A comparison of multiple shRNA expression methods for combinatorial RNAi

RNAi gene therapies for HIV-1 will likely need to employ multiple shRNAs to counter resistant strains. We evaluated 3 shRNA co-expression methods to determine their suitability for present use; multiple expression vectors, multiple expression cassettes and single transcripts comprised of several dsRNA units (aka domains) with each being designed to a different target. Though the multiple vector strategy was effective with 2 shRNAs, the increasing number of vectors required is a major shortcoming. With single transcript configurations we only saw adequate activity from 1 of 10 variants tested, the variants being comprised of 2 - 3 different target domains. Whilst single transcript configurations have the most advantages on paper, these configurations can not yet be rapidly and reliably re-configured for new targets. However, our multiple cassette combinations of 2, 3 and 4 (29 bp) shRNAs were all successful, with suitable activity maintained in all positions and net activities comparable to that of the corresponding single shRNAs. We conclude that the multiple cassette strategy is the most suitably developed for present use as it is easy to design, assemble, is directly compatible with pre-existing shRNA and can be easily expanded

Acute and Chronic Stress Associations With Blood Pressure: An Ecological Momentary Assessment Study on an App-Based Platform

ObjectiveThis study examined the within- and between-person associations of acute and chronic stress with blood pressure (BP) and heart rate (HR) using an app-based research platform.MethodsWe examined data from 31,964 adults (aged 18-90 years) in an app-based ecological momentary assessment study that used a research-validated optic sensor to measure BP.ResultsWithin-person associations revealed that moments with (versus without) acute stress exposure were associated with higher systolic (SBP; b = 1.54) and diastolic BP (DBP; b = 0.79) and HR ( b = 1.53; p values < .001). During moments with acute stress exposure, higher acute stress severity than usual was associated with higher SBP ( b = 0.26), DBP ( b = 0.09), and HR ( b = 0.40; p values < .05). During moments without acute stress, higher background stress severity than usual was associated with higher BP and HR (SBP: b = 0.87, DBP: b = 0.51, HR: b = 0.69; p values < .001). Between-person associations showed that individuals with more frequent reports of acute stress exposure or higher chronic stress severity had higher SBP, DBP, and HR ( p values < .05). Between-person chronic stress severity moderated within-person physiological responses to stress such that individuals with higher chronic stress severity had higher average BP and HR levels but showed smaller responses to momentary stress.ConclusionsTechnological advancements with optic sensors allow for large-scale physiological data collection, which provides a better understanding of how stressors of different timescales and severity contribute to momentary BP and HR in daily life

Self-injurious behaviours are associated with alterations in the somatosensory system in children with autism spectrum disorder.

Children with autism spectrum disorder (ASD) frequently engage in self-injurious behaviours, often in the absence of reporting pain. Previous research suggests that altered pain sensitivity and repeated exposure to noxious stimuli are associated with morphological changes in somatosensory and limbic cortices. Further evidence from postmortem studies with self-injurious adults has indicated alterations in the structure and organization of the temporal lobes; however, the effect of self-injurious behaviour on cortical development in children with ASD has not yet been determined. Thirty children and adolescents (mean age = 10.6 ± 2.5 years; range 7-15 years; 29 males) with a clinical diagnosis of ASD and 30 typically developing children (N = 30, mean age = 10.7 ± 2.5 years; range 7-15 years, 26 males) underwent T1-weighted magnetic resonance and diffusion tensor imaging. No between-group differences were seen in cerebral volume, surface area or cortical thickness. Within the ASD group, self-injury scores negatively correlated with thickness in the right superior parietal lobule t = 6.3, p \u3c 0.0001, bilateral primary somatosensory cortices (SI) (right: t = 4.4, p = 0.02; left: t = 4.48, p = 0.004) and the volume of the left ventroposterior (VP) nucleus of the thalamus (r = -0.52, p = 0.008). Based on these findings, we performed an atlas-based region-of-interest diffusion tensor imaging analysis between SI and the VP nucleus and found that children who engaged in self-injury had significantly lower fractional anisotropy (r = -0.4, p = 0.04) and higher mean diffusivity (r = 0.5, p = 0.03) values in the territory of the left posterior limb of the internal capsule. Additionally, greater incidence of self-injury was associated with increased radial diffusivity values in bilateral posterior limbs of the internal capsule (left: r = 0.5, p = 0.02; right: r = 0.5, p = 0.009) and corona radiata (left: r = 0.6, p = 0.005; right: r = 0.5, p = 0.009). Results indicate that self-injury is related to alterations in somatosensory cortical and subcortical regions and their supporting white-matter pathways. Findings could reflect use-dependent plasticity in the somatosensory system or disrupted brain development that could serve as a risk marker for self-injury

