A method for simultaneous detection of small and long RNA biotypes by ribodepleted RNA-Seq

RNA sequencing offers unprecedented access to the transcriptome. Key to this is the identification and quantification of many different species of RNA from the same sample at the same time. In this study we describe a novel protocol for simultaneous detection of coding and non-coding transcripts using modifications to the Ion Total RNA-Seq kit v2 protocol, with integration of QIASeq FastSelect rRNA removal kit. We report highly consistent sequencing libraries can be produced from both frozen high integrity mouse hippocampal tissue and the more challenging post-mortem human tissue. Removal of rRNA using FastSelect was extremely efficient, resulting in less than 1.5% rRNA content in the final library. We identified > 30,000 unique transcripts from all samples, including protein-coding genes and many species of non-coding RNA, in biologically-relevant proportions. Furthermore, the normalized sequencing read count for select genes significantly negatively correlated with Ct values from qRT-PCR analysis from the same samples. These results indicate that this protocol accurately and consistently identifies and quantifies a wide variety of transcripts simultaneously. The highly efficient rRNA depletion, coupled with minimized sample handling and without complicated and high-loss size selection protocols, makes this protocol useful to researchers wishing to investigate whole transcriptomes.

Nerve-mediated amputation-induced stem cell activation primes distant appendages for future regeneration events in axolotl

Animals exhibit extreme diversity in regenerative ability. This likely reflects different, lineage-specific selective pressures in their evolutionary histories, but how specific molecular features of regenerative programs help solve species-specific challenges has not been examined in detail. Here we discover that, in the highly-regenerative axolotl salamander, a conserved, body-wide stem cell activation response triggered in response to limb removal primes undisturbed limbs for regeneration upon subsequent amputation. This response should be particularly useful to salamanders, which frequently lose limbs in response to cannibalism. We further demonstrate the body-wide response requires both peripheral nervous system input at these distant sites and mTOR signaling. We defined gene expression changes within the nerves and nearby tissues, harboring responsive stem cells, leading to identification of candidate genetic pathways influencing distant stem cell activation following amputation. Functional experimentation confirmed a requirement for adrenergic signaling in amputation-induced activation of distant stem cells. These findings reveal a direct link between systemic cellular activation responses to local tissue damage and overall regenerative ability. Similar systemic activation responses to tissue removal have been observed in animals with widely differing regenerative abilities (e.g., planaria to mice), suggesting that it is the responses downstream of these signals, likely sculpted by differing evolutionary pressures, that ultimately distinguish regenerators from non-regenerators.

Handling of hilar vasculature in living donor right hepatectomy using extrahepatic Glissonean approach

Donor hepatectomy is one of the most important procedures in LDLT because it affects the safety of donors and the outcome of the recipients. We standardized a method of securing the important vessels at the hepatic hilum while advancing the dissection toward the central direction. This research introduces our technique of handling hilar vasculature in living donor hepatectomy, using the extrahepatic Glissonean approach, and discusses its efficacy. At first, after the extrahepatic right Glissonean approach, the resected hepatic artery and portal vein are secured on the same line as with the secured the glisson. The resected hepatic artery and portal vein are followed in the central direction, and the surrounding area is dissected. The dissection is continued up to the main brunch of hepatic artery and portal vein. The bile duct can be secured by subtracting the hepatic artery and portal vein from the tape that secured the Glissonean pedicle. The bile duct, hepatic artery, and the portal vein are dissected in this order, before dissecting the right hepatic vein, completing the surgery. This method of dissection approaching the extrahepatic Glisson is carried out towards the central direction suggest to acquire minimal tissue removal and to shorten operative time. This could result in adequate perfusion to the remaining liver and donor safety, taken together effective results on recipient.

CERAMIC LAMINATE VENEERS: A 3-YEAR CLINICAL FOLLOW-UP

Ceramic laminate veneers have been widely used in esthetic restorations, with advantages such as color stability, resistance to degradation, and excellent esthetic results, all of which contribute to long-term stability. The gold standard technique consists of bonding a thin ceramic laminate veneer to enamel. The present paper describes a clinical case with 3-year follow-up. Six ceramic laminate veneers were planned, fabricated and bonded to six sound anterior maxillary teeth showing diastema, thereby improving shape and size. The procedure followed a laminate veneer treatment protocol, using a mock-up, shade selection, and an adhesive system. The case was followed for 3 years. In conclusion, minimally invasive restorations with ceramic laminate veneers remained stable after 3 years of follow-up and are a good option for patients with diastema or microdontia and requires only a minimum amount of sound tooth tissue removal.

Health professionals' perceptions of maggot debridement therapy

Background: Maggot debridement therapy (MDT), or the use of maggots in dead tissue removal, has been shown to be beneficial in wound healing. Yet MDT in the US is often only used once conventional debridement methods have failed. Method: In this study, nine health professionals, experienced in MDT, were interviewed in order to identify and analyse the perceived societal barriers to MDT acceptance and usage in the US. Results: Through qualitative analysis, using the grounded theory framework, this study found that among those interviewed, insurance reimbursement restrictions and stigmatisation of medicinal maggots were the factors driving resistance to MDT use. Conclusion: Specifically, the ‘yuck’ factor and the perception of MDT as an ‘ancient’ modality contributed towards MDT stigma; in addition, lack of outpatient insurance coverage deterred MDT use. These findings provide useful information regarding the perceptual and systemic barriers that prevent greater acceptance of MDT. Ultimately, these barriers must be understood if we are to facilitate MDT implementation and improve MDT usage in the future.