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Using a convergent synthetic route to enable multiple points of diversity, a series of glucocorticoid receptor modulators (GRM) were profiled for potency, selectivity, and drug-like properties

Supplementary Materials and Methods; Supplementary Figure S1. Binding kinetics of hedgehog antibodies to human and mouse hedgehog proteins using surface plasmon resonance. Supplementary Figure S2. Hedgehog ligands stimulate mGLI1 reporter activity and osteoblast differentiation in a dose-dependent manner. Supplementary Figure S3. Selective inhibition of mGLI1 or mPTC1 induction in C3H10T1/2 cells by hedgehog antibodies. Supplementary Figure S4. MEDI-5304 inhibits GLI1 expression in Colo205 xenograft stromal cells but not tumor cells. Supplementary Figure S5. Hedgehog pathway component RNA expression in primary pancreatic tumor explant model P479 and effects of MEDI-5304 on tumorspheres derived from pancreatic explant model 947. Supplementary Figure S6. Pharmacokinetics of MEDI-5304 in rats. Supplementary Figure S7. Exploratory toxicology of MEDI-5304 in rats. Supplementary Figure S8. Pharmacokinetics of MEDI-5304 from preliminary toxicology study in cynomolgus monkeys. Supplementary Figure S9. Pharmacodynamics of MEDI-5304 in cynomolgus monkeys.

Amphibians are facing extinction on a global scale and maintaining small populations of threatened or endangered species in captivity is essential. In connection with potential captive breeding of Panha’s crocodile newt, Tylototriton panhai, we report a detailed husbandry protocol and describe breeding and mating behaviour. After six years in captivity, a group of two adult females and six males, tentatively identified as T. panhai, successfully reproduced for the first time on 24th June 2020 after a heavy rainstorm and two subsequent days of precipitation when water temperatures in a large aquaterrarium placed outdoors were 18-21 °C. The complete courtship behaviour consisted of five main stages: i/ approach; ii/ nuptial dance; iii/ amplexus; iv/ spermatophore deposition; v/ fertilisation. The two females laid a total of 84 eggs (41 and 43 eggs each) that were deposited on land in clutches of 4-22 eggs or even singly. Egg size averaged 12.6 ± 0.4 mm and the mean size of the embryo capsule 5.1 ± 0.1 (n=84). The egg hatch success rate was ~80% and on hatching the larvae moved to the water at the bottom of the aquaterrarium. By 7-9 days after hatching the total larval length was 11.89-13.78 mm (n=67). Diagnostic morphological characters are provided for stages 30-46. Metamorphosis occurred at 99-102 days and efts started to move to land at an average total length of 56.2 mm and weight of 0.6-0.9 g.

Amphibians are facing extinction on a global scale and maintaining small populations of threatened or endangered species in captivity is essential. In connection with potential captive breeding of Panha’s crocodile newt, Tylototriton panhai, we report a detailed husbandry protocol and describe breeding and mating behaviour. After six years in captivity, a group of two adult females and six males, tentatively identified as T. panhai, successfully reproduced for the first time on 24th June 2020 after a heavy rainstorm and two subsequent days of precipitation when water temperatures in a large aquaterrarium placed outdoors were 18-21 °C. The complete courtship behaviour consisted of five main stages: i/ approach; ii/ nuptial dance; iii/ amplexus; iv/ spermatophore deposition; v/ fertilisation. The two females laid a total of 84 eggs (41 and 43 eggs each) that were deposited on land in clutches of 4-22 eggs or even singly. Egg size averaged 12.6 ± 0.4 mm and the mean size of the embryo capsule 5.1 ± 0.1 (n=84). The egg hatch success rate was ~80% and on hatching the larvae moved to the water at the bottom of the aquaterrarium. By 7-9 days after hatching the total larval length was 11.89-13.78 mm (n=67). Diagnostic morphological characters are provided for stages 30-46. Metamorphosis occurred at 99-102 days and efts started to move to land at an average total length of 56.2 mm and weight of 0.6-0.9 g

