Depleting myeloid-biased haematopoietic stem cells rejuvenates aged immunity.

Ageing of the immune system is characterized by decreased lymphopoiesis and adaptive immunity, and increased inflammation and myeloid pathologies1,2. Age-related changes in populations of self-renewing haematopoietic stem cells (HSCs) are thought to underlie these phenomena3. During youth, HSCs with balanced output of lymphoid and myeloid cells (bal-HSCs) predominate over HSCs with myeloid-biased output (my-HSCs), thereby promoting the lymphopoiesis required for initiating adaptive immune responses, while limiting the production of myeloid cells, which can be pro-inflammatory4. Ageing is associated with increased proportions of my-HSCs, resulting in decreased lymphopoiesis and increased myelopoiesis3,5,6. Transfer of bal-HSCs results in abundant lymphoid and myeloid cells, a stable phenotype that is retained after secondary transfer; my-HSCs also retain their patterns of production after secondary transfer5. The origin and potential interconversion of these two subsets is still unclear. If they are separate subsets postnatally, it might be possible to reverse the ageing phenotype by eliminating my-HSCs in aged mice. Here we demonstrate that antibody-mediated depletion of my-HSCs in aged mice restores characteristic features of a more youthful immune system, including increasing common lymphocyte progenitors, naive T cells and B cells, while decreasing age-related markers of immune decline. Depletion of my-HSCs in aged mice improves primary and secondary adaptive immune responses to viral infection. These findings may have relevance to the understanding and intervention of diseases exacerbated or caused by dominance of the haematopoietic system by my-HSCs.

Artificial Intelligence for Real-Time Prediction of the Histology of Colorectal Polyps by General Endoscopists.

BACKGROUND: Real-time prediction of histologic features of small colorectal polyps may prevent resection and/or pathologic evaluation and therefore decrease colonoscopy costs. Previous studies showed that computer-aided diagnosis (CADx) was highly accurate, though it did not outperform expert endoscopists. OBJECTIVE: To assess the diagnostic performance of histologic predictions by general endoscopists before and after assistance from CADx in a real-life setting. DESIGN: Prospective, multicenter, single-group study. (ClinicalTrials.gov: NCT04437615). SETTING: 6 centers across the United States. PARTICIPANTS: 1252 consecutive patients undergoing colonoscopy and 49 general endoscopists with variable experience in real-time prediction of polyp histologic features. INTERVENTION: Real-time use of CADx during routine colonoscopy. MEASUREMENTS: The primary end points were the sensitivity and specificity of CADx-unassisted and CADx-assisted histologic predictions for adenomas measuring 5 mm or less. For clinical purposes, additional estimates according to location and confidence level were provided. RESULTS: The CADx device made a diagnosis for 2695 polyps measuring 5 mm or less (96%) in 1252 patients. There was no difference in sensitivity between the unassisted and assisted groups (90.7% vs. 90.8%; P = 0.52). Specificity was higher in the CADx-assisted group (59.5% vs. 64.7%; P < 0.001). Among all 2695 polyps measuring 5 mm or less, 88.2% and 86.1% (P < 0.001) in the CADx-assisted and unassisted groups, respectively, could be resected and discarded without pathologic evaluation. Among 743 rectosigmoid polyps measuring 5 mm or less, 49.5% and 47.9% (P < 0.001) in the CADx-assisted and unassisted groups, respectively, could be left in situ without resection. LIMITATION: Decision making based on CADx might differ outside a clinical trial. CONCLUSION: CADx assistance did not result in increased sensitivity of optical diagnosis. Despite a slight increase, the specificity of CADx-assisted diagnosis remained suboptimal. PRIMARY FUNDING SOURCE: Olympus America Corporation served as the clinical study sponsor.

Comparing methylation levels assayed in GC-rich regions with current and emerging methods.

DNA methylation is an epigenetic mechanism that regulates gene expression, and for mammals typically occurs on cytosines within CpG dinucleotides. A significant challenge for methylation detection methods is accurately measuring methylation levels within GC-rich regions such as gene promoters, as inaccuracies compromise downstream biological interpretation of the data. To address this challenge, we compared methylation levels assayed using four different Methods Enzymatic Methyl-seq (EM-seq), whole genome bisulphite sequencing (WGBS), Infinium arrays (Illumina MethylationEPIC, "EPIC"), and Oxford Nanopore Technologies nanopore sequencing (ONT) applied to human DNA. Overall, all methods produced comparable and consistent methylation readouts across the human genome. The flexibility offered by current gold standard WGBS in interrogating genome-wide cytosines is surpassed technically by both EM-seq and ONT, as their coverages and methylation readouts are less prone to GC bias. These advantages are tempered by increased laboratory time (EM-seq) and higher complexity (ONT). We further assess the strengths and weaknesses of each method, and provide recommendations in choosing the most appropriate methylation method for specific scientific questions or translational needs.

