-binding compound 2,7-bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[ lmn][3,8]phenanthroline-1,3,6,8(2 H,7 H)-tetraone (CM03). This compound has been designed by computer modeling, is a potent inhibitor of cell growth in PDAC cell lines, and has anticancer activity in PDAC models, with a superior profile compared to gemcitabine, a commonly used therapy. Whole-transcriptome RNA-seq methodology has been used to analyze the effects of this quadruplex-binding small molecule on global gene expression. This has revealed the down-regulation of a large number of genes, rich in putative quadruplex elements and involved in essential pathways of PDAC survival, metastasis, and drug resistance. The changes produced by CM03 represent a global response to the complexity of human PDAC and may be applicable to other currently hard-to-treat cancers.

Fragment-based approaches to TB drugs.

Tuberculosis is an infectious disease associated with significant mortality and morbidity worldwide, particularly in developing countries. The rise of antibiotic resistance in Mycobacterium tuberculosis (Mtb) urgently demands the development of new drug leads to tackle resistant strains. Fragment-based methods have recently emerged at the forefront of pharmaceutical development as a means to generate more effective lead structures, via the identification of fragment molecules that form weak but high quality interactions with the target biomolecule and subsequent fragment optimization. This review highlights a number of novel inhibitors of Mtb targets that have been developed through fragment-based approaches in recent years.

Novel Polyamine-Naphthalene Diimide Conjugates Targeting Histone Deacetylases and DNA for Cancer Phenotype Reprogramming.

A series of hybrid compounds was designed to target histone deacetylases and ds-/G-quadruplex DNAs by merging structural features deriving from Scriptaid and compound 1. Compound 6 binds different DNA arrangements, inhibits HDACs both in vitro and in cells, and is able to induce a reduction of cell proliferation. Moreover, compound 6 displays cell phenotype-reprogramming properties since it prevents the epithelial to mesenchymal transition in cancer cells, inducing a less aggressive and migratory phenotype, which is one of the goals of present innovative strategies in cancer therapies.

Targeting the Nrf2/Amyloid-Beta Liaison in Alzheimer's Disease: A Rational Approach.

Amyloid is a prominent feature of Alzheimer's disease (AD). Yet, a linear linkage between amyloid-ß peptide (Aß) and the disease onset and progression has recently been questioned. In this context, the crucial partnership between Aß and Nrf2 pathways is acquiring paramount importance, offering prospects for deciphering the Aß-centered disease network. Here, we report on a new class of antiaggregating agents rationally designed to simultaneously activate transcription-based antioxidant responses, whose lead 1 showed interesting properties in a preliminary investigation. Relying on the requirements of Aß recognition, we identified the catechol derivative 12. In SH-SY5Y neuroblastoma cells, 12 combined remarkable free radical scavenger properties to the ability to trigger the Nrf2 pathway and induce the Nrf2-dependent defensive gene NQO1 by means of electrophilic activation of the transcriptional response. Moreover, 12 prevented the formation of cytotoxic stable oligomeric intermediates, being significantly more effective, and per se less toxic, than prototype 1. More importantly, as different chemical features were exploited to regulate Nrf2 and Aß activities, the two pathways could be tuned independently. These findings point to compound 12 and its derivatives as promising tools for investigating the therapeutic potential of the Nrf2/Aß cellular network, laying foundation for generating new drug leads to confront AD.

Naphthalene diimide-polyamine hybrids as antiproliferative agents: Focus on the architecture of the polyamine chains.

Naphthalene diimides (NDIs) have been widely used as scaffold to design DNA-directed agents able to target peculiar DNA secondary arrangements endowed with relevant biochemical roles. Recently, we have reported disubstituted linear- and macrocyclic-NDIs that bind telomeric and non-telomeric G-quadruplex with high degree of affinity and selectivity. Herein, the synthesis, biological evaluation and molecular modelling studies of a series of asymmetrically substituted NDIs are reported. Among these, compound 9 emerges as the most interesting of the series being able to bind telomeric G-quadruplex (ΔTm = 29 °C at 2.5 µM), to inhibit the activity of DNA processing enzymes, such as topoisomerase II and TAQ-polymerase, and to exert antiproliferative effects in the NCI panel of cancer cell lines with GI50 values in the micro-to nanomolar concentration range (i.e. SR cell line, GI50 = 76 nM). Molecular mechanisms of cell death have been investigated and molecular modelling studies have been performed in order to shed light on the antiproliferative and DNA-recognition processes.

Combined inhibition of the EGFR/AKT pathways by a novel conjugate of quinazoline with isothiocyanate.

Epidermal growth factor receptor inhibitors (EGFR-TKIs) represent a class of compounds widely used in anticancer therapy. An increasing number of studies reports on combination therapies in which the block of the EGFR-TK activity is associated with inhibition of its downstream pathways, as PI3K-Akt. Sulforaphane targets the PI3K-Akt pathway whose dysregulation is implicated in many functions of cancer cells. According to these considerations, a series of multitarget molecules have been designed by combining key structural features derived from an EGFR-TKI, PD168393, and the isothiocyanate sulforaphane. Among the obtained molecules 1-6, compound 6 emerges as a promising lead compound able to exert antiproliferative and proapoptotic effects in A431 epithelial cancer cell line by covalently binding to EGFR-TK, and reducing the phosphorylation of Akt without affecting the total Akt levels.

How formative courses about damage control surgery and non-operative management improved outcome and survival in unstable politrauma patients in a Mountain Trauma Center.

