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Therapy-induced cellular senescence is a state of stable growth arrest induced by common cancer treatments such as chemotherapy and radiation. In an oncogenic context, therapy-induced senescence can have different consequences. By blocking cellular proliferation and by facilitating immune cell infiltration, it functions as tumor suppressive mechanism. By fueling the proliferation of bystander cells and facilitating metastasis, it acts as a tumor promoting factor. This dual role is mainly attributed to the differential expression and secretion of a set of pro-inflammatory cytokines and tissue remodeling factors, collectively known as the Senescence-Associated Secretory Phenotype (SASP). Here, we describe cell-autonomous and non-cell-autonomous mechanisms that senescent cells activate in response to chemotherapy and radiation leading to tumor suppression and tumor promotion. We present the current state of knowledge on the stimuli that affect the activation of these opposing mechanisms and the effect of senescent cells on their micro-environment eg. by regulating the functions of immune cells in tumor clearance as well as strategies to eliminate senescent tumor cells before exerting their deleterious side-effects.doi.org/10.1016/j.semcancer.2021.03.021 - 2021

Many instrumentation developments in crystallization have concentrated on massive parallelization assays and reduction of sample volume per experiment to find initial crystallization conditions. Yet improving the size and diffraction quality of the crystals for diffraction studies often requires decoupling of crystal nucleation and growth. This in turn requires the control of variables such as precipitant and protein concentration, equilibration rate, and temperature, which are all difficult parameters to control in the existing setups. The success of the temperature-controlled batch method, originally developed to grow very large crystals for neutron crystallography, demonstrated that the rational optimization of crystal growth has potential in structural biology. A temperature-controlled dialysis button has been developed for our previous device, and a prototype of an integrated apparatus for the rational optimization of crystal growth by mapping and manipulating temperature-precipitant concentration phase diagrams has been constructed. The presented approach differs from the current paradigm, since it involves serial instead of parallel experiments, exploring multiple crystallization conditions with the same protein sample. The sample is not consumed in the experiment and the conditions can be changed in a reversible fashion, using dialysis with a flowing precipitant reservoir as well as precise temperature control. The control software allows visualization of the crystals, as well as control of the temperature and composition of the crystallization solution. The rational crystallization optimization strategies presented here allow tailoring of crystal size, morphology and diffraction quality, significantly reducing the time, effort and amount of expensive protein material required for structure determination.DOI: 10.1107/S1600576716004635 - 2016

The ultimate purpose of diffraction data collection is to produce a data set which will result in the required structural information about the molecule of interest. This usually entails collecting a complete and accurate set of re ̄ection intensities to as high a resolution as possible. In practice, the characteristics of the crystal and properties of the X-ray source can be limiting factors to the data-set quality that can be achieved and a careful strategy has to be used to extract the maximum amount of information from the data within the experimental constraints. In the particular case of data intended for phasing using anomalous dispersion, the synchrotron beamline properties are relevant to determine how many wavelengths (one or more) should be used for the experiment and what the wavelength values should be. This will in turn affect the detailed strategy for data collection, including decisions about the data-collection sequence and how much data to collect at each wavelength. Collection of multiwavelength anomalous dispersion (MAD) data at three different wavelengths can provide very accurate experimental phases. Two-wavelength MAD experiments may offer the best compromise between phase quality and minimizing the effects of radiation damage to the sample. However, MAD experiments are demanding in terms of beamline wavelength range, easy tunability, stability and reproducibility. When the beamline cannot fulfill these demands, single-wavelength experiments may be a better option.doi.org/10.1107/S0907444903017700 -2003

Gene therapy has become a revolution and its breakthrough is a corner stone in modern science. This treatment has rising advantages with limited negative aspects. Gene therapy is a therapeutic method in which, transfer of DNA to an individual to manipulate a defective gene is performed and to mitigate a disease which is not responding to pharmacological therapy. The gene therapy strategies are divided into two main categories such as direct in-vivo gene delivery of manipulated viral vector vehicle into the host and ex-vivo genetically engineered stem cells. In this review, we tried to cover all aspects of gene therapy studies; starting with the concept of gene, its treatment, gene delivery system and types, clinical trial either by vitro or In-Vivo -Clinical Trials and Clinical Intoxication of Gene Therapy. Therefore, the promise of successful treatment with gene therapy could positively affect millions of lives. The main aim of this review is to address the principles of gene therapy, various methods involved in the gene therapy, clinical applications and its merits and demeritsdoi.org/10.1016/j.gene.2020.145246 - 2022

The impostor “syndrome” refers to the notion that some individuals feel as if they ended up in esteemed roles and positions not because of their competencies, but because of some oversight or stroke of luck. Such individuals therefore feel like frauds or “impostors.” Despite the fact that impostor feelings are often linked to marginalized groups in society, to date, research predominantly approaches this phenomenon as an issue of the individual: pointing toward individuals for the roots and solutions of the “syndrome.” Drawing from a rich body of social and organizational psychology research, in this perspectives piece, we propose a shift in how scholars conceptualize and empirically examine this phenomenon. Instead of framing the insecurities of individuals belonging to marginalized groups solely as a problem that arises within these individuals, we argue that it is critical for future research to consider the important role of the environment in eliciting their impostor feelings as well. By doing so, we can address the contextual roots of individuals’ impostor feelings, and offer more structural and effective solutions.doi: 10.3389/fpsyg.2020.575024 - 2020

Onasemnogene abeparvovec was recently approved for the treatment of spinal muscular atrophy (SMA) in children younger than two years; however, clinical trials were primarily completed in children younger than seven months, so practical experience dosing older children began in summer 2019. Here, we look at the safety and efficacy of onasemnogene in seven infants older than seven months who were treated at our center. Overall, onasemnogene appears to be efficacious in children older than seven months and well tolerated. Side effects were similar to those previously reported, although more common and in some cases more severe and more prolonged than seen in the original trials. The impact of age, weight, and other confounding factors on development of side effects still needs to be elucidated.doi.org/10.1016/j.pediatrneurol.2021.01.012 - 2021

