Direct delivery of Cas-embedded cytosine base editors as ribonucleoprotein complexes for efficient and accurate editing of clinically relevant targets.

Recently, cytosine base editors (CBEs) have emerged as a promising therapeutic tool for specific editing of single nucleotide variants and disrupting specific genes associated with disease. Despite this promise, the currently available CBEs have the significant liabilities of off-target and bystander editing activities, partly due to the mechanism by which they are delivered, causing limitations in their potential applications. In this study, we engineered optimized, soluble and stable Cas-embedded CBEs (CE_CBEs) that integrate several recent advances, which were efficiently formulated for direct delivery into cells as ribonucleoprotein (RNP) complexes.

Efficient convergent synthesis of 1,3-diazepinone nucleosides by ring-closing metathesis and direct glycosylation.

A new and highly efficient ring-closing metathesis-based strategy was developed for the synthesis of the cyclic urea 1,3-diazepinone, presenting significant improvement upon previous methods. Using a direct glycosylation approach, analogues of the potent cytidine deaminase (CDA) inhibitor diazepinone riboside were then synthesized including 2'-deoxyribo-, 2'-deoxy-2'-fluoroarabino-, and 2'-deoxy-2',2'-difluoro-diazepinone nucleosides, all with moderate to good yield and excellent anomeric selectivity. Crucially, in contrast to the previous multistep linear synthesis of 2'-deoxyribo- and 2'-deoxy-2'-fluoroarabino-diazepinone nucleosides, this is the first report of direct glycosylation to access these nucleosides.

Virion-associated influenza hemagglutinin clusters upon sialic acid binding visualized by cryo-electron tomography.

Influenza viruses are enveloped, negative sense single-stranded RNA viruses covered in a dense layer of glycoproteins. Hemagglutinin (HA) accounts for 80-90% of influenza glycoprotein and plays a role in host cell binding and membrane fusion. While previous studies have characterized structures of receptor-free and receptor-bound HA in vitro, the effect of receptor binding on HA organization and structure on virions remains unknown.

FcRn-enhancing mutations lead to increased and prolonged levels of the HIV CCR5-blocking monoclonal antibody leronlimab in the fetuses and newborns of pregnant rhesus macaques.

Prenatal administration of monoclonal antibodies (mAbs) is a strategy that could be exploited to prevent viral infections during pregnancy and early life. To reach protective levels in fetuses, mAbs must be transported across the placenta, a selective barrier that actively and specifically promotes the transfer of antibodies (Abs) into the fetus through the neonatal Fc receptor (FcRn). Because FcRn also regulates Ab half-life, Fc mutations like the M428L/N434S, commonly known as LS mutations, and others have been developed to enhance binding affinity to FcRn and improve drug pharmacokinetics.

Next generation APOBEC3 inhibitors: Optimally designed for potency and nuclease stability.

APOBEC3 (or A3) enzymes have emerged as potential therapeutic targets due to their role in introducing heterogeneity in viruses and cancer, often leading to drug resistance. Inhibiting these enzymes has remained elusive as initial phosphodiester (PO) linked DNA based inhibitors lack stability and potency. We have enhanced both potency and nuclease stability, of 2'-deoxy-zebularine (dZ), substrate-based oligonucleotide inhibitors for two critical A3's: A3A and A3G.

Generative Modelling of Cortical Receptor Distributions from Cytoarchitectonic Images in the Macaque Brain.

Neurotransmitter receptor densities are relevant for understanding the molecular architecture of brain regions. Quantitative in vitro receptor autoradiography, has been introduced to map neurotransmitter receptor distributions of brain areas. However, it is very time and cost-intensive, which makes it challenging to obtain whole-brain distributions.

Expanding the genetic toolbox for the obligate human pathogen .

Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen To overcome this limitation and enable the straightforward investigation of gene functions in , we have developed a comprehensive genetic toolset. By adapting and combining different tools previously applied in other Gram-positive bacteria, we have created new replicative and integrative plasmids for gene expression and genetic manipulation, constitutive and inducible promoters as well as fluorescence reporters for .

Phylogenetic reduction of the magnocellular red nucleus in primates and inter-subject variability in humans.

Introduction: The red nucleus is part of the motor system controlling limb movements. While this seems to be a function common in many vertebrates, its organization and circuitry have undergone massive changes during evolution. In primates, it is sub-divided into the magnocellular and parvocellular parts that give rise to rubrospinal and rubro-olivary connection, respectively.

