Emerging technologies in personalized medicine.

A variety of technologies are emerging to help clinicians provide patient-specific diagnosis and therapies. This special edition of the Molecular Aspects of Medicine is a collection of mini reviews covering a broad range of topics, from systems to model patient variants and discover therapies (Microphysiological systems with patient derived tissue and CRISPR-humanized animal models), to new modalities in diagnostics and therapeutics (Extracellular Vesicles, RNA therapeutics, microbiome and molecular dynamics).

Clinical variants in expressing human STXBP1 reveal a novel class of pathogenic variants and classify variants of uncertain significance.

Purpose: Modeling disease variants in animals is useful for drug discovery, understanding disease pathology, and classifying variants of uncertain significance (VUS) as pathogenic or benign.

Methods: Using Clustered Regularly Interspaced Short Palindromic Repeats, we performed a Whole-gene Humanized Animal Model procedure to replace the coding sequence of the animal model's ortholog with the coding sequence for the human gene. Next, we used Clustered Regularly Interspaced Short Palindromic Repeats to introduce precise point variants in the Whole-gene Humanized Animal Model-humanized locus from 3 clinical categories (benign, pathogenic, and VUS).

Phenotypic screening models for rapid diagnosis of genetic variants and discovery of personalized therapeutics.

Precision medicine strives for highly individualized treatments for disease under the notion that each individual's unique genetic makeup and environmental exposures imprints upon them not only a disposition to illness, but also an optimal therapeutic approach. In the realm of rare disorders, genetic predisposition is often the predominant mechanism driving disease presentation. For such, mostly, monogenic disorders, a causal gene to phenotype association is likely.

The use of CRISPR to generate a whole-gene humanized and the examination of P301L and G272V clinical variants, along with the creation of deletion null alleles of and loci.

To study important genes involved in Frontotemporal Dementia ( , and ), we created deletion alleles in the three orthologous genes ( , , and ). Simultaneously, we replaced the gene with the predicted orthologous human gene, often called whole-gene humanization, which allows direct assessment of conserved gene function, as well as the opportunity to examine consequences of clinical disease-associated patient variations. Each gene was manipulated using a different selection strategy, including a novel strategy using an mutation rescue technique.

Systems to model the personalized aspects of microbiome health and gut dysbiosis.

The human gut microbiome is a complex and dynamic microbial entity that interacts with the environment and other parts of the body including the brain, heart, liver, and immune system. These multisystem interactions are highly conserved from invertebrates to humans, however the complexity and diversity of human microbiota compositions often yield a context that is unique to each individual. Yet commonalities remain across species, where a healthy gut microbiome will be rich in symbiotic commensal biota while an unhealthy gut microbiota will be experiencing abnormal blooms of pathobiont bacteria.

Longevity Genes Revealed by Integrative Analysis of Isoform-Specific daf-16/FoxO Mutants of Caenorhabditis elegans.

FoxO transcription factors promote longevity across taxa. How they do so is poorly understood. In the nematode Caenorhabditis elegans, the A- and F-isoforms of the FoxO transcription factor DAF-16 extend life span in the context of reduced DAF-2 insulin-like growth factor receptor (IGFR) signaling.

Single-copy insertion of transgenes in Caenorhabditis elegans.

At present, transgenes in Caenorhabditis elegans are generated by injecting DNA into the germline. The DNA assembles into a semistable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline.

Aminoethylation in model peptides reveals conditions for maximizing thiol specificity.

Control of pH in aminoethylation reactions is critical for maintaining high selectivity towards cysteine modification. Measurement of aminoethylation rate constants by liquid chromatography mass spectrometry demonstrates reaction selectivity of cysteine>>amino-terminus>>histidine. Lysine and methionine were not reactive at the conditions used.

Chemical-modification rescue assessed by mass spectrometry demonstrates that gamma-thia-lysine yields the same activity as lysine in aldolase.

The role of active site residues in fructose 1,6-bisphosphate aldolase is investigated by chemical-modification rescue. An active-site mutation, K107C, is constructed in a background where the four solvent-accessible cysteine residues are converted to alanine. The resulting mutant, tetK107C, when reacted with bromoethylamine (BrEA), shows a 40-fold increase in activity (to 80% that of wild type).