N6-Methyladenosine (m6A), the most abundant internal mRNA modification, affects multiple steps in gene expression. Mechanistically, the binding of YTHDF2 to m6A on mRNAs elicits rapid mRNA degradation by recruiting several RNA degrading enzymes. Here, we show that N1-methyladenosine (m1A), another type of RNA modification, accelerates rapid m6A RNA degradation. We identify HRSP12 as an RNA-binding protein that recognizes m1A. The binding of HRSP12 to m1A promotes efficient interaction of YTHDF2 with m6A, consequently facilitating endoribonucleolytic cleavage via the RNase P/MRP complex. Transcriptome- wide analyses also reveal that mRNAs harboring both m1A and m6A are downregulated in an HRSP12-dependent manner compared with mRNAs harboring m6A only. Accordingly, a subset of endogenous circular RNAs that harbor m6A and associate with YTHDF2 in an HRSP12-dependent manner is also subjected to m1A-facilitated rapid degradation. Together, our observations provide compelling evidence for crosstalk between different RNA modifications.doi.org/10.1016/j.celrep.2022.111317 - 2022

N6-Methyladenosine (m6A), the most abundant internal mRNA modification, affects multiple steps in gene expression. Mechanistically, the binding of YTHDF2 to m6A on mRNAs elicits rapid mRNA degradation by recruiting several RNA degrading enzymes. Here, we show that N1-methyladenosine (m1A), another type of RNA modification, accelerates rapid m6A RNA degradation. We identify HRSP12 as an RNA-binding protein that recognizes m1A. The binding of HRSP12 to m1A promotes efficient interaction of YTHDF2 with m6A, consequently facilitating endoribonucleolytic cleavage via the RNase P/MRP complex. Transcriptome-wide analyses also reveal that mRNAs harboring both m1A and m6A are downregulated in an HRSP12-dependent manner compared with mRNAs harboring m6A only. Accordingly, a subset of endogenous circular RNAs that harbor m6A and associate with YTHDF2 in an HRSP12-dependent manner is also subjected to m1A-facilitated rapid degradation. Together, our observations provide compelling evidence for crosstalk between different RNA modifications.doi.org/10.1016/j.celrep.2022.111317 - 2022

Despite the success of monotherapies based on blockade of programmed cell death 1 (PD-1) in human melanoma, most patients do not experience durable clinical benefit. Pre-existing T-cell infiltration and/or the presence of PD-L1 in tumours may be used as indicators of clinical response; however, blood-based profiling to understand the mechanisms of PD-1 blockade has not been widely explored. Here we use immune profiling of peripheral blood from patients with stage IV melanoma before and after treatment with the PD-1-targeting antibody pembrolizumab and identify pharmacodynamic changes in circulating exhausted-phenotype CD8 T cells (Tex cells). Most of the patients demonstrated an immunological response to pembrolizumab. Clinical failure in many patients was not solely due to an inability to induce immune reinvigoration, but rather resulted from an imbalance between T-cell reinvigoration and tumour burden. The magnitude of reinvigoration of circulating Tex cells determined in relation to pretreatment tumour burden correlated with clinical response. By focused profiling of a mechanistically relevant circulating T-cell subpopulation calibrated to pretreatment disease burden, we identify a clinically accessible potential on-treatment predictor of response to PD-1 blockade.doi:10.1038/nature22079 - 2017

Tackling microbial resistance requires continuous efforts for the development of new molecules with novel mechanisms of action and potent antimicrobial activity. Our group has previously identified metal-based compounds, [Ag(1,10-phenan- throline-5,6-dione)2]ClO4 (Ag-phendione) and [Cu(1,10-phenanthroline-5,6-dione)3](ClO4)2.4H2O (Cu-phendione), with efficient antimicrobial action against multidrug-resistant species. Herein, we investigated the ability of Ag-phendione and Cu-phendione to bind with double-stranded DNA using a combination of in silico and in vitro approaches. Molecular dock- ing revealed that both phendione derivatives can interact with the DNA by hydrogen bonding, hydrophobic and electrostatic interactions. Cu-phendione exhibited the highest binding affinity to either major (− 7.9 kcal/mol) or minor (− 7.2 kcal/ mol) DNA grooves. In vitro competitive quenching assays involving duplex DNA with Hoechst 33258 or ethidium bromide demonstrated that Ag-phendione and Cu-phendione preferentially bind DNA in the minor grooves. The competitive eth- idium bromide displacement technique revealed Cu-phendione has a higher binding affinity to DNA (Kapp = 2.55 × 106 M−1) than Ag-phendione (Kapp = 2.79 × 105 M−1) and phendione (Kapp = 1.33 × 105 M−1). Cu-phendione induced topoisomerase I-mediated DNA relaxation of supercoiled plasmid DNA. Moreover, Cu-phendione was able to induce oxidative DNA inju- ries with the addition of free radical scavengers inhibiting DNA damage. Ag-phendione and Cu-phendione avidly displaced propidium iodide bound to DNA in permeabilized Pseudomonas aeruginosa cells in a dose-dependent manner as judged by flow cytometry. The treatment of P. aeruginosa with bactericidal concentrations of Cu-phendione (15 μM) induced DNA fragmentation as visualized by either agarose gel or TUNEL assays. Altogether, these results highlight a possible novel DNA-targeted mechanism by which phendione-containing complexes, in part, elicit toxicity toward the multidrug-resistant pathogen P. aeruginosa.https://doi.org/10.1007/s00775-021-01922-3 - 2022