Herein, we provided a morphological description combined with a phylogenetic analysis of the northernmost Thai Tylototriton population, which was found in Doi Pha Hom Pok National Park (DPHPNP), Chiang Mai Province, northern Thailand. Three adult males were collected from a small breeding pond during our survey in July 2020. Based on molecular (two specimens) and morphological (three specimens) evidence, all three specimens from DPHPNP were identified as Tylototriton verrucosus Anderson, 1871, and were characterized by having a blackish background, orange to light orange-brown color markings, segmented vertebral ridge, prominent rib nodules ranging from 13–15 warts, and a rough skin with granules. Hence, this second record of T. verrucosus populations in Thailand confirms a range extension of this species in northern Indochina. Additionally, the morphological variation and conservation status are discussed for the populations from Thailand.

Perkembangan teknologi memiliki dampak yang besar terhadap beberapa sektor kehidupan salah satunya adalah sektor perbankan. Dalam memanfaatkan kemajuan teknologi bank meningkatkan layanan dengan cara memberikan diferensiasi dari segi pelayanan maupun segi produk yang lebih baik dibandingkan dengan para pesaingnya. Penelitian ini bertujuan untuk mengetahui penggunaan aplikasi mobile banking sebagai aspek kepuasan nasabah yang terdiri dari kualitas layanan, kualitas informasi dan kualitas sistem. Bank XYZ memberikan fasilitas mobile banking yang terbaik kepada nasabah yang diberi nama OCTO Mobile. Pada OCTO Mobile terdapat berbagai macam layanan mulai dari m-transfer, m-payment, m-commerce dan lain sebagainya. Permasalahan yang terjadi karena adanya keluhan dari nasabah tentang fitur OCTO Mobile yang dianggap kurang user friendly dan terkadang terjadi error. Masalah tersebut kemudian dianalisis dengan menggunakan model yang terdiri dari kualitas layanan, kualitas informasi, dan kualitas sistem [1]. Data yang akan diteliti seluruh nasabah pengguna mobile banking. Pengelolaan data dalam penelitian ini menggunakan alat bantu SPSS. Hasil analisis diperoleh hasil uji Ttest yang menunjukkan bahwa kualitas layanan berpengaruh positif dan signifikan terhadap kepuasan nasabah, kualitas informasi memiliki pengaruh positif dan signifikan terhadap kepuasan nasabah dan kualitas sistem berpengaruh positif dan signifikan terhadap kepuasan nasabah. Sedangkan dalam hasil uji Ftest menunjukkan bahwa secara bersama-sama variabel kualitas layanan, kualitas informasi dan kualitas sistem berpengaruh secara positif dan signifikan terhadap kepuasan nasabah.

We describe a new species of the newt genus Tylototriton from Umphang Wildlife Sanctuary, Tak Province, western Thailand based on molecular and morphological evidence and named here as Tylototriton umphangensissp. nov. The new species is assigned to the subgenus Tylototriton and differs from other species in having dark-brown to blackish-brown body and limbs, truncate snout, prominent antero-medial ends of the expansion of the dentary bones, laterally protruding quadrate regions, indistinct and small rib nodules, a well-segmented vertebral ridge, and rough dorsolateral bony ridges, which are steeper anterior, and curved medially at the posterior ends. The molecular data show that Tylototriton umphangensissp. nov. differs from T. uyenoi sensu stricto by a 5% genetic sequence divergence of the mitochondrial NADH dehydrogenase subunit 2 region gene. The new species and T. uyenoi are both endemic to Thailand, distributed along the Northwest Thai (Dawna) Uplands of Indochina. To clarify the species boundary between Tylototriton umphangensissp. nov. and T. uyenoi, additional field research is needed in adjacent areas. Tylototriton umphangensissp. nov. is restricted to evergreen hill forests in Umphang Wildlife Sanctuary. We suggest that the new species should be classified as Endangered (EN) in the IUCN Red List.

