Cooperativity between Cas9 and hyperactive AID establishes broad and diversifying mutational footprints in base editors.

The partnership of DNA deaminase enzymes with CRISPR-Cas nucleases is now a well-established method to enable targeted genomic base editing. However, an understanding of how Cas9 and DNA deaminases collaborate to shape base editor (BE) outcomes has been lacking. Here, we support a novel mechanistic model of base editing by deriving a range of hyperactive activation-induced deaminase (AID) base editors (hBEs) and exploiting their characteristic diversifying activity. Our model involves multiple layers of previously underappreciated cooperativity in BE steps including: (i) Cas9 binding can potentially expose both DNA strands for 'capture' by the deaminase, a feature that is enhanced by guide RNA mismatches; (ii) after strand capture, the intrinsic activity of the DNA deaminase can tune window size and base editing efficiency; (iii) Cas9 defines the boundaries of editing on each strand, with deamination blocked by Cas9 binding to either the PAM or the protospacer and (iv) non-canonical edits on the guide RNA bound strand can be further elicited by changing which strand is nicked by Cas9. Leveraging insights from our mechanistic model, we create novel hBEs that can remarkably generate simultaneous C > T and G > A transitions over >65 bp with significant potential for targeted gene diversification.

A New Methodology for Incorporating Chiral Linkers into Stapled Peptides.

Stapled peptides have arisen as a new class of chemical probe and potential therapeutic agents for modulating protein-protein interactions. Here, we report the first two-component i,i+7 stapling methodology that makes use of two orthogonal, on-resin stapling reactions to incorporate linkers bearing a chiral centre into a p53-derived stapled peptide. Post-stapling modifications to the chain were performed on-resin and enabled rapid access to various peptide derivatives from a single staple. The stapled peptides have increased helicity, protease stability and in vitro binding affinities to MDM2 compared to the equivalent unstapled peptide. This approach can be used to generate a library of diverse stapled peptides with different properties starting from a single stapled peptide, with scope for much greater functional diversity than that provided by existing stapling methodologies.

Synthesis, antimalarial properties and 2D-QSAR studies of novel triazole-quinine conjugates.

Click chemistry technique led to novel 1,2,3-triazole-quinine conjugates 8a-g, 10a-o, 11a-h and 13 utilizing benzotriazole-mediated synthetic approach with excellent yields. Some of the synthesized analogs (11a, 11d-h) exhibited antimalarial properties against Plasmodium falciparum strain 3D7 with potency higher than that of quinine (standard reference used) through in vitro standard procedure bio-assay. Statistically significant BMLR-QSAR model describes the bio-properties, validates the observed biological observations and identifies the most important parameters governing bio-activity.

Synthesis and antibacterial evaluation of amino acid-antibiotic conjugates.

Amino acid conjugates of quinolone, metronidazole and sulfadiazine antibiotics were synthesized in good yields using benzotriazole methodology. All the conjugates were screened for their antibacterial activity using methods adapted from the Clinical and Laboratory Standards Institute. Antibiotic conjugates were tested for activity in four medically relevant organisms; Staphylococcus aureus (RN4220), Escherichia coli (DH5α), Pseudomonas aeruginosa (PAO1), and Bacillus subtilis (168). Several antibiotic conjugates show promising results against several of the strains screened.

Structure-Guided Design of a Potent and Specific Inhibitor against the Genomic Mutator APOBEC3A.

Nucleic acid structure plays a critical role in governing the selectivity of DNA- and RNA-modifying enzymes. In the case of the APOBEC3 family of cytidine deaminases, these enzymes catalyze the conversion of cytosine (C) to uracil (U) in single-stranded DNA, primarily in the context of innate immunity. DNA deamination can also have pathological consequences, accelerating the evolution of viral genomes or, when the host genome is targeted by either APOBEC3A (A3A) or APOBEC3B (A3B), promoting tumor evolution leading to worse patient prognosis and chemotherapeutic resistance. For A3A, nucleic acid secondary structure has emerged as a critical determinant of substrate targeting, with a predilection for DNA that can form stem loop hairpins. Here, we report the development of a specific nanomolar-level, nucleic acid-based inhibitor of A3A. Our strategy relies on embedding the nucleobase 5-methylzebularine, a mechanism-based inhibitor, into a DNA dumbbell structure, which mimics the ideal substrate secondary structure for A3A. Structure-activity relationship studies using a panel of diverse inhibitors reveal a critical role for the stem and position of the inhibitor moiety in achieving potent inhibition. Moreover, we demonstrate that DNA dumbbell inhibitors, but not nonstructured inhibitors, show specificity against A3A relative to the closely related catalytic domain of A3B. Overall, our work demonstrates the feasibility of leveraging secondary structural preferences in inhibitor design, offering a blueprint for further development of modulators of DNA-modifying enzymes and potential therapeutics to circumvent APOBEC-driven viral and tumor evolution.

Harnessing Alternative Substrates to Probe TET Family Enzymes.