A method and a device for the promotion of crystal growth by keeping the crystallization solution metastable during the growth process are described. This is achieved by controlled temperature variation of the crystallization solution using parameters determined in situ during the growth process. The technique finds application in the growth of large high-quality crystals for neutron crystallography. Thus, it has been applied to grow large crystals of several proteins of interest such as human gamma-crystallin E, PA-IIL lectin from Pseudomonas aeruginosa, yeast inorganic pyrophosphatase, urate oxidase from Aspergillus flavus and human carbonic anhydrase II.doi:10.1107/S0907444906054230 - 2007

Over the past six decades, steadily increasing progress in the application of the principles and techniques of the physical sciences to the study of biological systems has led to remarkable insights into the molecular basis of life. Of particular significance has been the way in which the determination of the structures and dynamical properties of proteins and nucleic acids has so often led directly to a profound understanding of the nature and mechanism of their functional roles. The increasing number and power of experimental and theoretical techniques that can be applied successfully to living systems is now ushering in a new era of structural biology that is leading to fundamentally new information about the maintenance of health, the origins of disease, and the development of effective strategies for therapeutic intervention. This article provides a brief overview of some of the most powerful biophysical methods in use today, along with references that provide more detailed information about recent applications of each of them. In addition, this article acts as an introduction to four authoritative reviews in this volume. The first shows the ways that a multiplicity of bio-physical methods can be combined with computational techniques to define the architectures of complex biological systems, such as those involving weak interactions within ensembles of molecular components. The second illustrates one aspect of this general approach by describing how recent advances in mass spectrometry, particularly in combination with other techniques, can generate fundamentally new insights into the properties of membrane proteins and their functional interactions with lipid molecules. The third review demonstrates the increasing power of rapidly evolving diffraction techniques, employing the very short bursts of X-rays of extremely high intensity that are now accessible as a result of the construction of free-electron lasers, in particular to carry out time-resolved studies of biochemical reactions. The fourth describes in detail the application of such approaches to probe the mechanism of the light-induced changes associated with bacteriorhodopsin’s ability to convert light energy into chemical energy.doi.org/10.1146/annurev-biochem-013118- 111947 - 2019

RNAs have emerged as a major target for diagnostics and therapeutics approaches. Regulatory nonprotein-coding RNAs (ncRNAs) in particular display remarkable ver- satility. They can fold into complex structures and interact with proteins, DNA, and other RNAs, thus modulating activity, localization, or interactome of multi-protein complexes. Thus, ncRNAs confer regulatory plasticity and represent a new layer of reg- ulatory control. Interestingly, long noncoding RNAs (lncRNAs) tend to acquire complex secondary and tertiary structures and their function—in many cases—is dependent on structural conservation rather than primary sequence conservation. Whereas for many proteins, structure and its associated function are closely con- nected, for lncRNAs, the structural domains that determine functionality and its inter- actome are still not well understood. Numerous approaches for analyzing the struc- tural configuration of lncRNAs have been developed recently. Here, will provide an overview of major experimental approaches used in the field, and discuss the poten- tial benefit of using combinatorial strategies to analyze lncRNA modes of action based on structural information.DOI: 10.1002/bies.202000027 - 2020

DNA is a strikingly flexible molecule and can form a variety of secondary structures, including the triple helix, which is the subject of this review. The DNA triplex may be formed naturally, during homologous recombination, or can be formed by the introduction of a synthetic triplex forming oligonucleotide (TFO) to a DNA duplex. As the TFO will bind to the duplex with sequence specificity, there is significant interest in developing TFOs with potential therapeutic applications, including using TFOs as a delivery mechanism for compounds able to modify or damage DNA. However, to combine triplexes with functionalised compounds, a full understanding of triplex structure and chemical modification strategies, which may increase triplex stability or in vivo degradation, is essential – these areas will be discussed in this review. Ruthenium polypyridyl complexes, which are able to photooxidise DNA and act as luminescent DNA probes, may serve as a suitable photophysical payload for a TFO system and the developments in this area in the context of DNA triplexes will also be reviewed.DOI: 10.1039/d2sc01793h - 2022

Amplification of pro-oncogenic kinases is a common genetic alteration driving tumorigenic phenotypes. Cancer cells rely on the amplified kinases to sustain cell proliferation, survival, and growth, presenting an opportunity to develop therapies targeting the amplified kinases. Utilizing small molecule catalytic inhibitors as therapies to target amplified kinases is plagued by de novo resistance driven by increased expression of the target, and amplified kinases can drive tumorigenic phenotypes independent of catalytic activity. Here, we discuss the emergence of proteolysis-targeting chimeras that provide an opportunity to target these oncogenic drivers effectively.doi.org/10.1124/molpharm.121.000306 - 2022

Glioblastomas are incurable tumors infiltrating the brain. A subpopulation of glioblastoma cells forms a functional and therapy-resistant tumor cell network interconnected by tumor microtubes (TMs). Other sub-populations appear unconnected, and their biological role remains unclear. Here, we demonstrate that whole-brain colonization is fueled by glioblastoma cells that lack connections with other tumor cells and astrocytes yet receive synaptic input from neurons. This subpopulation corresponds to neuronal and neural-progenitor-like tumor cell states, as defined by single-cell transcriptomics, both in mouse models and in the human disease. Tumor cell invasion resembled neuronal migration mechanisms and adopted a Levy-like movement pattern of probing the environment. Neuronal activity induced complex calcium signals in glioblastoma cells followed by the de novo formation of TMs and increased invasion speed. Collectively, superimposing molecular and functional single-cell data revealed that neuronal mechanisms govern glioblastoma cell invasion on multiple levels. This explains how glioblastoma’s dissemination and cellular heterogeneity are closely interlinked.doi.org/10.1016/j.cell.2022.06.054 - 2022

