The Varus Knee Reveals Differential Expression Patterns of miRNAs in Spared vs. Non-spared Compartments

Introduction MicroRNAs (miRNAs) function by repressing cellular protein levels to provide a sophisticated level of gene regulation that coordinates a broad spectrum of biological processes. MiRNA inhibition of mRNA translation has emerged as an important regulator of chondrogenic and osteogenic development, osteoblast, osteoclast and chondrocyte cell growth and differentiation, and tissue homeostasis in the adult skeleton. MiRNAs control many layers of regulation in adult tissues connected to both normal biological and pathologic cellular activities. The study of miRNAs in skeletal disorders is in its infancy. Osteoarthritis (OA) is a disease that progresses from degeneration of the articular cartilage to remodeling of the underlying subchondral bone over many years. While miRNAs have been identified with the inflammatory pathogenesis of rheumatoid arthritis (RA), only a few studies have been performed on OA tissue (1,2) . Here we performed a systematic analysis of the articular cartilage from varus OA knee replacements, comparing multiple tissue samples from the lateral (spared) and medial (diseased) compartments. Before proceeding to a miRNA profiling, each sample was analyzed for expression of a small set of miRNAs that have been reported in association with RA, OA and cartilage formation. These preliminary findings have identified a spectrum of changes in surface cartilage between control and diseased tissue. Methods Human tissues: 6 individual articular cartilage samples were harvested from a total of 5 osteoarthritic varus human knees. Cartilage samples were exempt from IRB review as they are discarded materials. Samples were removed with a biopsy punch and were approximately 6 x 2mm (diameter x thickness). Cartilage specimens were harvested from the more normal-appearing lateral (‘spared’) compartments and from the more OA-affected, medial compartments of the knees. This sampling technique allows direct comparison of more significantly OA-affected cartilage samples with those of lower OA grade from the same set of individuals. Knee ages ranged from 53-74 years old and averaged 65 years old. RNA and miRNA Isolation: Each osteochondral specimen was placed in RNA Later (Sigma) immediately following surgical removal, in order to preserve the integrity of the total RNA. Specimens were transported to the lab where individual samples were removed carefully with RNase-treated tools and were transferred intofresh RNA Later solution and incubated overnight at 4C to allow penetration and maximal inhibition of RNase activity. Samples were then removed from RNA Later, blotted briefly and frozen in liquid N2, and then pulverized using a Bessman tissue pulverizer (Fisher). The pulverized samples were immediately placed into Trizol (InVitrogen) and homogenized using a polytron device. Total RNA was isolated to include small RNAs of \u3e17 nucleotides, according to the manufacturer’s protocol (InVitrogen). Purified RNA was obtained using precipitated total RNAs filtered through glass columns according to the manufacturer’s protocol (Zymo Research). RNAs were reverse-transcribed into DNA using 900ng of each purified RNA sample using the TaqMan microRNA Reverse Transcription Kit (Applied Biosystems). TaqMan qPCR analysis for small RNAs was performed using the following human primer-probe sets from Applied Biosystems: hsa-miRs-: 9, 22, 27a, 29a and 34a. Human U6 was used to normalize all qPCR data and data was plotted as normalized relative values. Normalized relative values were averaged for each of the complement of medial vs. lateral samples. Results MiRs were found to be either up- or down-regulated in a manner that suggests a mechanism of de-repression of pro-inflammatory cytokine signaling and repression of pro-inflammatory events in medial vs. lateral varus knee OA cartilage samples, respectively. MiRs 9, 27a, and 29a were found to up-regulated in lateral varus knee cartilage samples vs. medial varus knee cartilage samples (Fig.1. A,C,E,F). Conversely, miRs 22 and 34a were found to upregulated in medial vs. lateral cartilage samples (Fig1, B, D). Discussion The functional characterization of global gene expression patterns through miRNAs in OA is lacking. Particularly, the roles of miRs in OA disease development, as biomarkers, and in disease outcomes are at question. A few large-scale microarray approaches have previously identified expression signatures of potential OA-involved miRNAs (2). By comparing cartilage samples that derive from more advanced (medial) vs. less advanced (lateral) OA stages in varus human knees, we seek to combine miRNA expression analysis with clinicopathologic features. MiRs -9, -22 and -34a are known to be involved in regulating pro-inflammatory events in OA. Higher levels of miRs, -9, -27a & -140 in less-affected lateral compartment cartilage are consistent with previous reports of reduced TNFa, MMP-13 & ADAMTS-5 expression events, respectively (Fig.1, F, E & A) (3,4,5). MiRs -22 and -34a have been shown to be associated with promoting tissue catabolism by their presence and are here shown to be increased in more affected medial compartment cartilage (Fig.1, B, D) (4). In addition, miR-34a deficiency has been previously shown to inhibit chondrocyte apoptosis, consistent with the lower expression level found in lateral cartilage (Fig.1, D) (6). MiR-29a was found in a previous microarray analysis to be the highest-fold down-regulated miRNA in OA vs. normal cartilage, consistent with our finding of under-expression in medial cartilage samples (Fig.1, C) (1). The goal of these studies is to begin to understand how miRNAs can both contribute to and protect against OA. Here we show that the comparison of cartilage-derived miRNAs in medial and lateral compartment pairs from the same knee may facilitate validation of candidate OA miRNAs. Significance The aims of this project are to provide an internally-controlled platform of study for the miRNAs of OA using the natural disease differences inherent in spared vs. non-spared cartilage compartments from a varus OA knee. Such efforts may provide an alternative methodology when compared to the significant barrier of obtaining age-matched, non-OA control knee cartilage. References 1.) Iliopoulus D. PLoS One. 2008;3(11):e3740. Epub 2008 Nov 17. 2.) Goldring MB. Curr Opin Rheumatol. 2011 Jul 22. [Epub]. 3.) Yu C. J Int Med Res. 2011;39(1):1-9. 4.) Alcaraz MJ. Biochem Pharmacol. 2010 Jul 1;80(1):13-21. 5.) Miyaki S. Genes Dev. 2010 Jun 1;24(11):1173-85. 6.) Abouheif MM. Rheumatology. 2010 Nov;49(11):2054-60