TET family enzymes normally oxidize 5-methylcytosine (5mC) in DNA, and play critical roles in shaping the epigenome. Despite their importance, assessing TET activity can be difficult, particularly given the challenge of studying modifications to single nucleobases within complex DNA substrates. We recently demonstrated that in addition to acting on 5mC, TET enzymes can act promiscuously on unnatural nucleobases. Here, we describe how these alternative unnatural substrates can be employed in facile assays to detect and quantify TET activity. DNA containing unnatural 5-vinylcytosine (vC) can be used as a direct endpoint reporter of TET activity, a method that can potentially be adapted to high-throughput platforms. Complementarily, DNA containing unnatural 5-ethynylcytosine (eyC) can trap and inactivate TET enzymes upon reaction, a strategy that can be used to extract active TET enzymes from a complex cellular milieu. We present a detailed PCR-based protocol to synthesize DNA probes with either natural or unnatural modifications, and methods for using these probes to track TET activity either in vitro or in cell extracts.

TET-TDG Active DNA Demethylation at CpG and Non-CpG Sites.

In mammalian genomes, cytosine methylation occurs predominantly at CG (or CpG) dinucleotide contexts. As part of dynamic epigenetic regulation, 5-methylcytosine (mC) can be erased by active DNA demethylation, whereby ten-eleven translocation (TET) enzymes catalyze the stepwise oxidation of mC to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC), thymine DNA glycosylase (TDG) excises fC or caC, and base excision repair yields unmodified cytosine. In certain cell types, mC is also enriched at some non-CG (or CH) dinucleotides, however hmC is not. To provide biochemical context for the distribution of modified cytosines observed in biological systems, we systematically analyzed the activity of human TET2 and TDG for substrates in CG and CH contexts. We find that while TET2 oxidizes mC more efficiently in CG versus CH sites, this context preference can be diminished for hmC oxidation. Remarkably, TDG excision of fC and caC is only modestly dependent on CG context, contrasting its strong context dependence for thymine excision. We show that collaborative TET-TDG oxidation-excision activity is only marginally reduced for CA versus CG contexts. Our findings demonstrate that the TET-TDG-mediated demethylation pathway is not limited to CG sites and suggest a rationale for the depletion of hmCH in genomes rich in mCH.

Efficient Routes to a Diverse Array of Amino Alcohol-Derived Chiral Fragments.

Efficient syntheses of chiral fragments derived from chiral amino alcohols are described. Several unique scaffolds were readily accessed in 1-5 synthetic steps leading to 45 chiral fragments, including oxazolidinones, morpholinones, lactams, and sultams. These fragments have molecular weights ranging from 100 to 255 Da and are soluble in water (0.085 to >15 mM).

Epidemiology of childhood ocular trauma in a northeastern Colombian region.

OBJECTIVE: To describe the epidemiology of ocular trauma in children 15 years and younger who underwent evaluation during a 5-year period in the emergency department of a tertiary referral center in northeastern Colombia. METHODS: We retrospectively reviewed the medical records of children 15 years and younger. Records of 393 children with 415 incidents of eye injury were included in the study, of whom 22 were initially treated for bilateral ocular trauma. RESULTS: Most patients (64.9%) were boys. The highest proportion of injuries (44.4%) occurred at home, followed by streets and roads (28.6%). Blunt (35.1%) and sharp (22.6%) objects represented the most frequent causes of trauma. Closed-globe injuries were far more frequent than open-globe injuries for boys (82.4% vs 17.6%) and girls (83.8% vs 16.2%). Of those with closed-globe injuries, 253 injuries (80.0%) registered an initial visual acuity of greater than 20/60, whereas 31 open-globe injuries (52.5%) registered an initial visual acuity of less than 20/400. Most closed-globe injuries (223 [92.1%]) did not cause any final visual impairment in the affected eye, whereas 26 open-globe injuries (55.3%) caused severe visual impairment or blindness. CONCLUSIONS: Most of the accidents reported in this study could have been avoided; the data demonstrate a clear need for primary prevention and control measures.

Alteraciones funcionales y morfológicas de la glandula tiroides en pacientes con enfermedad de Hodgkin tratados con radioterapia y quimioterapia / Functional alterations and morphology of the thyroide glans in patients with of hodgkin disease treaty with radiotherapy and chimioterapy

Determinar la frecuencia de disfunción tiroidea, patología tiroidea autoinmune y enfermedad nodular en pacientes tratados con radioterapia (RT) y quimioterapia (QT) por enfermedad de Hodgkin (E.H), estableciendo correlación con los cambios al ecosonograma tiroidea en dichos pacientes. Se evaluaron 25 pacientes tratados por E.H entre 1985 y 1996. Veintiuno recibieron Rt más Qt, 2 Rt y 2 Qt. La región cervical fue irradiada en 22 pacientes. El intervalo medio de tiempo entre el tratamiento y la evaluación fue de 66 meses. en cada paciente y en sus respectivos controles se determinó TSH, T4L, anticuerpos antimicrosales y ecosonograma tiroideo. 8 pacientes (32 por ciento)presentaron hipotiroidismo, subclínico, 2 (8 por ciento) hipotiroidimo franco, 3 8 (12 por ciento) enfermedad nodular y 3 (12 por ceinto) tiroiditis de Hashimoto. El volumen tiroideo por ultrasonografía mostró reducción significativa en comparación con los controles. El ecopatrón heterogéneo se observó en 7 de los 10 pacientes con hipotiroidismo. La elevada frecuencia de aptologías tiroideas en estos pacientes hace la evaluación periódica y prolongada, incorporando el ecosonograma, como herramienta útil en el diagnóstico precoz de estas alteraciones