Systemic and Ovarian Impacts of Heat Stress in the Porcine Model.

Heat stress (HS) in mammals results from an imbalance in heat accumulation and dissipation. Fertility impairments consequential to HS have been recognized for decades in production animals, and more recently, observations have been extended to other species including women. There are several systemic impacts of HS that can independently affect reproduction including metabolic endotoxemia, reduced plane of nutrition and endocrine disruption. At the level of the ovary, molecular pathways are altered by HS such as inflammation, JAK-STAT, PI3K, oxidative stress, cell death and heat shock response. Taken together, impaired ovarian function contributes to seasonal infertility that results from HS. This review paper describes physiological and endocrine systemic impacts of HS that may independently and collaboratively impair fertility in the porcine model. The review then details ovarian intracellular events that are altered during HS and finally determine futures needs in this area of research.

Facile Oxidation of Ce(III) to Ce(IV) Using Cu(I) Salts.

The synthesis, luminescence, and electrochemical properties of the Ce(III) compound, [(C5Me5)2(2,6-iPr2C6H3O)Ce(THF)], 1, were investigated. Based on the electrochemical data, treatment of 1 with CuX (X = Cl, Br, I) results in the formation of the corresponding Ce(IV) complexes, [(C5Me5)2(2,6-iPr2C6H3O)Ce(X)]. Each complex has been characterized using NMR, IR, and UV-vis spectroscopy as well as structurally determined using X-ray crystallography. Additionally, the treatment of [(C5Me5)2(2,6-iPr2C6H3O)Ce(Br)] with AgF results in the formation of the putative [(C5Me5)2(2,6-iPr2C6H3O)Ce(F)]. The electronic structure of these Ce(IV)-X complexes was investigated by bond analyses and the Ce(IV)-F moiety using quantum chemistry NMR calculations.

Phenotypic, endocrinological, and metabolic effects of zearalenone exposure and additive effect of heat stress in prepubertal female pigs.

Independently, both heat stress (HS) and zearalenone (ZEN) compromise female reproduction, thus the hypothesis that ZEN would affect phenotypic, endocrine, and metabolic parameters in pigs with a synergistic and/or additive impact of HS was investigated. Prepubertal gilts (n = 6-7) were assigned to: thermoneutral (TN) vehicle control (TC; n = 6); TN ZEN (40 µg/kg; TZ; n = 6); pair-fed (PF; n = 6) vehicle control (PC; n = 6); PF ZEN (40 µg/kg; PZ; n = 6); HS vehicle control (HC; n = 7); and HS ZEN (40 µg/kg; HZ; n = 7) and experienced either constant 21.0 ± 0.10 °C (TN and PF) or 35.0 ± 0.2 °C (12 h) and 32.2 ± 0.1 °C (12 h) to induce HS for 7 d. Elevated rectal temperature (P < 0.01) and respiration rate (P < 0.01) confirmed induction of HS. Rectal temperature was decreased (P = 0.03) by ZEN. Heat stress decreased (P < 0.01) feed intake, body weight, and average daily gain, with absence of a ZEN effect (P > 0.22). White blood cells, hematocrit, and lymphocytes decreased (P < 0.04) with HS. Prolactin increased (P < 0.01) in PC and PZ and increased in HZ females (P < 0.01). 17ß-estradiol reduced (P < 0.01) in HC and increased in TZ females (P = 0.03). Serum metabolites were altered by both HS and ZEN. Neither HS nor ZEN impacted ovary weight, uterus weight, teat size or vulva area in TN and PF treatments, although ZEN increased vulva area (P = 0.02) in HS females. Thus, ZEN and HS, independently and additively, altered blood composition, impacted the serum endocrine and metabolic profile and increased vulva size in prepubertal females, potentially contributing to infertility.

Lumbar transforaminal epidural steroid injections with particulate vs. nonparticulate steroid: an evidence-informed review on shifting gear to a personalized medicine paradigm.

PURPOSE OF REVIEW: To provide an evidence-informed review weighing the pros and cons of particulate vs. nonparticulate steroids for lumbar transforaminal epidural steroid injections (TFESI). RECENT FINDINGS: The relative use of nonparticulate vs. particulate steroids for lumbar TFESI has risen recently in light of catastrophic consequences reported for the latter during cervical TFESI. Among various causes of spinal cord infarct, an exceedingly rare event in the lower lumbar spine, embolization of particulate steroid is among the least likely. Case reports have documented cases of spinal cord infarct during lower lumbar TFESI with both particulate and nonparticulate steroids, with database reviews finding no difference in complication rates. There is some evidence for superiority of particulate over nonparticulate steroids in well-designed studies, which could lead to increase steroid exposure (i.e. more injections) and treatment failure resulting in surgical and/or opioid management when nonparticulate steroids are utilized. SUMMARY: Similar to a paradigm shift in medicine, a personalized approach based on a shared decision model and the consequences of treatment failure, should be utilized in deciding which steroid to utilize. Alternatives to ESI include high-volume injections with nonsteroid solutions, and the use of hypertonic saline, which possesses anti-inflammatory properties and has been shown to be superior to isotonic saline in preliminary clinical studies.