The research interest in the field of inorganic medicinal chemistry had a large increase after the serendipitous discovery of the cytotoxic activity of cisplatin by Rosenberg at the end of 1960s. Since then, cisplatin has entered clinical practice and become one of the most common treatments for solid tumors. Unfortunately, the use of cisplatin and its derivatives is associated with undesired side effects, such as general toxicity and intrinsic and acquired drug-resistance. For these reasons, alternative metallodrugs based on non- Pt metals have been synthetized, characterized and tested for biological activity in recent years. Although, initially, DNA seemed the exclusive target for metallodrugs [6], successive studies have revealed that various metabolites, peptides and proteins have a central role in the recognition, transport and mechanism of action of these compounds. Thus, a deeper understanding of the molecular bases of the interaction of metallodrugs with these molecules is needed.doi.org/10.3390/ijms23073504 - 2022

Undoubtedly, metallomic approaches based on mass spectrometry have evolved into essential tools supporting the drug development of novel metal-based anticancer drugs. This article will comment on the state-of-the-art instrumentation and highlight some of the recent analytical advances beyond routine, especially focusing on the latest developments in inductively coupled plasma-mass spectrometry (ICP-MS). Mass spectrometry-based bioimaging and single-cell methods will be presented, paving the way to exciting investigations of metal-based anticancer drugs in heterogeneous and structurally, as well as functionally complex solid tumor tissues.doi.org/10.1016/j.cbpa.2020.12.005 - 2021

Currently there are 850,000 people with Alzheimer’s disease in the UK, with an estimated rise to 1.1 million by 2025. Alzheimer’s disease is characterised by the accumulation of amyloid-beta plaques and hyperphosphorylated tau in the brain causing a progressive decline in cognitive impairment. Small non-coding microRNA (miRNA) sequences have been found to be deregulated in the peripheral blood of Alzheimer patients. A systematic review was conducted to extract all miRNA found to be significantly deregulated in the peripheral blood. These deregulated miRNAs were cross-referenced against the miRNAs deregulated in the brain at Braak Stage III. This resulted in a panel of 10 miRNAs (hsa-mir-107, hsa-mir-26b, hsa-mir-30e, hsa-mir-34a, hsa-mir-485, hsa-mir200c, hsa-mir-210, hsa-mir-146a, hsa-mir-34c, and hsa-mir-125b) hypothesised to be deregulated early in Alzheimer’s disease, nearly 20 years before the onset of clinical symptoms. After network analysis of the 10 miRNAs, they were found to be associated with the immune system, cell cycle, gene expression, cellular response to stress, neuron growth factor signalling, wnt signalling, cellular senescence, and Rho GTPases.doi.org/10.1007/s12035-019-1500-y - 2019

miRNAs are small RNAs that guide Argonaute proteins to specific target mRNAs to repress their translation and stability. Canonically, miRNA targeting is reliant on base pairing of the seed region, nucleotides 2–7, of the miRNA to sites in mRNA 30 untranslated regions. Recently, the 30 half of the miRNA has gained attention for newly appreciated roles in regulating target specificity and regulation. In addition, the extent of pairing to the miRNA 30 end can influence the stability of the miRNA itself. These findings highlight the importance of sequences beyond the seed in controlling the function and existence of miRNAs.doi.org/10.1016/j.tig.2018.12.005 - 2018

Manufacture of oligonucleotide active pharmaceutical ingredients (APIs) typically consists of solid-phase synthesis, deprotection and cleavage, purification and filtration, and isolation from aqueous solutions through lyophilization. In the first step of drug product manufacture, the API is dissolved in water again and excipients are added. While isolation of oligonucleotide APIs can be meaningful in many cases, there may be cases where keeping the API in solution provides benefit, and multiple technical aspects must be taken into account and balanced when determining the appropriate API form. A significant factor is whether an API in solution will contain additional components. While APIs in solution containing additional components (so-called formulated APIs) are well established for biological products, there are regulatory guidelines in place that represent hurdles for industry to using a formulated API approach for oligonucleotide drugs. The present communication outlines conditions where a formulated API approach can be chosen in compliance with existing guidelines. Relevant aspects pertaining to risk management, GMP standards, facility design, control strategies, and regulatory submission content are discussed. In addition, the authors propose that existing guidelines be modernized to enable the use of a formulated API approach for additional reasons than the ones described in the existing regulatory framework. The manuscript aims to promote a dialog with regulators in this field.doi.org/10.1007/s43441-022-00384-2 - 2022

Metallodrugs are extensively used to treat and diagnose distinct disease types. The unique physical-chemical properties of metal ions offer tantalizing opportunities to tailor effective scaffolds for selectively targeting specific biomolecules. Modern experimental techniques have collected a large body of structural data concerning the interactions of metallodrugs with their biomolecular targets, although being unable to exhaustively assess the molecular basis of their mechanism of action. In this scenario, the complementary use of accurate computational methods allows uncovering the minutiae of metallodrugs/targets interactions and their underlying mechanism of action at an atomic level of detail. This knowledge is increasingly perceived as an invaluable requirement to rationally devise novel and selective metallodrugs. Building on literature studies, selected largely from the last 2 years, this compendium encompasses a cross-section of the current role, advances, and challenges met by computer simulations to decipher the mechanistic intricacies of prototypical metallodrugs.doi.org/10.1016/j.cbpa.2020.07.005 -2020

Adoptive T cell transfer (ACT) is a new area of transfusion medicine involving the infusion of lymphocytes to mediate antitumor, antiviral, or anti-inflammatory effects. The field has rapidly advanced from a promising form of immuno-oncology in preclinical models to the recent commercial approvals of chimeric antigen receptor (CAR) T cells to treat leukemia and lymphoma. This Review describes opportunities and challenges for entering mainstream oncology that presently face the CAR T field, with a focus on the challenges that have emerged over the past several years.DOI: 10.1126/science.aar6711 - 2018

Nucleic acid-based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid-based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid-based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide-based therapeutics.DOI 10.15252/emmm.202013243 - 2020

