The rockefeller university

Analysing environmental DNA (eDNA) in seawater can aid in monitoring marine fish populations. However, the extent to which current methods optimize fish eDNA detection from water samples is unknown. Here, we test modifications to laboratory components of an eDNA metabarcoding protocol targeting marine finfish. As compared to baseline methods, amplifying a smaller proportion of extracted DNA yielded fewer species, and, conversely, amplifying a larger proportion identified more taxa. Higher-read species were amplified more reproducibly and with less variation in read number than were lower-read species. Among pooled samples, 20-fold deeper sequencing recovered one additional fish species out of a total of 63 species. No benefit was observed with additional PCR cycles, alternative primer concentrations, or fish-selective primers. Experiments using an exogenous DNA standard to assess absolute eDNA concentration suggested that, for a given proportion of a DNA sample, current laboratory methods for metabarcoding marine fish eDNA are near to maximally sensitive. Our results support the unofficial standard collection volume of one liter for eDNA assessment of commonly encountered marine fish species. We conclude that eDNA rarity poses the main challenge to current methods.

Canonically, EZH2 serves as the catalytic subunit of PRC2, which mediates H3K27me3 deposition and transcriptional repression. Here, we report that in acute leukaemias, EZH2 has additional noncanonical functions by binding cMyc at non-PRC2 targets and uses a hidden transactivation domain (TAD) for (co)activator recruitment and gene activation. Both canonical (EZH2-PRC2) and noncanonical (EZH2-TAD-cMyc-coactivators) activities of EZH2 promote oncogenesis, which explains the slow and ineffective antitumour effect of inhibitors of the catalytic function of EZH2. To suppress the multifaceted activities of EZH2, we used proteolysis-targeting chimera (PROTAC) to develop a degrader, MS177, which achieved effective, on-target depletion of EZH2 and interacting partners (that is, both canonical EZH2-PRC2 and noncanonical EZH2-cMyc complexes). Compared with inhibitors of the enzymatic function of EZH2, MS177 is fast-acting and more potent in suppressing cancer growth. This study reveals noncanonical oncogenic roles of EZH2, reports a PROTAC for targeting the multifaceted tumorigenic functions of EZH2 and presents an attractive strategy for treating EZH2-dependent cancers.

Nup358, a protein of the nuclear pore complex, facilitates a nuclear positioning pathway that is essential for many biological processes, including neuromuscular and brain development. Nup358 interacts with the dynein adaptor Bicaudal D2 (BicD2), which in turn recruits the dynein machinery to position the nucleus. However, the molecular mechanisms of the Nup358/BicD2 interaction and the activation of transport remain poorly understood. Here for the first time, we show that a minimal Nup358 domain activates dynein/dynactin/BicD2 for processive motility on microtubules. Using nuclear magnetic resonance titration and chemical exchange saturation transfer, mutagenesis, and circular dichroism spectroscopy, a Nup358 alpha-helix encompassing residues 2162-2184 was identified, which transitioned from a random coil to an alpha-helical conformation upon BicD2 binding and formed the core of the Nup358-BicD2 interface. Mutations in this region of Nup358 decreased the Nup358/BicD2 interaction, resulting in decreased dynein recruitment and impaired motility. BicD2 thus recognizes Nup358 through a 'cargo recognition alpha-helix,' a structural feature that may stabilize BicD2 in its activated state and promote processive dynein motility.

Progress in genome sequencing now enables the large-scale generation of reference genomes. Various international initiatives aim to generate reference genomes representing global biodiversity. These genomes provide unique insights into genomic diversity and architecture, thereby enabling comprehensive analyses of population and functional genomics, and are expected to revolutionize conservation genomics.

Any realistic evolutionary theory has to consider 1) the dynamics of organisms that reproduce and possess heritable traits, 2) the appearance of stochastic variations in these traits, and 3) the selection of those organisms that better survive and reproduce. These elements shape the "evolutionary forces" that characterize the evolutionary dynamics. Here, we introduce a general model of reproduction-variation-selection dynamics. By treating these dynamics as a nonequilibrium thermodynamic process, we make precise the notion of the forces that characterize evolution. One of these forces, in particular, can be associated with the robustness of reproduction to variations. Some of the detailed predictions of our model can be tested by quantitative laboratory experiments, similar to those performed in the past on evolving populations of proteins or viruses.

Ethical considerations are central to all medicine though, likely, nowhere more essential than in the practice of reproductive endocrinology and infertility. Through in vitro fertilization (IVF), this is the only field in medicine involved in creating human life. IVF has, indeed, so far led to close to 10 million births worldwide. Yet, relating to substantial changes in clinical practice of IVF, the medical literature has remained surprisingly quiet over the last two decades. Major changes especially since 2010, however, call for an updated commentary. Three key changes deserve special notice: Starting out as a strictly medical service, IVF in recent years, in efforts to expand female reproductive lifespans in a process given the term "planned" oocyte cryopreservation, increasingly became more socially motivated. The IVF field also increasingly underwent industrialization and commoditization by outside financial interests. Finally, at least partially driven by industrialization and commoditization, so-called add-ons, the term describing mostly unvalidated tests and procedures added to IVF since 2010, have been held responsible for worldwide declines in fresh, non-donor live birthrates after IVF, to levels not seen since the mid-1990s. We here, therefore, do not offer a review of bioethical considerations regarding IVF as a fertility treatment, but attempt to point out ethical issues that arose because of major recent changes in clinical IVF practice.