Crocodile newts (Echinotriton and Tylototriton) are emblematic of the biodiversity of South-East Asia. In this study, we provide an account of their evolution, diversity and phylogeography based on mitochondrial (16.2 kb) and nuclear sequences (3.2 kb), combining barcoding data from nearly 1200 specimens collected over ~180 localities, representative of all lineages known to date. While phylogenies of nuclear data lacked resolution, the mitochondrial tree showed dynamic allopatric speciation that followed climate cooling during the last 10 million years, a pattern shared with many other tropical and subtropical amphibians. In crocodile newts, this implies weak dispersal and local adaptation as contributing factors of isolation, and was accompanied by macro- and micro-evolutionary changes in reproductive behaviours. Specifically, our resolved phylogeny of Tylototriton allowed the tracing of a major shift from conserved habits of terrestrial clutch-spawning towards more plastic strategies involving both aquatic and land spawning of single eggs. Using this framework, we then revised the taxonomy of crocodile newts by describing a new subgenus and two new species. Our study provides a basis to guide future phylogeographic, speciation and conservation research on these iconic amphibians.

Numerous amphibian species occupy microhabitats with buffered thermal and moisture conditions, typically under forest canopies. Here, we assessed whether the insular endemic Salamandra corsica also uses this type of habitat, which could have important implications for its conservation. We used data loggers to record the air temperature and air humidity at 4 h intervals over a period of 2 years, at 13 stations with a confirmed presence of S. corsica. These data were compared with those recorded at Corsican weather stations and those generated by a climate model (WorldClim 2). The weather station data showed significant deviations from the probe data, by an average of − 1.26 °C (minimum temperature), + 2.61 °C (maximum temperature), and − 0.04% (relative humidity). Similarly, the WorldClim 2 data showed significant deviations from the probe data, by an average of − 2.49 °C (minimum temperature) and + 1.69 °C (maximum temperature). These discrepancies reflect the use of densely vegetated and topographically complex habitats by S. corsica, which reduce temperature fluctuations. Overall, our results highlight the importance of natural vegetal cover in the conservation of populations of this endemic salamander.

Glucocorticoid receptor modulators (GRM) are the first-line treatment for many immune diseases, but unwanted side effects restrict chronic dosing. However, targeted delivery of a GRM payload via an immunology antibody-drug conjugate (iADC) may deliver significant efficacy at doses that do not lead to unwanted side effects. We initiated our α-TNF-GRM ADC project focusing on identifying the optimal payload and a linker that afforded stable attachment to both the payload and antibody, resulting in the identification of the synthetically accessible maleimide-Gly-Ala-Ala linker. DAR 4 purified ADCs were shown to be more efficacious in a mouse contact hypersensitivity model than the parent α-TNF antibody. Analysis of P1NP and corticosterone biomarkers showed there was a sufficient therapeutic window between efficacy and unwanted effects. In a chronic mouse arthritis model, α-TNF-GRM ADCs were more efficacious than both the parent α-TNF mAb and an isotype control bearing the same GRM payload.

A new subspecies within the Salamandra algira species complex from north-western Africa is described. Previous molecular analysis showed that the populations of S. algira splendens from north-western Morocco consisted of two well supported clades: clade 1 distributed in the Rif Mountains, from Chefchaouen (type locality) to Al Hoceima, and clade 2, located southern from clade 1 being isolated in the northern and central Middle Atlas Mountains. Clade 2 is herein described as a distinct subspecies: Salamandra algira atlanticassp. nov. based on morphological data, allopatric range and molecular divergence. This new subspecies shows an uncorrected pairwise distance of 0.0265 from clade 1 based on cytochrome b DNA sequences. Salamandra algira atlanticassp. nov. is a slender and large sized salamander with a highly variable colouration pattern. It can be distinguished from S. algira splendens by the greater proportion of coral red in the background colouration, being the only known subspecies of S. algira in which coral red can exceed the proportion of black. Variable number (0–5) of yellow to golden yellow dorsal blotches, but usually in lower numbers than the nominotypical subspecies. Salamandra algira atlanticassp. nov. inhabits subhumid to humid forests and karstic systems at mid to high elevations. We briefly discuss the phylogenetic and taxonomic issues among the genus Salamandra which encompasses more valid species than currently recognised.

