Intrinsically Unstructured Sequences in the mRNA 3' UTR Reduce the Ability of Poly(A) Tail to Enhance Translation.

The 5' cap and 3' poly(A) tail of mRNA are known to synergistically stimulate translation initiation via the formation of the cap•eIF4E•eIF4G•PABP•poly(A) complex. Most mRNA sequences have an intrinsic propensity to fold into extensive intramolecular secondary structures that result in short end-to-end distances. The inherent compactness of mRNAs might stabilize the cap•eIF4E•eIF4G•PABP•poly(A) complex and enhance cap-poly(A) translational synergy. Here, we test this hypothesis by introducing intrinsically unstructured sequences into the 5' or 3' UTRs of model mRNAs. We found that the introduction of unstructured sequences into the 3' UTR, but not the 5' UTR, decreases mRNA translation in cell-free wheat germ and yeast extracts without affecting mRNA stability. The observed reduction in protein synthesis results from the diminished ability of the poly(A) tail to stimulate translation. These results suggest that base pair formation by the 3' UTR enhances the cap-poly(A) synergy in translation initiation.

Genetic quality: a complex issue for experimental study reproducibility.

Laboratory animal research involving mice, requires consideration of many factors to be controlled. Genetic quality is one factor that is often overlooked but is essential for the generation of reproducible experimental results. Whether experimental research involves inbred mice, spontaneous mutant, or genetically modified strains, exercising genetic quality through careful breeding, good recordkeeping, and prudent quality control steps such as validation of the presence of mutations and verification of the genetic background, will help ensure that experimental results are accurate and that reference controls are representative for the particular experiment. In this review paper, we will discuss various techniques used for the generation of genetically altered mice, and the different aspects to be considered regarding genetic quality, including inbred strains and substrains used, quality check controls during and after genetic manipulation and breeding. We also provide examples for when to use the different techniques and considerations on genetic quality checks. Further, we emphasize on the importance of establishing an in-house genetic quality program.

ICLAS LAQ Network for the Promotion of Animal Quality in Research.

ICLAS Laboratory Animal Quality Network (LAQN) programs currently consist of the Performance Evaluation Program (PEP), which focuses on microbial monitoring by and for laboratory animal diagnostic laboratories, and the Genetic Reference Monitoring Program (GENRef), which provides assay-ready reference DNA for genetic testing of mouse strains. Since 2008, PEP has grown to become a truly international program with participating laboratories in 5 continents. Launched in 2016, GENRef currently distributes DNA from 12 common inbred mouse strains for use in genetic monitoring of locally inbred colonies as well as for genetic testing of stocks, particularly genetically engineered stocks, of uncertain origins. GENRef has the capacity to include additional strains as well as additional species. PEP and GENRef provide the reagents at cost, as a resource to the international scientific community, in the interest of improving research quality in an environment of growing concern for research quality, rigor, and reproducibility.

Human C6orf211 encodes Armt1, a protein carboxyl methyltransferase that targets PCNA and is linked to the DNA damage response.

Recent evidence supports the presence of an L-glutamyl methyltransferase(s) in eukaryotic cells, but this enzyme class has been defined only in certain prokaryotic species. Here, we characterize the human C6orf211 gene product as "acidic residue methyltransferase-1" (Armt1), an enzyme that specifically targets proliferating cell nuclear antigen (PCNA) in breast cancer cells, predominately methylating glutamate side chains. Armt1 homologs share structural similarities with the SAM-dependent methyltransferases, and negative regulation of activity by automethylation indicates a means for cellular control. Notably, shRNA-based knockdown of Armt1 expression in two breast cancer cell lines altered survival in response to genotoxic stress. Increased sensitivity to UV, adriamycin, and MMS was observed in SK-Br-3 cells, while in contrast, increased resistance to these agents was observed in MCF7 cells. Together, these results lay the foundation for defining the mechanism by which this post-translational modification operates in the DNA damage response (DDR).

The influence of sequential debridement in total knee arthroplasty on the flexion axis of the knee using computer-aided navigation.