Ketamine is used clinically as an anaesthetic and a fast-acting antidepressant, and recreationally for its dissociative properties, raising concerns of addiction as a possible side effect. Addictive drugs such as cocaine increase the levels of dopamine in the nucleus accumbens. This facilitates synaptic plasticity in the mesolimbic system, which causes behavioural adaptations and eventually drives the transition to compulsion1–4. The addiction liability of ketamine is a matter of much debate, in part because of its complex pharmacology that among several targets includes N-methyl-d-aspartic acid (NMDA) receptor (NMDAR) antagonism5,6. Here we show that ketamine does not induce the synaptic plasticity that is typically observed with addictive drugs in mice, despite eliciting robust dopamine transients in the nucleus accumbens. Ketamine nevertheless supported reinforcement through the disinhibition of dopamine neurons in the ventral tegmental area (VTA). This effect was mediated by NMDAR antagonism in GABA (γ-aminobutyric acid) neurons of the VTA, but was quickly terminated by type-2 dopamine receptors on dopamine neurons. The rapid off-kinetics of the dopamine transients along with the NMDAR antagonism precluded the induction of synaptic plasticity in the VTA and the nucleus accumbens, and did not elicit locomotor sensitization or uncontrolled self-administration. In summary, the dual action of ketamine leads to a unique constellation of dopamine-driven positive reinforcement, but low addiction liability.doi.org/10.1038/s41586-022-04993-7 - 2022

Reaction generality is crucial in determining the overall impact and usefulness of synthetic methods. Typical generalization protocols require a priori mechanistic understanding and suffer when applied to complex, less understood systems. We developed an additive mapping approach that rapidly expands the utility of synthetic methods while generating concurrent mechanistic insight. Validation of this approach on the metallaphotoredox decarboxylative arylation resulted in the discovery of a phthalimide ligand additive that overcomes many lingering limitations of this reaction and has important mechanistic implications for nickel-catalyzed cross-couplings.DOI: 10.1126/science.abn1885 - 2022

Highly proliferative tissues such as the gut, skin and bone marrow lose millions of cells each day to normal attrition and challenge from different biological adversities. To achieve a lifespan beyond the longevity of individual cell types, tissue-specific stem cells sustain these tissues throughout the life of a human. For example, the lifespan of erythrocytes is about 100 days and adults make about two million new erythrocytes every second. A small pool of hematopoietic stem cells (HSCs) in the bone marrow is responsible for the lifetime maintenance of these populations. However, there are changes that occur within the HSC pool during aging. Biologically, these changes manifest as blunted immune responses, decreased bone marrow cellularity, and increased risk of myeloid diseases. Understanding the molecular mechanisms underlying dysfunction of aging HSCs is an important focus of biomedical research. With advances in modern health care, the average age of the general population is ever increasing. If molecular or pharmacological interventions could be discovered that rejuvenate aging HSCs, it could reduce the burden of age related immune system compromise as well as open up new opportunities for treatment of hematological disorders with regenerative therapy.doi:10.1053/j.seminhematol.2016.10.006 - 2017

Epigenetic editing refers to the locus-specific targeting of epigenetic enzymes to rewrite the local epigenetic landscape of an endogenous genomic site, often with the aim of transcriptional reprogramming. Implementing clustered regularly interspaced short palindromic repeat–dCas9 greatly accelerated the advancement of epigenetic editing, yielding preclinical therapeutic successes using a variety of epigenetic enzymes. Here, we review the current applications of these epigenetic editing tools in mammals and shed light on biochemical improvements that facilitate versatile applications.doi.org/10.1016/j.cbpa.2020.04.020 - 2020

Cancer progression is an evolutionary process. During this process, evolving cancer cell populations encounter restrictive ecological niches within the body, such as the primary tumor, circulatory system, and diverse metastatic sites. Efforts to prevent or delay cancer evolution—and progression—require a deep understanding of the underlying molecular evolutionary processes. Herein we discuss a suite of concepts and tools from evolutionary and ecological theory that can inform cancer biology in new and meaningful ways. We also highlight current challenges to applying these concepts, and propose ways in which incorporating these concepts could identify new therapeutic modes and vulnerabilities in cancer.doi:10.1093/molbev/msz242 - 2019

Structural DNA nanotechnology is poised to transform targeted therapeutic and theranostic delivery agents. Some of the most promising biomedical applications of DNA nanostructures include carriers for biosensing, imaging, and drug delivery. Additionally, the unique ability to precisely position inorganic and organic molecules on DNA-based substrates enables the spatially optimized high density interfacing of ligands with cell membrane receptors. To realize clinically viable biomedical products made from DNA nanostructures, it is necessary to fully understand the behavior of these systems inside and outside the cellular environment. To that end, cohesive and conclusive information on the physiological fate of DNA nanostructures at various time points – from the cell culture to the cell cytosol – is still lacking. In this highlight, we bring to attention efforts to understand DNA nanostructure behavior in vitro as well as some widespread disparities among studies on the subject. We also call for a discussion on the implementation of common standards and controls to address these disparities and consequently unify the scientific community’s endeavours to build foundational knowledge on DNA nanostructure–cellular interaction.DOI: 10.1039/d0tb00395f - 2020

The direct incorporation of macrocyclic cyclidene complexes into DNA via automated synthesis results in a new family of metal-functionalized DNA derivatives that readily demonstrate their utility through the ability of one redox-active copper(II)-containing strand to distinguish electrochemically between all four canonical DNA nucleobases at a single site within a target sequence of DNA.DOI: 10.1021/jacs.5b11319 - 2015