AIM: Aim of this study is to analyze how the starting of Course of Trauma in our hospital improved survival and organization in management of polytraumatized patients. MATERIAL OF STUDY: We analysed all major trauma patients (Injury Severity Score (Injury Severity Score (ISS)> 15) treated at Emergency Department of the Santa Chiara Hospital between January 2011 and December 2014. The training courses (TC) were named "management of polytrauma" (MP) and "clinical cases discussion" (CCD), and started in November 2013. We divided the patients between two groups: before November 2013 (pre-TC group) and after November 2013 (post-TC group). RESULTS: MTG's courses (EMC accredited), CCD and MP courses started in November 2013. The target of these courses was the multidisciplinary management of polytrauma patient; the courses were addressed to general surgeons, anaesthesiologists, radiologists, orthopaedics and emergency physicians. Respectively 110 and 78 doctors were formed in CCD's and MP's courses. Patients directly transported to our trauma centre rose from 67.5% to 83% (p<0,005), and E-FAST grew from 15.6% in the pre-TC group to 51.3% in the post-TC group. Time of access in operatory theatre decreased from 62 to 44 minutes. Early Mortality (within 48 hours from the hospital arrival) was 9% in the pre-TC group and 4.5% in the post-tc group (p<0.005). DISCUSSION: Be needed to complete our goal. Further analysis and possible comparison with other trauma centers be needed to complete our goal CONCLUSIONS: Our results show that in our experience the multidisciplinary approach to polytrauma patients increased early survival and improved outcome with an evidence of worker's satisfaction. However, the best practice would ask to start with the approval of procedures and guidelines by the hospital governance, followed by clinical practice changes, in order to create a dedicated emergency and trauma surgery group. KEY WORD: Damage Control Surgery, Non Operative Management, Trauma Course, Trauma Team, Trauma Center.

An optimized polyamine moiety boosts the potency of human type II topoisomerase poisons as quantified by comparative analysis centered on the clinical candidate F14512.

Combined computational-experimental analyses explain and quantify the spermine-vectorized F14512's boosted potency as a topoII poison. We found that an optimized polyamine moiety boosts drug binding to the topoII/DNA cleavage complex, rather than to the DNA alone. These results provide new structural bases and key reference data for designing new human topoII poisons.

Tumor vascular targeted liposomal-bortezomib minimizes side effects and increases therapeutic activity in human neuroblastoma.

Neuroblastoma is a childhood cancer with poor long-term prognosis in advanced stages. A major aim in neuroblastoma therapy is to develop targeted drug delivery systems to ameliorate drug therapeutic index and efficacy. In this study, a novel bortezomib (BTZ) liposomal formulation was set-up and characterized. Since BTZ is freely permeable across the lipidic bilayer, an amino-lactose (LM) was synthesized as complexing agent to entrap BTZ inside the internal aqueous compartment of stealth liposomes. High encapsulation efficiency was achieved by a loading method based on the formation of boronic esters between the boronic acid moiety of BTZ and the hydroxyl groups of LM. Next, NGR peptides were linked to the liposome surface as a targeting-ligand for the tumor endothelial cell marker, aminopeptidase N. Liposomes were characterized for size, Z-potential, polydispersity index, drug content, and release. Lyophilization in the presence of cryoprotectants (trehalose, sucrose) was also examined in terms of particle size changes and drug leakage. BTZ was successfully loaded into non-targeted (SL[LM-BTZ]) and targeted (NGR-SL[LM-BTZ]) liposomes with an entrapment efficiency of about 68% and 57%, respectively. These nanoparticles were suitable for intravenous administration, presenting an average diameter of 170nm and narrow polydispersity. Therefore, orthotopic NB-bearing mice were treated with 1.0 or 1.5mg/kg of BTZ, either in free form or encapsulated into liposomes. BTZ loaded liposomes showed a significant reduction of drug systemic adverse effects with respect to free drug, even at the highest dose tested. Moreover, mice treated with 1.5mg/kg of NGR-SL[LM-BTZ] lived statistically longer than untreated mice (P=0.0018) and SL[LM-BTZ]-treated mice (P=0.0256). Our results demonstrate that the novel vascular targeted BTZ formulation is endowed with high therapeutic index and low toxicity, providing a new tool for future applications in neuroblastoma clinical studies.

A G-quadruplex-binding compound showing anti-tumour activity in an in vivo model for pancreatic cancer.

We report here that a tetra-substituted naphthalene-diimide derivative (MM41) has significant in vivo anti-tumour activity against the MIA PaCa-2 pancreatic cancer xenograft model. IV administration with a twice-weekly 15 mg/kg dose produces ca 80% tumour growth decrease in a group of tumour-bearing animals. Two animals survived tumour-free after 279 days. High levels of MM41 are rapidly transported into cell nuclei and were found to accumulate in the tumour. MM41 is a quadruplex-interactive compound which binds strongly to the quadruplexes encoded in the promoter sequences of the BCL-2 and k-RAS genes, both of which are dis-regulated in many human pancreatic cancers. Levels of BCL-2 were reduced by ca 40% in tumours from MM41-treated animals relative to controls, consistent with BCL-2 being a target for MM41. Molecular modelling suggests that MM41 binds to a BCL-2 quadruplex in a manner resembling that previously observed in co-crystal structures with human telomeric quadruplexes. This supports the concept that MM41 (and by implication other quadruplex-targeting small molecules) can bind to quadruplex-forming promoter regions in a number of genes and down-regulate their transcription. We suggest that quadruplexes within those master genes that are up-regulated drivers for particular cancers, may be selective targets for compounds such as MM41.