The effects of osteophyte debridement, bony cuts, and soft tissue releases on the functional flexion axis of the knee can be assessed by evaluating 3D kinematics following each step of a total knee arthroplasty. Using a navigated knee system with dedicated software, the functional flexion axis (helical axis) can be determined after each step. Five paired fresh-frozen cadaveric knees were used with a CT scan performed on each specimen identifying implanted fiducial markers. Kinematics data were recorded during each step of sequential osseous cuts and soft tissue releases for both an unloaded and loaded limb by each of three surgeons. The functional helical (flexion/extension) axis was identified for all specimens. The internal/external rotation angle (theta) of the helical axis differed from the transepicondylar axis by -8.3 degrees to +6.7 degrees for the unloaded condition. theta ranged from -7.2 degrees to +7.4 degrees with distraction. Soft tissue releases had no effect on theta; until a bony cut of the articular surface, which increased theta from -0.3 degrees to +9.7 degrees. Implantation of cruciate retaining prosthetic components subsequently reduced the theta range -7.3 degrees to +4.0 degrees. Thus, soft tissue releases had minimal effect on theta of the helical axis except for resection of the proximal tibia. Implantation of the CR prosthesis reduced è close to that of the intact knee. In a minority of knees, the helical axis did not coincide exactly with the transepicondylar axis. Interspecimen and left/right variability of theta were significant, although interinvestigator variability and an applied distraction force were insignificant.

Subjective and functional outcomes following surgical repair of complete ruptures of the proximal hamstring complex.

Complete ruptures of the proximal hamstring tendon insertion are less common than typical muscle tendon junction strain injuries, with a far more debilitating natural history. The present study was designed to assess the subjective and functional outcomes of a group of patients following surgical repair of both acute and chronic complete proximal hamstring tendon ruptures. Twenty-five patients with an average age of 44 years comprised the study cohort. Clinical and diagnostic tests confirmed the diagnosis. All patients underwent primary repair of the tendon to the ischium with suture anchors and were protected postoperatively for 4 to 6 weeks. Following a progressively phased strengthening and functional exercise program, strength was isokinetically assessed. The Proximal Hamstring Injury Questionnaire was completed by all patients. All patients reported being satisfied with their outcomes following surgical repair. Strength scores for patients who were 6 months to <12 months (N=10) postoperative averaged an 87% (range, 69%-108%) strength return. Patients who were =or> 12 months postoperative (N=15) averaged a 98% strength return (range, 72%-176%). Patients sustaining a severe injury to the hamstring muscle caused by an external load should be suspected of having a proximal hamstring tendon disruption. Magnetic resonance imaging examination is necessary to confirm the diagnosis and assess the pathologic anatomy. Surgical repair of both acute and chronic cases yielded good results with a high degree of patient satisfaction and objective functional recovery.

Fatigue performance of composite analogue femur constructs under high activity loading.

Synthetic mechanical analogue bone models are valuable tools for consistent analysis of implant performance in both equilibrium and fatigue biomechanical testing. Use of these models has previously been limited by the poor fatigue performance when tested under realistic service loads. An objective was to determine whether a new analogue bone model (Fourth-Generation) using enhanced analogue cortical bone provides significantly improved resistance to high load fracture and fatigue as compared to the current (Third-Generation) bone models in clinically relevant in situ type testing of total hip implants. Six Third-Generation and six Fourth-Generation mechanical analogue proximal femur models were implanted with a cemented mock hip arthroplasty. Each specimen was loaded at 5 Hz in simulated one-legged stance under load control with a maximum compressive load of 2670 N and load ratio of 0.1. Average complete structural failure in Third-Generation femurs occurred at 3.16 million cycles; all specimens exhibited substantial displacement and crazing at well below 3 million cycles. In contrast, all Fourth-Generation femurs sustained 10 million cycles without complete structural failure and showed little change in actuator deflection. The Fourth-Generation femur model performance was sufficient to allow the model to be used in biomechanically relevant load bearing levels with an intramedullary device without model compromise that would affect test results.