Randomized trial of polychromatic blue-enriched light for circadian phase shifting, melatonin suppression, and alerting responses.

Wavelength comparisons have indicated that circadian phase-shifting and enhancement of subjective and EEG-correlates of alertness have a higher sensitivity to short wavelength visible light. The aim of the current study was to test whether polychromatic light enriched in the blue portion of the spectrum (17,000 K) has increased efficacy for melatonin suppression, circadian phase-shifting, and alertness as compared to an equal photon density exposure to a standard white polychromatic light (4000 K). Twenty healthy participants were studied in a time-free environment for 7 days. The protocol included two baseline days followed by a 26-h constant routine (CR1) to assess initial circadian phase. Following CR1, participants were exposed to a full-field fluorescent light (1 × 10 14 photons/cm 2 /s, 4000 K or 17,000 K, n = 10/condition) for 6.5 h during the biological night. Following an 8 h recovery sleep, a second 30-h CR was performed. Melatonin suppression was assessed from the difference during the light exposure and the corresponding clock time 24 h earlier during CR1. Phase-shifts were calculated from the clock time difference in dim light melatonin onset time (DLMO) between CR1 and CR2. Blue-enriched light caused significantly greater suppression of melatonin than standard light ((mean ± SD) 70.9 ± 19.6% and 42.8 ± 29.1%, respectively, p \u3c 0.05). There was no significant difference in the magnitude of phase delay shifts. Blue-enriched light significantly improved subjective alertness (p \u3c 0.05) but no differences were found for objective alertness. These data contribute to the optimization of the short wavelength-enriched spectra and intensities needed for circadian, neuroendocrine and neurobehavioral regulation

Liposomal Bupivacaine in Total Knee Arthroplasty: Preliminary Results of a Two-Surgeon, Retrospective Study