Evaluating comorbidities in total hip and knee arthroplasty: available instruments

Each year millions of patients are treated for joint pain with total joint arthroplasty, and the numbers are expected to rise. Comorbid disease is known to influence the outcome of total joint arthroplasty, and its documentation is therefore of utmost importance in clinical evaluation of the individual patient as well as in research. In this paper, we examine the various methods for obtaining and assessing comorbidity information for patients undergoing joint replacement. Multiple instruments are reliable and validated for this purpose, such as the Charlson Index, Index of Coexistent Disease, and the Functional Comorbidity Index. In orthopedic studies, the Charnley classification and the American Society of Anesthesiologists physical function score (ASA) are widely used. We recommend that a well-documented comorbidity index that incorporates some aspect of mental health is used along with other appropriate instruments to objectively assess the preoperative status of the patient

SLC35A2â CDG: Functional characterization, expanded molecular, clinical, and biochemical phenotypes of 30 unreported Individuals

Pathogenic de novo variants in the Xâ linked gene SLC35A2 encoding the major Golgiâ localized UDPâ galactose transporter required for proper protein and lipid glycosylation cause a rare type of congenital disorder of glycosylation known as SLC35A2â congenital disorders of glycosylation (CDG; formerly CDGâ IIm). To date, 29 unique de novo variants from 32 unrelated individuals have been described in the literature. The majority of affected individuals are primarily characterized by varying degrees of neurological impairments with or without skeletal abnormalities. Surprisingly, most affected individuals do not show abnormalities in serum transferrin Nâ glycosylation, a common biomarker for most types of CDG. Here we present data characterizing 30 individuals and add 26 new variants, the single largest study involving SLC35A2â CDG. The great majority of these individuals had normal transferrin glycosylation. In addition, expanding the molecular and clinical spectrum of this rare disorder, we developed a robust and reliable biochemical assay to assess SLC35A2â dependent UDPâ galactose transport activity in primary fibroblasts. Finally, we show that transport activity is directly correlated to the ratio of wildâ type to mutant alleles in fibroblasts from affected individuals.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150498/1/humu23731_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150498/2/humu23731-sup-0001-Supp_Mat__2019.2.10_.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150498/3/humu23731.pd

A prospective investigation of swallowing, nutrition, and patient-rated functional impact following altered fractionation radiotherapy with concomitant boost for oropharyngeal cancer

Altered fractionation radiotherapy for head and neck cancer has been associated with improved locoregional control, overall survival, and heightened toxicity compared with conventional treatment. Swallowing, nutrition, and patient-perceived function for altered fractionation radiotherapy with concomitant boost (AFRT-CB) for T1–T3 oropharyngeal squamous cell carcinoma (SCC) have not been previously reported. Fourteen consecutive patients treated with AFRT-CB for oropharyngeal SCC were recruited from November 2006 to August 2009 in a tertiary hospital in Brisbane, Australia. Swallowing, nutrition, and patient-perceived functional impact assessments were conducted pretreatment, at 4–6 weeks post-treatment, and at 6 months post-treatment. Deterioration from pretreatment to 4–6 weeks post-treatment in swallowing, nutrition, and functional impact was evident, likely due to the heightened toxicity associated with AFRT-CB. There was significant improvement at 6 months post-treatment in functional swallowing, nutritional status, patient-perceived swallowing, and overall function, consistent with recovery from acute toxicity. However, weight and patient perception of physical function and side effects remained significantly worse than pretreatment scores. The ongoing deficits related to weight and patient-perceived outcomes at 6 months revealed that this treatment has a long-term impact on function possibly related to the chronic effects of AFRT-CB

An original phylogenetic approach identified mitochondrial haplogroup T1a1 as inversely associated with breast cancer risk in BRCA2 mutation carriers