The Mediterranean region possesses a rich diversity of salamanders, which also exhibit a high degree of ecological diversification. It is assumed that the presence of salamanders is dependent on the level of vegetation cover in terrestrial habitats, but the strength of this association is likely to vary among species. In this study, we investigated the patterns of habitat utilization for Mediterranean salamanders based on 589 records of 33 species. We tested the hypothesis that the association between salamander presence and vegetation density varies among genera or reproductive modes (i.e. terrestrial vs aquatic). The results show that vegetation cover has similar influences on terrestrial and aquatic groups but important differences are identified for lotic and lentic aquatic species. Our findings also indicate that the aquatic lotic, terrestrial, and small-body species deviate significantly from that expected from the background range of variation.

Two Moroccan subspecies of Salamandra algira were recently observed in caves - S. algira splendens and S. algira spelaea. Here, I report the first observations of cave breeding in S. algira splendens, in the Chaâra and Friouato caves of the north-eastern Middle Atlas (Taza region, Morocco). Twenty-two salamander and newt species are known to be facultative or obligate cave-dwellers in the Mediterranean ecoregion. They can inhabit or breed in caves so avoiding hot and very dry summer conditions. Caves with groundwater and stable humidity act as thermal refugia, winter or summer shelters, hiding places, feeding areas or, in some cases, as regular breeding sites.

Mexico is a hotspot of salamander diversity and harbours 18 species of the genus Ambystoma (Amphibia: Urodela) widely distributed from the Sierra Madre Occidental to the Trans-Mexican Volcanic Belt. These species are highly threatened by various factors and some of them are considered nearly extinct in the wild. The Michoacán stream salamander, Ambystoma ordinarium and the yellow-peppered salamander, A. flavipiperatum are two endemic and scarce species from Mexico living in isolated and declining populations. Few observations have been made on them in their natural habitat since their original description. Here we report new data regarding the biology and habitats of A. ordinarium observed at Rio Bello, 2,120 m a.s.l., Morelia city, Michoacán state, and for A. flavipiperatum at Sierra de Quila, 2,165 m a.s.l., 100 km south from Guadalajara city, Jalisco state. For A. ordinarium we found one neotenic adult male and two dead adult females in a small shallow stream located within a fragmented fir, oak and pine forest. For A. flavipiperatum we recorded an adult male and a female both neotenic, one clutch of eggs, and eleven larvae inhabiting a slow-moving stream located through a riparian habitat surrounded by large pineoak forest. Our observations confirm that both species are highly endangered, capable of facultative neoteny and occur in small slow-moving streams surrounded by coniferous forests. We discuss their morphology, geographical distribution and conservation status.

Niche conservatism underlies the process of speciation in amphibians on the global scale. However, high tropical diversity is partly attributable to niche shifts within evolutionary lineages. Crocodile newts (genera Echinotriton and Tylototriton) are a monophyletic group endemic to south-east Asia, and are one of the few lineages of urodeles that appear in tropical regions. Here, we examined the patterns of ecological diversification within the crocodile newts based on 207 records of 23 species. The niche space was described according to topographic, climatic variables (temperature, rainfall, and seasonality) and forest cover. Our analysis showed that the occurrence of most of these species was strongly associated with the precipitation regime and seasonality. The diversification patterns of the crocodile newts are complex, showing both conservatism and divergence. These patterns can be related to the Tibetan uplift phase during the Neogene, resulting in the emergence of new montane habitats and subsequent changes in the palaeo-drainage system and climate. The range of ecological diversification within a lineage is possibly the result of several interacting processes, including environmental heterogeneity, the regional climatic history, and physiological constraints on species.