INTRODUCTION: Liposomal bupivacaine (LB) is a slowly degrading preparation that provides local anesthesia for up to 72 hours. It targets the site directly responsible for pain sensation, with no associated motor blockade. In total knee arthroplasty (TKA), it may have superior outcomes to anesthesia with regional nerve block. METHODS: Our surgeons began using LB in TKA patients in 2013. All patients following each surgeon’s LB start date were the experimental group. An equivalent number of patients prior to 2013 served as the control group. All control group patients received a preoperative femoral nerve block, and all experimental group patients received peri- and intra-articular LB, delivered intraoperatively. All other surgical and anesthesia interventions were the same. We used retrospective chart review to identify patient demographics, time to first ambulation, time to discharge, and incidence of postoperative nausea and vomiting. We also recorded opioid consumption intraoperatively, in the recovery room, and on the floor. RESULTS: There were 161 patients in each group. We found no significant difference between the two groups with regard to gender, age, weight, preoperative opioid exposure, side of procedure, type of anesthesia (general vs. spinal), or ASA status. On average, LB patients consumed 29.2% less opioid after leaving the recovery room (p DISCUSSION AND CONCLUSION: With increasing focus on the cost of TKA, there is strong incentive to manage costs associated with the procedure. Additionally, all patients, regardless of comorbid conditions, can benefit from limited exposure to narcotic pain medication. Our results indicate that LB decreases total systemic opioid requirement, time to first ambulation, and time to discharge from the hospital

An analytic expression for the electronic correlation term of the kinetic functional

We propose an analytic formula for the non-local Fisher information functional, or electronic kinetic correlation term, appearing in the expression of the kinetic density functional. Such an explicit formula is constructed on the basis of well-founded physical arguments and a rigorous mathematical prescription

Post-traumatic stress disorder following childbirth: an update of current issues and recommendations for future research

Objective: This paper aimed to report the current status of research in the field of post-traumatic stress disorder following childbirth (PTSD FC), and to update the findings of an earlier 2008 paper. Background: A group of international researchers, clinicians and service users met in 2006 to establish the state of clinical and academic knowledge relating to PTSD FC. A paper identified four key areas of research knowledge at that time. Methods: Fourteen clinicians and researchers met in Oxford, UK to update the previously published paper relating to PTSD FC. The first part of the meeting focused on updating the four key areas identified previously, and the second part on discussing new and emerging areas of research within the field. Results: A number of advances have been made in research within the area of PTSD FC. Prevalence is well established within mothers, several intervention studies have been published, and there is growing interest in new areas: staff and pathways; prevention and early intervention; impact on families and children; special populations; and post-traumatic growth. Conclusion: Despite progress, significant gaps remain within the PTSD FC knowledge base. Further research continues to be needed across all areas identified in 2006, and five areas were identified which can be seen as ‘new and emerging’. All of these new areas require further extensive research. Relatively little is still known about PTSD FC

Mutations underlying Episodic Ataxia type-1 antagonize Kv1.1 RNA editing

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Scientific Reports 7 (2017): 41095, doi:10.1038/srep41095.Adenosine-to-inosine RNA editing in transcripts encoding the voltage-gated potassium channel Kv1.1 converts an isoleucine to valine codon for amino acid 400, speeding channel recovery from inactivation. Numerous Kv1.1 mutations have been associated with the human disorder Episodic Ataxia Type-1 (EA1), characterized by stress-induced ataxia, myokymia, and increased prevalence of seizures. Three EA1 mutations, V404I, I407M, and V408A, are located within the RNA duplex structure required for RNA editing. Each mutation decreased RNA editing both in vitro and using an in vivo mouse model bearing the V408A allele. Editing of transcripts encoding mutant channels affects numerous biophysical properties including channel opening, closing, and inactivation. Thus EA1 symptoms could be influenced not only by the direct effects of the mutations on channel properties, but also by their influence on RNA editing. These studies provide the first evidence that mutations associated with human genetic disorders can affect cis-regulatory elements to alter RNA editing.This work was supported by the Vanderbilt Molecular Endocrinology Training Program (T32DK007563; E.A.F.K.), a Ruth L. Kirschstein National Research Service Award (F31NS087911; E.A.F.K), a Vanderbilt Dissertation Enhancement Grant (E.A.F.K.), and the Vanderbilt Joel G. Hardman Chair in Pharmacology (R.B.E). Additional support for J.J.C.R. included NINDS (R0111223855, R01NS64259) and the Cystic Fibrosis Foundation Therapeutics (Rosent14XXO). Infrastructural support for J.J.C.R. was provided by NIGMS (P20GM103642), NIMH (G12-MD007600), and NSF (DBI 0115825, DBI 1337284)

