Validity of the Rule of Threes and Anatomical Relationships in the Thoracic Spine.

CONTEXT: The location of the more superficial thoracic spinous processes is used to help osteopathic physicians locate the deeper and more difficult-to-palpate thoracic transverse processes. In 1979, Mitchell et al proposed the thoracic rule of threes to describe the relationship of the spinous processes to the transverse processes in the thoracic spine. This rule is currently taught at osteopathic medical schools. The rule of threes separates the thoracic vertebrae into 3 distinct groups, each with a different relationship between transverse processes and spinous processes. In 2006, Geelhoed et al proposed a new relationship between the spinous processes and transverse processes for all thoracic vertebrae (ie, Geelhoed's rule). OBJECTIVE: To determine which anatomical relationship-the rule of threes or Geelhoed's rule-is most accurate in locating the transverse processes and to define anatomical relationships between thoracic spinous and transverse processes. METHODS: The thoracic spinous and transverse processes of 44 formalin-embalmed human cadavers were dissected, marked, and photographed. Six different measurements per vertebra were made between spinous processes and transverse processes in the thoracic spine. Geelhoed's protocol was used to determine the validity of each rule. The measurements were analyzed for additional relationships between thoracic spinous processes and transverse processes. Group 1 consisted of vertebrae T1 to T3 and T12; group 2 consisted of T4 to T6 and T11; and group 3 consisted of T7 to T10. RESULTS: Of the 528 vertebrae measured, 0% of the first group vertebrae, 10.8% of the second group vertebrae, and 69.3% of the third group vertebrae followed the rule of threes. In total, 26.7% of vertebrae followed the rule of threes, whereas 62.3% of vertebrae followed Geelhoed's rule. Additional relationships worth noting include the distance between the transverse process and the adjacent caudal transverse process on the same side is approximately 25.4 mm (1 inch), and the distance between the transverse processes of the same vertebra is approximately 50.8 mm (2 inches) for male T3-T10 vertebrae and female T1-T12 vertebrae. CONCLUSION: According to our findings, the rule of threes is not as accurate anatomically as Geelhoed's rule in locating the transverse processes of the thoracic spine. This study suggests osteopathic medical schools should teach Geelhoed's rule rather than the rule of threes.

Single nucleotide seed modification restores in vivo tolerability of a toxic artificial miRNA sequence in the mouse brain.

Huntington's disease is a fatal neurodegenerative disease caused by polyglutamine-expansion in huntingtin (HTT). Recent work showed that gene silencing approaches, including RNA interference (RNAi), improve disease readouts in mice. To advance RNAi to the clinic, we designed miHDS1, with robust knockdown of human HTT and minimized silencing of unintended transcripts. In Rhesus macaque, AAV delivery of miHDS1 to the putamen reduced HTT expression with no adverse effects on neurological status including fine and gross motor skills, no immune activation and no induction of neuropathology out to 6 weeks post injection. Others showed safety of a different HTT-targeting RNAi in monkeys for 6 months. Application of miHDS1 to Huntington's patients requires further safety testing in normal rodents, despite the fact that it was optimized for humans. To satisfy this regulatory requirement, we evaluated normal mice after AAV.miHDS1 injection. In contrast to monkeys, neurological deficits occurred acutely in mice brain and was attributed to off-target silencing through interactions of miHDS1 with the 3'UTR of other transcripts. While we resolved miHDS1 toxicity in mouse brain and maintained miHDS1-silencing efficacy, these studies highlight that optimizing nucleic acid-based medicines for safety in humans presents challenges for safety testing in rodents or other distantly related species.

Functional microRNAs and target sites are created by lineage-specific transposition.

Transposable elements (TEs) account for nearly one-half of the sequence content in the human genome, and de novo germline transposition into regulatory or coding sequences of protein-coding genes can cause heritable disorders. TEs are prevalent in and around protein-coding genes, providing an opportunity to impart regulation. Computational studies reveal that microRNA (miRNA) genes and miRNA target sites reside within TE sequences, but there is little experimental evidence supporting a role for TEs in the birth of miRNAs, or as platform for gene regulation by miRNAs. In this work, we validate miRNAs and target sites derived from TE families prevalent in the human genome, including the ancient long interspersed nuclear element 2 (LINE2/L2), mammalian-wide interspersed repeat (MIR) retrotransposons and the primate-specific Alu family. We show that genes with 3' untranslated region (3' UTR) MIR elements are enriched for let-7 targets and that these sites are conserved and responsive to let-7 expression. We also demonstrate that 3' UTR-embedded Alus are a source of miR-24 and miR-122 target sites and that a subset of active genomic Alus provide for de novo target site creation. Finally, we report that although the creation of miRNA genes by Alu elements is relatively uncommon relative to their overall genomic abundance, Alu-derived miR-1285-1 is efficiently processed from its genomic locus and regulates genes with target sites contained within homologous elements. Taken together, our data provide additional evidence for TEs as a source for miRNAs and miRNA target sites, with instances of conservation through the course of mammalian evolution.