Proper segregation of chromosomes during mitosis depends on "amphitelic attachments"-load-bearing connections of sister kinetochores to the opposite spindle poles via bundles of microtubules, termed as the "K-fibers." Current models of spindle assembly assume that K-fibers arise largely from stochastic capture of microtubules, which occurs at random times and locations and independently at sister kinetochores. We test this assumption by following the movements of all kinetochores in human cells and determine that most amphitelic attachments form synchronously at a specific stage of spindle assembly and within a spatially distinct domain. This biorientation domain is enriched in bundles of antiparallel microtubules, and perturbation of microtubule bundling changes the temporal and spatial dynamics of amphitelic attachment formation. Structural analyses indicate that interactions of kinetochores with microtubule bundles are mediated by noncentrosomal short microtubules that emanate from most kinetochores during early prometaphase. Computational analyses suggest that momentous molecular motor-driven interactions with antiparallel bundles rapidly convert these short microtubules into nascent K-fibers. Thus, load-bearing connections to the opposite spindle poles form simultaneously on sister kinetochores. In contrast to the uncoordinated sequential attachments of sister kinetochores expected in stochastic models of spindle assembly, our model envisions the formation of amphitelic attachments as a deterministic process in which the chromosomes connect with the spindle poles synchronously at a specific stage of spindle assembly and at a defined location determined by the spindle architecture. Experimental analyses of changes in the kinetochore behavior in cells with perturbed activity of molecular motors CenpE and dynein confirm the predictive power of the model.

hypothalamic paraventricular nucleus (PVN) plays a key role in hypertension, however the signaling pathways that contribute to the adaptability of the PVN during hypertension are uncertain. We present evidence that signaling at the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) GluA1 receptor contributes to increased blood pressure in a model of neurogenic hypertension induced by 14-day slow pressor angiotensin II (AngII) infusion in male mice. It was found that AngII hypertension was associated with an increase in plasma membrane affiliation of GluA1, but decreased GluA2, in dendritic profiles of PVN neurons expressing the TNFa type 1 receptor, a modulator of AMPA receptor trafficking. The increased plasma membrane GluA1 was paralleled by heightened AMPA currents in PVN-spinal cord projection neurons from AngII-infused male mice. Significantly, elevated AMPA currents in AngII-treated mice were blocked by 1-Naphthyl acetyl spermine trihydrochloride, pointing to the involvement of GluA2-lacking GluA1 receptors in the heightened AMPA signaling in PVN neurons. A further functional role for GluA1 in the PVN was demonstrated by the attenuated hypertensive response following silencing of GluA1 in the PVN of AngII-infused male mice. In female mice, AngII-infusion did not impact blood pressure or plasma membrane localization of GluA1 . Post-translational modifications that increase the plasma membrane localization of AMPA GluA1 and heighten the rapid excitatory signaling actions of glutamate in PVN neurons may serve as a molecular substrate underlying sex differences in hypertension. (c) 2022 IBRO. Published by Elsevier Ltd. All rights reserved.

Severe viral infections of the skin can occur in patients with inborn errors of immunity (IEI). We report an all-in-one whole-transcriptome sequencing-based method by RNA-Seq on a single skin biopsy for concomitantly identifying the cutaneous virome and the underlying IEI. Skin biopsies were obtained from healthy and lesional skin from patients with cutaneous infections suspected to be of viral origin. RNA-Seq was utilized as the first-tier strategy for unbiased human genome-wide rare variant detection. Reads unaligned to the human genome were utilized for the exploration of 926 viruses in a viral genome catalog. In 9 families studied, the patients carried pathogenic variants in 6 human IEI genes, including IL2RG, WAS, CIB1, STK4, GATA2, and DOCK8. Gene expression profiling also confirmed pathogenicity of the human variants and permitted genome-wide homozygosity mapping, which assisted in identification of candidate genes in consanguineous families. This automated, online, all-in-one computational pipeline, called VirPy, enables simultaneous detection of the viral triggers and the human genetic variants underlying skin lesions in patients with suspected IEI and viral dermatosis.

Embryonic cells grow in environments that provide a plethora of physical cues, including mechanical forces that shape the development of the entire embryo. Despite their prevalence, the role of these forces in embryonic development and their integration with chemical signals have been mostly neglected, and scrutiny in modern molecular embryology tilted, instead, towards the dissection of molecular pathways involved in cell fate determination and patterning. It is now possible to investigate how mechanical signals induce downstream genetic regulatory networks to regulate key developmental processes in the embryo. Here, we review the insights into mechanical control of early vertebrate development, including the role of forces in tissue patterning and embryonic axis formation. We also highlight recent in vitro approaches using individual embryonic stem cells and self-organizing multicellular models of human embryos, which have been instrumental in expanding our understanding of how mechanics tune cell fate and cellular rearrangements during human embryonic development. Cells in the embryo are subject to autonomous and external mechanical forces that help steer embryonic tissue patterning. Technical developments, such as in vitro models of early embryos, allow probing of the roles of mechanical forces in animal and human embryonic development.