Zebrafish embryonic explants undergo genetically encoded self-assembly

Embryonic stem cell cultures are thought to self-organize into embryoid bodies, able to undergo symmetry-breaking, germ layer specification and even morphogenesis. Yet, it is unclear how to reconcile this remarkable self-organization capacity with classical experiments demonstrating key roles for extrinsic biases by maternal factors and/or extraembryonic tissues in embryogenesis. Here, we show that zebrafish embryonic tissue explants, prepared prior to germ layer induction and lacking extraembryonic tissues, can specify all germ layers and form a seemingly complete mesendoderm anlage. Importantly, explant organization requires polarized inheritance of maternal factors from dorsal-marginal regions of the blastoderm. Moreover, induction of endoderm and head-mesoderm, which require peak Nodal-signaling levels, is highly variable in explants, reminiscent of embryos with reduced Nodal signals from the extraembryonic tissues. Together, these data suggest that zebrafish explants do not undergo bona fide self-organization, but rather display features of genetically encoded self-assembly, where intrinsic genetic programs control the emergence of order

Live tracking of moving samples in confocal microscopy for vertically grown roots

Roots navigate through soil integrating environmental signals to orient their growth. The Arabidopsis root is a widely used model for developmental, physiological and cell biological studies. Live imaging greatly aids these efforts, but the horizontal sample position and continuous root tip displacement present significant difficulties. Here, we develop a confocal microscope setup for vertical sample mounting and integrated directional illumination. We present TipTracker – a custom software for automatic tracking of diverse moving objects usable on various microscope setups. Combined, this enables observation of root tips growing along the natural gravity vector over prolonged periods of time, as well as the ability to induce rapid gravity or light stimulation. We also track migrating cells in the developing zebrafish embryo, demonstrating the utility of this system in the acquisition of high-resolution data sets of dynamic samples. We provide detailed descriptions of the tools enabling the easy implementation on other microscopes

Вемурафениб улучшает выживаемость при меланоме кожи с мутацией BRAF V600E

В 40–60% случаев меланомы кожи выявляют мутации онкогена BRAF, следствием которых является конститутивная активация серинтреониновой киназы BRAF и, соответственно, митогенного сигнала по пути MAPK/ERK. Представлены результаты международных рандомизированных исследований I, II, III фазы, в которых оценивали эффективность и токсичность ингибитора RAF-киназной активности вемурафениба (PLX4032, Зелбораф) у больных метастатической меланомой кожи с мутацией BRAF V600E. В исследовании III фазы BRIM3 сравнивали режимы монотерапии вемурафенибом (960 мг перорально 2 раза в сутки — 337 пациентов и дакарбазином (1000 мг/м2 внутривенно каждые 3 нед — 338 пациентов). Первичными точками исследования были общая выживаемость и выживаемость без прогрессирования, вторичными — частота объективного ответа, его длительность, время до развития ответа. Анализ данных после 6 мес наблюдения показал, что применение вемурафениба приводило к относительному снижению риска смерти на 63% и риска прогрессирования заболевания на 74% по сравнению с дакарбазином (р < 0,001 для обоих показателей). Объективный ответ зафиксирован в 48% случаев в группе вемурафениба и в 5% случаев — дакарбазина. Наиболее частыми токсическими эффектами вемурафениба были артралгии, сыпь, общая слабость, алопеция, плоскоклеточная карцинома кожи и кератоакантома, фотосенсибилизация, тошнота, диарея; сокращение дозы, связанное с токсичностью, потребовалось у 38% пациентов. По результатам исследования сделан вывод об эффективности вемурафениба у нелеченных ранее больных метастатической меланомой кожи с мутацией BRAF V600E.40 to 60% of cutaneous melanomas carry mutations in BRAF that lead to constitutive activation of BRAF serine/threonine kinase and downstream signaling through the MAPK/ERK pathway. Phase I and II clinical trials of the BRAF kinase inhibitor vemurafenib (PLX4032, Zelboraf) have shown response rates of more than 50% in patients with metastatic melanoma with the BRAF V600E mutation. In a phase III randomized clinical trial (BRIM-3) comparing vemurafenib with dacarbazine in 675 patients with previously untreated, metastatic melanoma with the BRAF V600E mutation. Patients were randomly assigned to receive either vemurafenib (960 mg orally twice daily) or dacarbazine (1000 mg per square meter of body-surface area intravenously every 3 weeks). Coprimary end points were rates of overall and progression-free survival. Secondary end points included the response rate, response duration, and safety. At 6 months, vemurafenib was associated with a relative reduction of 63% in the risk of death and of 74% in the risk of either death or disease progression, as compared with dacarbazine (p < 0,001 for both comparisons). Response rates were 48% for vemurafenib and 5% for dacarbazine. Common adverse events associated with vemurafenib were arthralgia, rash, fatigue, alopecia, keratoacanthoma or squamous-cell carcinoma, photosensitivity, nausea, and diarrhea; 38% of patients required dose modification because of toxic effects. Vemurafenib produced improved rates of overall and progression-free survival in patients with previously untreated melanoma with the BRAF V600E mutation

Intratumoural immunotherapies for unresectable and metastatic melanoma: current status and future perspectives.