Culex tarsalis is a competent vector species for Cache Valley virus

Background: Cache Valley virus (CVV) is a mosquito-borne orthobunyavirus endemic in North America. The virus is an important agricultural pathogen leading to abortion and embryonic lethality in ruminant species, especially sheep. The importance of CVV in human public health has recently increased because of the report of severe neurotropic diseases. However, mosquito species responsible for transmission of the virus to humans remain to be determined. In this study, vector competence of three Culex species mosquitoes of public health importance, Culex pipiens, Cx. tarsalis and Cx. quinquefasciatus, was determined in order to identify potential bridge vector species responsible for the transmission of CVV from viremic vertebrate hosts to humans. Results: Variation of susceptibility to CVV was observed among selected Culex species mosquitoes tested in this study. Per os infection resulted in the establishment of infection and dissemination in Culex tarsalis, whereas Cx. pipiens and Cx. quinquefasciatus were highly refractory to CVV. Detection of viral RNA in saliva collected from infected Cx. tarsalis provided evidence supporting its role as a competent vector. Conclusions: Our study provided further understanding of the transmission cycles of CVV and identifies Cx. tarsalis as a competent vector

Associations between Self-Reported Gastrointestinal Illness and Water System Characteristics in Community Water Supplies in Rural Alabama: A Cross-Sectional Study

Background: Community water supplies in underserved areas of the United States may be associated with increased microbiological contamination and risk of gastrointestinal disease. Microbial and health risks affecting such systems have not been systematically characterized outside outbreak investigations. The objective of the study was to evaluate associations between self-reported gastrointestinal illnesses (GII) and household-level water supply characteristics.Methods: We conducted a cross-sectional study of water quality, water supply characteristics, and GII in 906 households served by 14 small and medium-sized community water supplies in Alabama’s underserved Black Belt region.Results: We identified associations between respondent-reported water supply interruption and any symptoms of GII (adjusted odds ratio (aOR): 3.01, 95% confidence interval (CI) = 1.65– 5.49), as well as low water pressure and any symptoms of GII (aOR: 4.51, 95% CI = 2.55– 7.97). We also identified associations between measured water quality such as lack of total chlorine and any symptoms of GII (aOR: 5.73, 95% CI = 1.09–30.1), and detection of E. coli in water samples and increased reports of vomiting (aOR: 5.01, 95% CI = 1.62–15.52) or diarrhea (aOR: 7.75, 95% CI = 2.06–29.15).Conclusions: Increased self-reported GII was associated with key water system characteristics as measured at the point of sampling in a cross-sectional study of small and medium water systems in rural Alabama in 2012 suggesting that these water supplies can contribute to endemic gastro-intestinal disease risks. Future studies should focus on further characterizing and managing microbial risks in systems facing similar challenges

A prospective study of the parent–baby bond in men and women 15 months after birth

Objective: To prospectively examine the impact of parental mental health (PTSD, depression and anxiety), the couple’s relationship quality and the infant temperament on the parent–baby bond in first-time mothers and fathers. Background: Evidence suggests that poor parental mental health, difficult infant temperament and/or lower quality of the couple’s relationship may impede the parent–baby bond. However, little research has included both parents or followed these measures across time. Methods: 75 women and 66 men completed questionnaire measures during pregnancy, 3 and 15 months postpartum, assessing mental health symptoms, the parent–baby bond, the couple’s relationship and infant characteristics. The response rates at different time-points were 90%, 77% and 70%. Results: The parent–baby bond was associated with parental mental health, the couple’s relationship and infant characteristics. The most important predictors of the parent–baby bond three months postpartum for both men and women were the couple’s relationship during pregnancy and their baby’s temperament at three months. At 15 months postpartum, after accounting for the parent–baby bond at 3 months, only concurrent infant temperament remained a significant predictor for women. However, men’s bond with their baby at 15 months was predicted by their relationship with their partner in pregnancy and concurrent affective symptoms. Few significant gender differences were found, apart from women reporting more mental health symptoms than men. Conclusion: This study highlights the significance of the couple’s relationship in pregnancy and the infant’s temperament on the development of the parent–baby bond. Future research is needed to examine this in larger more representative samples