We report the use of DNA origami nanostructures, functionalized with aptamers, as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner. We test the system against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) targets. We use direct stochastic optical reconstruction microscopy (dSTORM) and atomic force microscopy (AFM) to characterize the DNA origami nanostructures and structured illumination microscopy (SIM) to assess the binding of the origami to the bacteria. We show that treatment with lysozyme-functionalized origami slows bacterial growth more effectively than treatment with free lysozyme. Our study introduces DNA origami as a tool in the fight against antibiotic resistance, and our results demonstrate the specificity and efficiency of the nanostructure as a drug delivery vehicle.doi.org/10.1002/anie.202002740 - 2020

Nanopores are key in portable sequencing and research given their ability to transport elongated DNA or small bioactive molecules through narrow transmembrane channels. Transport of folded proteins could lead to similar scientific and technological benefits. Yet this has not been realised due to the shortage of wide and structurally defined natural pores. Here we report that a synthetic nanopore designed via DNA nanotechnology can accommodate folded proteins. Transport of fluorescent proteins through single pores is kinetically analysed using massively parallel optical readout with transparent silicon-on-insulator cavity chips vs. electrical recordings to reveal an at least 20-fold higher speed for the electrically driven movement. Pores nevertheless allow a high diffusive flux of more than 66 molecules per second that can also be directed beyond equillibria. The pores may be exploited to sense diagnostically relevant proteins with portable analysis technology, to create molecular gates for drug delivery, or to build synthetic cells.doi.org/10.1038/s41467-019-12639-y - 2019

Synthetic biology is among the most hyped research topics this century, and in 2010 it entered its teenage years. But rather than these being a problematic time, we’ve seen synthetic biology blossom and deliver many new technologies and landmark achievements.doi.org/10.1038/s41467-020-19092-2 - 2020

Deregulated cell proliferation is an established feature of cancer, and altered tumor metabolism has witnessed renewed interest over the past decade, including the study of how cancer cells rewire metabolic pathways to renew energy sources and ‘‘building blocks’’ that sustain cell division. Microenvironmental oxygen, glucose, and glutamine are regarded as principal nutrients fueling tumor growth. However, hostile tumor microenvironments render O2/nutrient supplies chronically insufficient for increased proliferation rates, forcing cancer cells to develop strategies for opportunistic modes of nutrient acquisition. Recent work shows that cancer cells overcome this nutrient scarcity by scavenging other substrates, such as proteins and lipids, or utilizing adaptive metabolic pathways. As such, reprogramming lipid metabolism plays important roles in providing energy, macromolecules for membrane synthesis, and lipid-mediated signaling during cancer progression. In this review, we highlight more recently appreciated roles for lipids, particularly cholesterol and its derivatives, in cancer cell metabolism within intrinsically harsh tumor microenvironments.doi.org/10.1016/j.molcel.2019.09.008 - 2019

The structural basis of minor groove recognition of a DNA duplex containing synthetic genetic information by hairpin pyrrole- imidazole polyamides is described. Hairpin polyamides induce a higher melting stabilization of a DNA duplex containing the unnatural P·Z base- pair when an imidazole unit is aligned with a P nucleotide. An NMR structural study showed that the incorporation of two isolated P·Z pairs enlarges the minor groove and slightly narrows the major groove at the site of this synthetic genetic information, relative to a DNA duplex consisting entirely of Watson−Crick base-pairs. Pyrrole-imidazole polyamides bind to a P·Z-containing DNA duplex to form a stable complex, effectively mimicking a G·C pair. A structural hallmark of minor groove recognition of a P·Z pair by a polyamide is the reduced level of allosteric distortion induced by binding of a polyamide to a DNA duplex. Understanding the molecular determinants that influence minor groove recognition of DNA containing synthetic genetic components provides the basis to further develop unnatural base-pairs for synthetic biology applications.DOI: 10.1021/jacs.8b12444 - 2019

Systemic administration of therapeutic agents has been the preferred approach to treat most pathological conditions, in particular for cancer therapy. This treatment modality is associated with side effects, off-target accumulation, toxicity, and rapid renal and hepatic clearance. Multiple efforts have focused on incorporating targeting moieties into systemic therapeutic vehicles to enhance retention and minimize clearance and side effects. However, only a small percentage of the nanoparticles administered systemically accumulate at the tumor site, leading to poor therapeutic efficacy. This has prompted researchers to call the status quo treatment regimen into question and to leverage new delivery materials and alternative administration routes to improve therapeutic outcomes. Recent approaches rely on the use of local delivery platforms that circumvent the hurdles of systemic delivery. Local administration allows delivery of higher “effective” doses while enhancing therapeutic molecules’ stability, minimizing side effects, clearance, and accumulation in the liver and kidneys following systemic administration. Hydrogels have proven to be highly biocompatible materials that allow for versatile design to afford sensing and therapy at the same time. Hydrogels’ chemical and physical versatility can be exploited to attain disease-triggered in situ assembly and hydrogel programmed degradation and consequent drug release, and hydrogels can also serve as a biocompatible depot for local delivery of stimuli-responsive therapeutic cargo. We will focus this Account on the hydrogel platform that we have developed in our lab, based on dendrimer amine and dextran aldehyde. This hydrogel is disease-responsive and capable of sensing the microenvironment and reacting in a graded manner to diverse pathologies to render different properties, including tissue adhesion, biocompatibility, hydrogel degradation, and embedded drug release profile. We also studied the degradation kinetics of our stimuli-responsive materials in vivo and analyzed the in vitro conditions under which in vitro−in vivo correlation is attained. Identifying key parameters in the in vivo microenvironment under healthy and disease conditions was key to attaining that correlation. The adhesive capacity of our dendrimer−dextran hydrogel makes it optimal for localized and sustained release of embedded drugs. We demonstrated that it affords the delivery of a range of therapeutics to combat cancer, including nucleic acids, small molecules, and antibody drugs. As a depot for local delivery, it allows a high dose of active biomolecules to be delivered directly at the tumor site. Immunotherapy, a recently blooming area in cancer therapy, may exploit stimuli-responsive hydrogels to impart systemic effects following localized therapy. Local delivery would enable release of the proper drug dose and improve drug bioavailability where needed at the same time creating memory and exerting the therapeutic effect systemically. This Account highlights our perspective on how local and systemic therapies provided by stimuli-responsive hydrogels should be used to impart more precise, long-lasting, and potent therapeutic outcomes.DOI: 10.1021/acs.accounts.6b00536 - 2017