The emergence of human intratumoural immunotherapy (HIT-IT) is a major step forward in the management of unresectable melanoma. The direct injection of treatments into melanoma lesions can cause cell lysis and induce a local immune response, and might be associated with a systemic immune response. Directly injecting immunotherapies into tumours achieves a high local concentration of immunostimulatory agent while minimising systemic exposure and, as such, HIT-IT agents are associated with lower toxicity than systemic immune checkpoint inhibitors (CPIs), enabling their potential use in combination with other therapies. Consequently, multiple HIT-IT agents, including oncolytic viruses, pattern-recognition receptor agonists, injected CPIs, cytokines and immune glycolipids, are under investigation. This review considers the current clinical development status of HIT-IT agents as monotherapy and in combination with systemic CPIs, and the practical aspects of administering and assessing the response to these agents. The future of HIT-IT probably lies in its use in combination with systemic CPIs; data from Phase 2 trials indicate a synergy between HIT-IT and CPIs. Data also suggest that the addition of HIT-IT to a CPI might generate responses in CPI-refractory tumours, thereby overcoming resistance and addressing a current unmet need in unresectable and metastatic melanoma for treatment options following progression after CPI treatment

目次

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions. The Video is licensed under a CC BY NC ND license

Fast and efficient genetic engineering of hematopoietic precursor cells for the study of dendritic cell migration

Dendritic cells (DCs) are sentinels of the adaptive immune system that reside in peripheral organs of mammals. Upon pathogen encounter, they undergo maturation and up-regulate the chemokine receptor CCR7 that guides them along gradients of its chemokine ligands CCL19 and 21 to the next draining lymph node. There, DCs present peripherally acquired antigen to naïve T cells, thereby triggering adaptive immunity

Nationwide Outcomes of Advanced Melanoma According to BRAFV600 Status

OBJECTIVE: The aim of this study was to evaluate treatment patterns and overall survival (OS) of patients with BRAFV600 wild-type and BRAFV600-mutant advanced melanoma in the Netherlands. METHODS: We selected patients of 18 years and over, diagnosed between 2016 and 2017 with unresectable stage IIIC or IV melanoma, registered in the Dutch Melanoma Treatment Registry. To assess the association of BRAFV600-mutation status with OS we used the Cox proportional-hazards model. RESULTS: A total of 642 BRAFV600 wild-type and 853 mutant patients were included in the analysis. Median OS did not differ significantly between both groups, 15.2 months (95% confidence interval [CI]: 13.2-19.2) versus 20.6 months (95% CI: 18.3-25.0). Survival rates at 6 and 12 months were significantly lower for BRAFV600 wild-type patients compared with BRAFV600-mutant patients, 72.0% (95% CI: 68.6-75.6) and 56.0% (95% CI: 52.2-60.0) versus 83.4% (95% CI: 80.9-85.9) and 65.7% (95% CI: 62.6-69.0). Two-year survival was not significantly different between both groups, 41.1% (95% CI: 37.2-45.3) versus 47.0% (95% CI: 43.6-60.6). Between 0 and 10 months, BRAFV600 wild-type patients had a decreased survival with a hazard ratio for OS of 2.00 (95% CI: 1.62-2.46) but this effect disappeared after 10 months. At 12 months, BRAFV600-mutant patients had started with second-line systemic treatment more often compared with BRAFV600 wild-type patients (50% vs. 19%). CONCLUSION: These results suggest that advanced BRAFV600 wild-type melanoma patients have worse survival than BRAFV600-mutated patients during the first 10 months after diagnosis because of less available treatment options

Profiles of phenotype resistance to antibiotic other than β-lactams in Klebsiella pneumoniae ESBLs-producers, carrying blaSHV genes

Extended spectrum β-lactamases production is one of the most common mechanism of resistance to extendedspectrum β-lactam antibiotics is increasing worldwide. Twenty five strains of Klebsiella pneumoniae isolated from clinicalspecimens were tested. Based on the phenotypic confirmatory test all these strains were defined as ESBL producers namedESBL(+). The plasmid DNA from each strains was used to investigate the presence of blaSHV genes responsible for extendedspectrum β-lactamases production. Moreover, susceptibility of these strains to antibiotic other than β-lactams in was tested

Profiles of phenotype resistance to antibiotic other than β-lactams in Klebsiella pneumoniae ESBLs-producers, carrying blaSHV genes.

Extended spectrum β-lactamases production is one of the most common mechanism of resistance to extended spectrum β-lactam antibiotics is increasing worldwide. Twenty five strains of Klebsiella pneumoniae isolated from clinical specimens were tested. Based on the phenotypic confirmatory test all these strains were defined as ESBL producers named ESBL(+). The plasmid DNA from each strains was used to investigate the presence of blaSHV genes responsible for extended spectrum β-lactamases production. Moreover, susceptibility of these strains to antibiotic other than β-lactams in was tested