The role of intraoperative electrocorticography (iECoG) and of its patterns in epilepsy surgery have shown contradictory results. Our aim was to describe iECoG patterns and their association with outcome in epilepsy surgery.We retrospectively analyzed 104 patients who underwent epilepsy surgery (2009-2015) with pre- and postresection iECoG. We described clinical findings, type of surgery, preresection iECoG patterns according to Palmini et al., 1995 (sporadic, continuous, burst, and recruiting interictal epileptiform discharges-IEDs) and postresection iECoG outcome (de novo, residual, and without IEDs). The Engel scale was used to evaluate the outcome. Descriptive statistics, Kaplan-Meier, the logistic regression model, and analysis of variance tests were used.We included 60.6% (63/104) females, with a mean age of 35 (±10.2) years at the time of epilepsy surgery. The etiologies were hippocampal sclerosis (63.5%), cavernomas (14.4%), cortical dysplasia (11.5%), and low-grade tumors (10.6%). The most common preresection iECoG pattern was sporadic IEDs (47%). Postresection iECoG patterns were de novo (55.7%), residual (27.8%), and without IEDs (16.3%). Mean follow-up was 19.2 months. Engel scale was as follows: Engel I (91 patients, 87.5%), Engel II (10 patients, 9.6%), and Engel III (three patients, 2.9%). Analysis by mixed-design analysis of variance showed a significant difference between etiology groups with a strong size effect (P = 0.021, η = 0.513) and also between preresection iECoG patterns (P = 0.008, η = 0.661).Preresection iECoG patterns and etiology influence Engel scale outcome in lesional epilepsy surgery.

The natural history of newly discovered species among the genus Tylototriton in South-Eastern Asia is poorly known to date, and most of the species are threatened by extinction. The description of their ecology is crucial to determine their survival status and successful reproduction for conservation purposes. In this study, we assessed the microhabitat preferences of Tylototriton yangi at the type locality, Gejiu, Yunnan province, China, with the Visual Encounter Survey method and by using new important materials. The main habitat is located within a humid subtropical climate and is composed of a large karstic mountain containing some patch of secondary mixed forests and plantations such as tomato fields, pepper and cabbage that are surrounded by scrubs, grasses and small ponds. We provided a complete diagnosis with new morphological data analysing ten adult individuals from the type series to establish a formal description which is absent from the first original description. We compared our results with other related taxa of the genus Tylototriton to updated key determinants between similar species. Thus, we analysed the population structure and the behaviour of T. yangi during a period of ten years, 2008–2017, to assess its survival status and to determine the main threat factors. A total of 186 specimens were found during the whole study predicting a small population inhabiting the type locality, Gejiu. Consequently, T. yangi is now regarded as a highly threatened species that need more attention of Chinese authorities and we suggest regarding it as Endangered due to its small distribution range, the low number of adult specimens observed and ongoing exploitation.

De las diversas complicaciones de la meningoencefalitis infecciosa, el síndrome de Guillain-Barré es raro. Presentamos el caso de una mujer de 37 años de edad, quien inicio con meningoencefalitis infecciosa, a los 9 días se observó empeoramiento de la fuerza y sensibilidad de las cuatro extremidades. Con sospecha clínica y electromiografía se diagnosticó polineuropatía desmielinizante secundariamente axonal sensitiva motora de las cuatro extremidades. No se aisló un patógeno específico. El manejo integral e inmunomodulador (IgG IV 0.4g/kg/dx5d) estuvo basado en los principios establecidos para las dos situaciones neurológicas observadas en la paciente. La paciente se recuperó totalmente a los 6 meses.