DNA has been a key target for cancer therapy, with a range of compounds able to bind and either impair its processing or induce damage. Targeting DNA with small molecules in a truly sequence specific way, to impair gene specific processes, remains out of reach. The ability of DNA to assume different structures from the classical double helix allows access to more specific ligand binding modes and, potentially, to new avenues of treatment. In this review, we illustrate the small molecules that have been reported to bind to three- and four-way junctions.doi.org/10.1016/j.bmc.2022.116897 - 2022

Aptamers are molecules identified from large combinatorial nucleic acid libraries by their high affinity to target molecules. Due to a variety of desired properties, aptamers are attractive alternatives to antibodies in molecular biology and medical applications. Aptamers are identified through an iterative selection–amplification process known as systematic evolution of ligands by exponential enrichment (SELEX). Although SELEX is typically carried out using purified target molecules, whole live cells are also employable as selection targets. This technology, Cell-SELEX, has several advantages. For example, generated aptamers are functional with a native conformation of the target molecule on live cells, and thus, cell surface transmembrane proteins would be targets even went their purifications in native conformations are difficult. In addition, cell-specific aptamers can be obtained without any knowledge about cell surface molecules on the target cells. Here, I review the progress of Cell-SELEX technology and discuss advantages of the technologyDOI: 10.1089/biores.2012.0253265 - 2012

We describe a method for direct tRNA sequencing using the Oxford Nanopore MinION. The principal technical advance is custom adapters that facilitate end-to-end sequencing of individual transfer RNA (tRNA) molecules at subnanometer precision. A second advance is a nanopore sequencing pipeline optimized for tRNA. We tested this method using purified E. coli tRNAfMet, tRNALys, and tRNAPhe samples. 76–92% of individual aligned tRNA sequence reads were full length. As a proof of concept, we showed that nanopore sequencing detected all 43 expected isoacceptors in total E. coli MRE600 tRNA as well as isodecoders that further define that tRNA population. Alignment-based comparisons between the three purified tRNAs and their synthetic controls revealed systematic nucleotide miscalls that were diagnostic of known modifications. Systematic miscalls were also observed proximal to known modifications in total E. coli tRNA alignments, including a highly conserved pseudouridine in the T loop. This work highlights the potential of nanopore direct tRNA sequencing as well as improvements needed to implement tRNA sequencing for human healthcare applications.doi.org/10.1021/acsnano.1c06488 - 2021

Ageing is a complex, multifaceted process leading to widespread functional decline that affects every organ and tissue, but it remains unknown whether ageing has a unifying causal mechanism or is grounded in multiple sources. Phenotypically, the ageing process is associated with a wide variety of features at the molecular, cellular and physiological level—for example, genomic and epigenomic alterations, loss of proteostasis, declining overall cellular and subcellular function and deregulation of signalling systems. However, the relative importance, mechanistic interrelationships and hierarchical order of these features of ageing have not been clarified. Here we synthesize accumulating evidence that DNA damage affects most, if not all, aspects of the ageing phenotype, making it a potentially unifying cause of ageing. Targeting DNA damage and its mechanistic links with the ageing phenotype will provide a logical rationale for developing unified interventions to counteract age-related dysfunction and disease.doi.org/10.1038/s41586-021-03307-7 - 2021

Organelles juxtaposition has been detected for decades, although only recently gained importance due to a pivotal role in the regulation of cellular processes dependent on membrane contact sites. Endoplasmic reticulum (ER) and mitochondria interaction is a prime example of organelles contact sites. Mitochondria-associated membranes (MAM) are proposed to harbor ER-mitochondria tether complexes, mainly when these organelles are less than 30 nm apart. Dysfunctions of proteins located at the MAM are associated with neurodegenerative diseases such as Parkinson’s, Alzheimer’s and amyotrophic lateral sclerosis, as well as neurodevelopmental disorders; hence any malfunction in MAM can potentially trigger cell death. This review will focus on the role of ER-mitochondria contact sites, regarding calcium homeostasis, lipid metabolism, autophagy, morphology and dynamics of mitochondria, mainly in the context of neurodegenerative diseases. Approaches that have been employed so far to study organelles contact sites, as well as methods that were not used in neurosciences yet, but are promising and accurate ways to unveil the functions of MAM during neurodegeneration, is also discussed in the present review.doi.org/10.1007/s10571-020-01008-9 - 2022

The DNA methyltransferase (DNMT) family comprises a conserved set of DNA-modifying enzymes that have a central role in epigenetic gene regulation. Recent studies have shown that the functions of the canonical DNMT enzymes — DNMT1, DNMT3A and DNMT3B — go beyond their traditional roles of establishing and maintaining DNA methylation patterns. This Review analyses how molecular interactions and changes in gene copy numbers modulate the activity of DNMTs in diverse gene regulatory functions, including transcriptional silencing, transcriptional activation and post-transcriptional regulation by DNMT2-dependent tRNA methylation. This mechanistic diversity enables the DNMT family to function as a versatile toolkit for epigenetic regulation.doi:10.1038/nrg.2017.80 - 2018

We propose a bimolecular approach for G-quadruplex alkylation, using a pro-reactive furan-containing ligand, activated by red-light irradiation of a proximate G4-binding photosensitizer. G4- over dsDNA alkylation can be achieved selectively and proves high-yielding at low ligand excess. HPLC and modelling studies allowed identifying potential residues involved in the alkylation.DOI: 10.1039/d0cc06030e - 2021