Introduction: Individuals carrying pathogenic mutations in the BRCA1 and BRCA2 genes have a high lifetime risk of breast cancer. BRCA1 and BRCA2 are involved in DNA double-strand break repair, DNA alterations that can be caused by exposure to reactive oxygen species, a main source of which are mitochondria. Mitochondrial genome variations affect electron transport chain efficiency and reactive oxygen species production. Individuals with different mitochondrial haplogroups differ in their metabolism and sensitivity to oxidative stress. Variability in mitochondrial genetic background can alter reactive oxygen species production, leading to cancer risk. In the present study, we tested the hypothesis that mitochondrial haplogroups modify breast cancer risk in BRCA1/2 mutation carriers. Methods: We genotyped 22,214 (11,421 affected, 10,793 unaffected) mutation carriers belonging to the Consortium of Investigators of Modifiers of BRCA1/2 for 129 mitochondrial polymorphisms using the iCOGS array. Haplogroup inference and association detection were performed using a phylogenetic approach. ALTree was applied to explore the reference mitochondrial evolutionary tree and detect subclades enriched in affected or unaffected individuals. Results: We discovered that subclade T1a1 was depleted in affected BRCA2 mutation carriers compared with the rest of clade T (hazard ratio (HR) = 0.55; 95% confidence interval (CI), 0.34 to 0.88; P = 0.01). Compared with the most frequent haplogroup in the general population (that is, H and T clades), the T1a1 haplogroup has a HR of 0.62 (95% CI, 0.40 to 0.95; P = 0.03). We also identified three potential susceptibility loci, including G13708A/rs28359178, which has demonstrated an inverse association with familial breast cancer risk. Conclusions: This study illustrates how original approaches such as the phylogeny-based method we used can empower classical molecular epidemiological studies aimed at identifying association or risk modification effects.Peer reviewe

The Use of a human papillomavirus 18 promoter for tissue-specific expression in cervical carcinoma cells

The use of tissue-specific promoter elements in the treatment of cervical cancer has been explored in this paper. The P105 promoter of human papillomavirus 18 (HPV18) was utilised to direct tissue-specific expression in a number of cell types. Expression was examined in three cervical carcinoma cell lines: HeLa (HPV18 positive), SiHa (HPV16 positive), and C33A cells (HPV negative); the epithelial cell line, H1299; and the foetal fibroblast cell line, MRC5, utilising a luciferase expression vector. Expression was highest in the cervical cell lines by a factor of at least 80. The effect of a number of mutations in the P105 promoter on expression levels was examined. Three deletion constructs of the long control region (LCR) were investigated: an 800 bp fragment (LCR800), a 400 bp fragment (LCR400), and a 200 bp fragment (LCR200), as well as the full length product LCR of HPV18 (LCR1000). The LCR800 construct of the HPV18 P105 promoter had the highest level of expression in the cervical cell lines and was also highest in the HPV18-positive HeLa cell line. Site-directed mutagenesis was then employed on the LCR800 construct to create four further constructs that each had inactivating mutations in one of the four E2 bindin g sites (E2BSs). Overall, this study indicated that the LCR800 construct of the HPV18 P105 promoter could be utilised as a tissuerestricted promoter in cervical cancer cells.16 page(s

Prenatal screening for trisomy 21: a comparative performance and cost analysis of different screening strategies

Abstract Background Prenatal screening for chromosome aneuploidies have constantly been evolving, especially with the introduction of cell-free fetal DNA (cfDNA) screening in the most recent years. This study compares the performance, costs and timing of test results of three cfDNA screening implementation strategies: contingent, reflex and primary. Methods We modelled enhanced first trimester screening (eFTS) as the first-tier test in contingent or reflex strategies. cfDNA test was performed contingent on or reflex from eFTS results. A comparison was made between cfDNA screening using sequencing technology and Rolling Circle Amplification (RCA)/imaging solution. All model assumptions were based on results from previous publications or information from the Ontario prenatal screening population. Results At an eFTS risk cut-off of ≥1/1000, contingent and reflex cfDNA screening have the same detection rate (DR) (94%) for trisomy 21. Reflex cfDNA screening using RCA/Imaging solution provided the lowest false positive rate and cost. The number of women requiring genetic counselling and diagnostic testing was significantly reduced and women received their cfDNA screening result 9 days sooner compared with the contingent model. While primary cfDNA screening improved the trisomy 21 DR by 3–5%, it was more costly and more women required diagnostic testing. Conclusion Reflex cfDNA screening is the most cost-effective prenatal screening strategy. It can improve the efficiency of prenatal aneuploidy screening by reducing the number of patient visits and providing more timely results