Objective The study aimed to establish the prevalence of seizure history (SH) and epilepsy in a rural community in Hidalgo, Mexico and determine the patients' beliefs and attitudes towards the disease and its initial medical treatment. Methodology A transverse, descriptive, door-to-door epidemiological study (April 2011–November 2012) was conducted with 863 inhabitants from Xocotitla, Huejutla, Hidalgo, Mexico (162 housing units). Patients with SH were identified with an adaptation of the WHO protocol for epidemiological studies of neurological diseases. Afterwards, the subjects identified with seizure history (SH) or epilepsy were interviewed with a 20-question Likert type questionnaire regarding the management and belief set of their SH. The interviews were conducted in Spanish and Nahuatl. Results The prevalence of epilepsy and isolated nonrecurring seizures was 38.2/1000 and 25.4/1000, respectively. Out of the total population of 863 inhabitants, 33/863 were identified with SH: only 39.3% were able to identify an epileptic seizure as such, 48.5% sought medical attention upon the first seizure, 33.3% used a traditional healer, 15.2% took no action, 3% sought a religious representative, 85% lacked any lab analysis, and 60% received no antiepileptic drugs. Only 39% received free local medical attention, 69.7% considered seizures and epilepsy to be a consequence of divine intervention, and 94% reported some type of discrimination. Conclusions A high prevalence of epilepsy and SH was found in this rural community in Mexico. Divine/religious beliefs, discrimination, scarce access to basic health services and inadequate medical management of epilepsy and SH persist.

El objetivo de este trabajo es analizar diversas construcciones sintácticas de las que, ya sea desde un punto de vista formal o discursivo, cabe suponer que expresan mandatos. A partir de sus rasgos formales, fueron seleccionadas diversas construcciones con verbos en imperativo y en subjuntivo, además de otras formalmente heterogéneas, pertenecientes al español mexicano de los siglos XIX y XX. Presentamos el análisis de diversos aspectos sintácticos, semánticos y pragmáticos a la luz de la teoría de los actos de habla para determinar en que medida el contexto discursivo en el que aparecen las oraciones influye en su interpretación. En particular, nos interesa saber si la presencia del imperativo implica la expresión de un mandato; examinar su alternancia con otras estructuras para expresar mandatos y también si se registra algún cambio diacrónico al respecto.

Background: Transcranial direct current stimulation (tDCS) has been evaluated in medication refractory epilepsy patients. The results have been inconclusive and protocols have varied between studies. Objective: To evaluate the safety and efficacy of two protocols of tDCS in adult patients with mesial temporal lobe epilepsy and hippocampal sclerosis (MTLE-HS). Methods: This is a randomized placebo-controlled, double-blinded clinical trial, with 3 arms, 3 sessions, 5 sessions and placebo stimulation. Frequency of seizures (SZs), interictal epileptiform discharges (IEDs) and adverse effects (AEs) were registered before and after treatment, and at 30 and 60 days follow-up. Descriptive statistics, k-related samples, Friedman's test, and relative risk (RR) estimation were used for analysis. Results: We included twenty-eight subjects (3d n = 12, 5d n = 8, placebo n = 8), 16/28 (57%) men, age 37.8(±10.9) years old. There was a significant reduction of the frequency of SZs at one (p = 0.001) and two (p = 0.0001) months following cathodal tDCS compared to baseline in the 3 arms (p = 0.0001). The mean reduction of SZ frequency at two months in both active groups was significantly higher than placebo (−48% vs. −6.25%, p

Introduction The cardinal sign of essential tremor (ET) and Parkinson Disease (PD) is tremor, which is part of the diagnostic criteria. But, in early stages of the disease and in atypical presentations it is difficult to evaluate the tremors, the clinical examination has an extensive variability and it has been poorly studied. Accelerometers can quantify and differentiate the tremor, but, rarely have they been used in daily living conditions. The objective was to design a supervised learning algorithm that can detect, quantify, and differentiate the movements of a portable accelerometer in ET and PD during controlled and daily living conditions. Methods A descriptive and comparative clinical study. It enrolled 30 ET and 30 PD patients. The clinical and sociodemographic variables (age, sex, HY dominant frequency and harmonic. An algorithm was designed (rate 0.1 with a 1000 trees). Inferential statistics was used, and a normalized confusion matrix was calculated, sensitivity, specificity, and accuracy. Results Performance was classified in the normalized confusion matrix which allowed the distinction of tremor from no tremor in ET (86%) and PD (71%). The mathematical tree algorithm of decisions showed in: PD sensitivity: 65.4–69.8%, specificity: 72.8–90.9% and accuracy: 71.3–85.1%. In ET sensitivity: 77.6–93.8%, specificity 80.5–96.2% and accuracy: 79.5–95.6%. Being better in ET than in PD. Conclusion The tree algorithm of decisions in accelerometers allows the detection, quantification, and differentiation of the tremors of ET and PD during daily living conditions.