Chiral communications exist in secondary structures of foldamers and copolymers via a network of noncovalent interactions within effective intermolecular force (IMF) range. It is not known whether long-range chiral communication exists between macromolecular tertiary structures such as peptide coiled-coils beyond the IMF distance. Harnessing the high sensitivity of single-molecule force spectroscopy, we investigate the chiral interaction between covalently linked DNA duplexes and peptide coiled-coils by evaluating the binding of a diastereomeric pair of three DNA-peptide conjugates. We find that right-handed DNA triple helices well accommodate peptide triple coiled-coils of the same handedness, but not with the left-handed coiled-coil stereoisomers. This chiral communication is effective in a range (<4.5 nm) far beyond canonical IMF distance. Small-angle X-ray scattering and molecular dynamics simulation indicate that the interdomain linkers are tightly packed via hydrophobic interactions, which likely sustains the chirality transmission between DNA and peptide domains. Our findings establish that long-range chiral transmission occurs in tertiary macromolecular domains, explaining the presence of homochiral pairing of superhelices in proteins.doi.org/10.1038/s41467-021-27708-4 - 2022

Atomic mutagenesis is the key to advance our understanding of RNA recognition and RNA catalysis. To this end, deazanucleosides are utilized to evaluate the participation of specific atoms in these processes. One of the remaining challenges is access to RNA-containing 1-deazaguanosine (c1G). Here, we present the synthesis of this nucleoside and its phosphoramidite, allowing first time access to c1G-modified RNA. Thermodynamic analyses revealed the base pairing parameters for c1G-modified RNA. Furthermore, by NMR spectroscopy, a c1G-triggered switch of Watson-Crick into Hoogsteen pairing in HIV-2 TAR RNA was identified. Additionally, using X-ray structure analysis, a guanine−phosphate backbone interaction affecting RNA fold stability was characterized, and finally, the critical impact of an active-site guanine in twister ribozyme on the phosphodiester cleavage was revealed. Taken together, our study lays the synthetic basis for c1G-modified RNA and demonstrates the power of the completed deazanucleoside toolbox for RNA atomic mutagenesis needed to achieve in-depth understanding of RNA recognition and catalysis.doi.org/10.1021/jacs.2c01877 - 2022

Membrane proteins play the central roles in a variety of cellular processes, ranging from nutrient uptake and signalling, to cell-cell communication. Their biological functions are directly related to how they fold and assemble; defects often lead to disease. Protein–protein interactions (PPIs) within the membrane are therefore of great interest as therapeutic targets. Here we review the progress in the application of membrane–insertable peptides for the disruption or stabilization of membrane–based PPIs. We describe the design and preparation of transmembrane peptide mimics; and of several categories of peptidomimetics used for study, including D-enantiomers, non–natural amino acids, peptoids, and β-peptides. Further aspects of the review describe modifications to membrane–insertable peptides, including lipidation and cyclization via hydrocarbon stapling. These approaches provide a pathway toward the development of metabolically stable, non-toxic, and efficacious peptide modulators of membrane–based PPIs. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid- control of protein activity edited by Dirk Schneider.doi.org/10.1016/j.bbamem.2016.08.013 - 2017

The increasing number of approved nucleic acid therapeutics demonstrates the potential to treat diseases by targeting their genetic blueprints in vivo. Conventional treatments generally induce therapeutic effects that are transient because they target proteins rather than underlying causes. In contrast, nucleic acid therapeutics can achieve long-lasting or even curative effects via gene inhibition, addition, replacement or editing. Their clinical translation, however, depends on delivery technologies that improve stability, facilitate internalization and increase target affinity. Here, we review four platform technologies that have enabled the clinical translation of nucleic acid therapeutics: antisense oligonucleotides, ligand-modified small interfering RNA conjugates, lipid nanoparticles and adeno-associated virus vectors. For each platform, we discuss the current state-of-the-art clinical approaches, explain the rationale behind its development, highlight technological aspects that facilitated clinical translation and provide an example of a clinically relevant genetic drug. In addition, we discuss how these technologies enable the development of cutting-edge genetic drugs, such as tissue-specific nucleic acid bioconjugates, messenger RNA and gene-editing therapeutics.doi.org/10.1038/s41565-021-00898-0 - 2021

Polymeric materials that can be readily modified with (bio)molecules of interest to render them suitable for intended applications are in demand for use in various areas of biomedical and materials science. Among various clickable reactive groups that can be incorporated into polymeric materials as handles for functionalization, ready availability, efficient reactivity under relatively mild conditions, as well as selectivity to specific functional groups makes the furan moiety an attractive choice. While the dynamic nature of furan-maleimide cycloadduct has been exploited for the fabrication of self-healing materials for decades, this reversibility has been only recently exploited to further expand the functional properties of polymeric materials. This review article focuses on recent examples that demonstrate that functional properties imparted to polymeric materials through incorporation of the furan group go beyond self-healing. Recent examples illustrating biomolecular immobilization, drug delivery/release, and dynamic material fabrication are highlighted to show the versatility of furan- containing polymeric materials in various biomedical applications.doi.org/10.1016/j.eurpolymj.2021.110514 - 2021

Pyrene derivatives can be incorporated into nucleic acid analogs in order to obtain switchable probes or supramolecular architectures. In this paper, peptide nucleic acids (PNAs) containing 1 to 3 1-pyreneacetic acid units (PNA1–6) with a sequence with prevalence of pyrimidine bases, complementary to cystic fibrosis W1282X point mutation were synthesized. These compounds showed sequence-selective switch-on of pyrene excimer emission in the presence of target DNA, due to PNA2DNA triplex formation, with stability depending on the number and positioning of the pyrene units along the chain. An increase in triplex stability and a very high mismatch-selectivity, derived from combined stacking and base-pairing interactions, were found for PNA2, bearing two distant pyrene units.doi:10.3762/bjoc.10.154 - 2014