Contributions of Hyperactive Mutations in M from SARS-CoV-2 to Drug Resistance.

The appearance and spread of mutations that cause drug resistance in rapidly evolving diseases, including infections by the SARS-CoV-2 virus, are major concerns for human health. Many drugs target enzymes, and resistance-conferring mutations impact inhibitor binding or enzyme activity. Nirmatrelvir, the most widely used inhibitor currently used to treat SARS-CoV-2 infections, targets the main protease (M) preventing it from processing the viral polyprotein into active subunits.

Characterization of the major autolysin (AtlC) of Staphylococcus carnosus.

Background: Autolysis by cellular peptidoglycan hydrolases (PGH) is a well-known phenomenon in bacteria. During food fermentation, autolysis of starter cultures can exert an accelerating effect, as described in many studies on cheese ripening. In contrast, very little is known about autolysis of starter cultures used in other fermentations.

Direct delivery of stabilized Cas-embedded base editors achieves efficient and accurate editing of clinically relevant targets.

Over the last 5 years, cytosine base editors (CBEs) have emerged as a promising therapeutic tool for specific editing of single nucleotide variants and disrupting specific genes associated with disease. Despite this promise, the currently available CBE's have the significant liabilities of off-target and bystander editing activities, in part due to the mechanism by which they are delivered, causing limitations in their potential applications. In this study we engineeredhighly stabilized Cas-embedded CBEs (sCE_CBEs) that integrate several recent advances, andthat are highly expressible and soluble for direct delivery into cells as ribonucleoprotein (RNP) complexes.

Human fertilization in vivo and in vitro requires the CatSper channel to initiate sperm hyperactivation.

The infertility of many couples rests on an enigmatic dysfunction of the man's sperm. To gain insight into the underlying pathomechanisms, we assessed the function of the sperm-specific multisubunit CatSper-channel complex in the sperm of almost 2,300 men undergoing a fertility workup, using a simple motility-based test. We identified a group of men with normal semen parameters but defective CatSper function.

Addressing the dNTP bottleneck restricting prime editing activity.

Prime editing efficiency is modest in cells that are quiescent or slowly proliferating where intracellular dNTP levels are tightly regulated. MMLV-reverse transcriptase - the prime editor polymerase subunit - requires high intracellular dNTPs levels for efficient polymerization. We report that prime editing efficiency in primary cells and in vivo is increased by mutations that enhance the enzymatic properties of MMLV-reverse transcriptase and can be further complemented by targeting SAMHD1 for degradation.

FruitFire: a luciferase based on a fruit fly metabolic enzyme.

Firefly luciferase is homologous to fatty acyl-CoA synthetases from insects that are not bioluminescent. Here, we determined the crystal structure of the fruit fly fatty acyl-CoA synthetase CG6178 to 2.5 Å.

Systematic Analyses of the Resistance Potential of Drugs Targeting SARS-CoV-2 Main Protease.

Drugs that target the main protease (M) of SARS-CoV-2 are effective therapeutics that have entered clinical use. Wide-scale use of these drugs will apply selection pressure for the evolution of resistance mutations. To understand resistance potential in M, we performed comprehensive surveys of amino acid changes that can cause resistance to nirmatrelvir (Pfizer), and ensitrelvir (Xocova) in a yeast screen.

HIV-1 protease inhibitors with a P1 phosphonate modification maintain potency against drug-resistant variants by increased interactions with flap residues.

Protease inhibitors are the most potent antivirals against HIV-1, but they still lose efficacy against resistant variants. Improving the resistance profile is key to developing more robust inhibitors, which may be promising candidates for simplified next-generation antiretroviral therapies. In this study, we explored analogs of darunavir with a P1 phosphonate modification in combination with increasing size of the P1' hydrophobic group and various P2' moieties to improve potency against resistant variants.

Crystal Structures of Inhibitor-Bound Main Protease from Delta- and Gamma-Coronaviruses.

With the spread of SARS-CoV-2 throughout the globe causing the COVID-19 pandemic, the threat of zoonotic transmissions of coronaviruses (CoV) has become even more evident. As human infections have been caused by alpha- and beta-CoVs, structural characterization and inhibitor design mostly focused on these two genera. However, viruses from the delta and gamma genera also infect mammals and pose a potential zoonotic transmission threat.