Aim. To describe the general aspects of cavernomas and epilepsy and review the available literature on the utility of electrocorticography (ECoG) in cerebral cavernoma surgery. Methods. We searched studies in PubMed, MedLine, Scopus, Web of Science, and Google Scholar (from January 1969 to December 2013) using the keywords “electrocorticography” or “ECoG” or “prognosis” or “outcome” and “cavernomas”. Original articles that reported utility of ECoG in epilepsy surgery were included. Four review authors independently selected the studies, extracted data, and assessed the methodological quality of the studies using the recommendations of the Cochrane Handbook for Systematic Reviews of Interventions, PRISMA guidelines, and Jadad Scale. A meta-analysis was not possible due to methodological, clinical, and statistical heterogeneity of included studies. We analysed six articles with a total of 219 patients. Results. The most common surgical approach was lesionectomy using ECoG in the temporal lobe with Engel I outcome range from 72.7 to 100%. Conclusions. Small controlled studies suggest that ECoG-guided resection offers the best functional results in seizure control for subjects undergoing cavernoma surgery, especially in the temporal lobe.

The hedgehog pathway has been implicated in the tumorigenesis, tumor progression, and metastasis of numerous human cancers. We generated the first fully human hedgehog antibody MEDI-5304 and characterized its antitumor activity and preclinical toxicology. MEDI-5304 bound sonic hedgehog (SHH) and Indian hedgehog (IHH) with low picomolar affinity and neutralized SHH and IHH activity in cellular mGLI1 reporter assays. The antibody inhibited transcription of hedgehog target genes and osteoblast differentiation of C3H10T1/2 cells. We evaluated the activity of MEDI-5304 in vivo in model systems that allowed us to evaluate two primary hypotheses of hedgehog function in human cancer, paracrine signaling between tumor and stromal cells and cancer stem cell (CSC) self-renewal. MEDI-5304 displayed robust pharmacodynamic effects in stromal cells that translated to antitumor efficacy as a single agent in an HT-29/MEF coimplantation model of paracrine hedgehog signaling. MEDI-5304 also improved responses to carboplatin in the HT-29/MEF model. The antibody, however, had no effect as a single agent or in combination with gemcitabine on the CSC frequency or growth of several primary pancreatic cancer explant models. These findings support the conclusion that hedgehog contributes to tumor biology via paracrine tumor-stromal signaling but not via CSC maintenance or propagation. Finally, the only safety study finding associated with MEDI-5304 was ondontodysplasia in rats. Thus, MEDI-5304 represents a potent dual hedgehog inhibitor suitable for continued development to evaluate efficacy and safety in human patients with tumors harboring elevated levels of SHH or IHH. Mol Cancer Ther; 13(2); 386–98. ©2013 AACR.

This chapter describes in vitro and in vivo methods to characterize a lead monoclonal antibody candidate in the drug discovery setting. Approaches to characterize monoclonal antibody specificity, heavy and light chain composition, and antibody mode of action including the ability to mediate secretion of effector molecules, inhibit cell proliferation, induce apoptosis, or elicit antibody effector function are described. ELISA and flow cytometry based methods, as well as in vitro assays to assess for cell proliferation, ADCC, and CDC are detailed.In addition, both subcutaneous and orthotopic in vivo tumor xenograft models to assess antibody efficacy are described. The xenograft tumor model is a valuable tool for assessing the therapeutic activity of a monoclonal antibody drug candidate. Xenograft models are generated by the implantation of tumor cells or tumor fragments of human origin into immune-compromised mice or rats. This allows for fast and efficient in vivo evaluation of an antibody drug candidate in human cancer models. Here, we describe the procedures for generating preclinical animal tumor models frequently employed in the preclinical drug discovery setting.

This chapter describes in vitro and in vivo methods to characterize a lead monoclonal antibody candidate in the drug discovery setting. Approaches to characterize monoclonal antibody specificity, heavy and light chain composition, and antibody mode of action including the ability to mediate secretion of effector molecules, inhibit cell proliferation, induce apoptosis, or elicit antibody effector function are described. ELISA and flow cytometry based methods, as well as in vitro assays to assess for cell proliferation, ADCC, and CDC are detailed.In addition, both subcutaneous and orthotopic in vivo tumor xenograft models to assess antibody efficacy are described. The xenograft tumor model is a valuable tool for assessing the therapeutic activity of a monoclonal antibody drug candidate. Xenograft models are generated by the implantation of tumor cells or tumor fragments of human origin into immune-compromised mice or rats. This allows for fast and efficient in vivo evaluation of an antibody drug candidate in human cancer models. Here, we describe the procedures for generating preclinical animal tumor models frequently employed in the preclinical drug discovery setting.

Fuente TESIUNAM

Breast cancer is one of the most common cancer types, with greater than 450,000 deaths reported per year worldwide. Through genome wide sequencing efforts, multiple genetic alterations have been identified, including mutations and amplifications in genes such as v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), GATA binding protein 3 (GATA3), phosphatidylinositol 3-kinase alpha catalytic subunit (PIK3CA) as well as novel genomic rearrangements such as the recently identified MAGI3-AKT3 fusion. Now that breast cancer can be characterized to an unprecedented level, one of the key challenges remaining is to identify and distinguish critical ‘driver’ events responsible for tumor progression, from neutral ‘passenger’ lesions. In order to achieve this, we utilized high resolution aCGH analysis of 50 purified breast cancer samples (made up of Her2+, estrogen receptor positive (ER+) and triple negative tumors with variable responses to SOC regimens), in combination with a Gain-of-Function transformation screen to identify and validate novel breast targets. 158 genomic regions were found to be recurrently amplified, consisting of 759 genes in total. The top 32 focally amplified genes, along with 12 cancer-relevant mutant alleles were prioritized and a library generated utilizing the pTRIPZ-tetracycline regulated inducible lentiviral vector system. These 44 genes were subsequently combined into 16 different target pools (5-13 targets per pool, co-expressing genes that were co-amplified in the same clinical specimen) and evaluated for their ability to transform immortalized breast epithelial MCF10A cells (both wild-type and p53 -/- cells). Through this screening approach, p21-activated kinase 1 (PAK1) was identified, whose kinase activity was required to robustly transform MCF10A cells through regulating multiple signalling pathways including MAPK. Several other putative oncogenes were also identified and will be presented here, including the glycosyltransferse asparagine-linked glycosylation 8 (ALG8). Interestingly, PAK1 and ALG8 are co-amplified in both breast (8%) and ovarian cancers (11%). Our target validation studies have suggested that ALG8 can support PAK1-induced transformation, as dramatic suppression of soft-agar colony growth was seen in co-amplified breast cancer cell lines upon combined siRNA treatment to both targets. Thus, this combined high resolution aCGH profiling and functional screening approach has enabled the successful identification of novel oncogenic targets in breast cancer. Citation Format: Krishna Vasudevan, Axel Hernandez, Zhongwu Lai, Yonghong Xiao, Nin Guan, Carolyn Hardy, Robert Godin, Christopher Denz, Minwei Ye, Elizabeth Lenkiewicz, Stephanie Savage, Michael T. Barrett, Donna Prunkard, Peter Rabinovitch, Mark Basik, Ewa Przybytkowski, Kevin Webster, Michael Zinda, Emma-Louise Jenkins. Identification and functional validation of novel genetically-linked breast cancer targets through pooled gain-of-function screening. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3133. doi:10.1158/1538-7445.AM2013-3133