MicroRNA21 inhibition affects porcine oocyte maturation and alters protein expression critical for metabolic pathway function.

MicroRNA21 (MIR21) abundance in porcine oocytes and cumulus cells increases during in vitro maturation. The mechanism by which MIR21 regulates oocyte maturation and the effect on the developmental competence of subsequent embryos remains unclear. The objective of this study was to assess the function of MIR21 during porcine oocyte maturation and its effect on embryonic development. Treatment with peptide nucleic acid MIR21 inhibitor (MIR21-PNA), designed to specifically bind to and prevent MIR21 activity during in vitro oocyte maturation, decreased cumulus cell expansion, and the oocyte ability to achieve metaphase II maturation stage when compared to control groups. Following parthenogenetic activation, the cleavage rate at 48 h in the MIR21-PNA group was decreased (p ≤ 0.03) relative to the control groups. Additionally, liquid chromatography-mass spectrometry (LC-MS/MS) of oocyte and cumulus cell total protein following MIR21-PNA treatment during in vitro maturation identified changes in signaling pathways with primary involvement of glucose metabolism (GM) pathways. Furthermore, there was no difference (p = 0.21) in oocyte maturation of control and MIR21-PNA treated oocytes when cultured in pyruvate lacking medium. Finally, MIR21-PNA treatment decreased (p = 0.04) glutathione and increased (p = 0.07) reactive oxygen species production in the oocyte. These data suggest that MIR21 influences porcine oocyte maturation by regulating GM pathways in the cumulus-oocyte complex.

Drug-Drug Interaction Between Orally Administered Hydrocodone-Acetaminophen and Inhalation of Cannabis Smoke: A Case Report.

Objective: To determine if a 2-day protocol measuring pharmacokinetic and pharmacodynamic characteristics can demonstrate drug-drug interactions when smoked cannabis is added to orally administered hydrocodone/acetaminophen combination products. Case Summary: A 51-year-old non-Hispanic white male with chronic pain diagnoses participated in a 2-day pilot protocol. The participant attended two 7-hour in-lab days where he received 10 blood draws each day and completed self-administered pain and anxiety surveys. For both days, the participant took his prescribed dose of hydrocodone/acetaminophen (1/2 tablet of 7.5 mg/325 mg combination product) with the addition of 1 smoked pre-rolled marijuana cigarette (labeled as 0.5 g; 22.17% Δ9-tetrahydrocannabinol; 0.12% cannabidiol) on Day 2. Blood specimens were analyzed using mass spectrometry to quantify the difference of plasma hydrocodone levels between Day 1 and Day 2. Results: Compared to Day 1, lower levels of pain and anxiety were reported during Day 2 with the addition of cannabis to oral hydrocodone/acetaminophen. Day 2 pharmacokinetic analysis also revealed more rapid absorption and overall lower levels of hydrocodone in plasma. Discussion: Lower hydrocodone plasma levels in Day 2 may indicate cannabis's effect on metabolism and reduce the risk of opioid toxicity. The quicker absorption rate of hydrocodone could explain lower pain and anxiety scores reported on the second day. Conclusion and Relevance: A 2-day protocol was able to capture differences across time in pharmacokinetic and pharmacodynamic measurements. Larger studies can be designed to better characterize the potential drug-drug interaction of cannabis and opioids.

Vaginal microbiota differences associated with pelvic organ prolapse risk during late gestation in commercial sows†.

During the last decade, sow mortality due to pelvic organ prolapse (POP) has increased. To better understand the biology associated with POP, sows were phenotypically assessed and assigned a perineal score (PS) based on presumed POP risk and categorized as PS1 (low), PS2 (moderate), or PS3 (high). The study objective was to identify changes in sow vaginal microbiota that may be associated with POP. The hypothesis is that vaginal microbiota differs between sows with variable risk for POP, and changes in microbiota during late gestation exist between sows with differing risk. Of the 2864 sows scored during gestation week 15, 1.0, 2.7, and 23.4% of PS1, PS2, and PS3 sows, respectively, subsequently experienced POP. Vaginal swabs subjected to 16S rRNA gene sequencing revealed differences in community composition (Bray-Curtis; P < 0.05) and individual operational taxonomic unit (OTU) comparisons between vaginal microbiota of PS1 and PS3 sows at gestation week 15. Further, differences (P < 0.05) in community composition and OTUs (Q < 0.05) were observed in PS3 sows that either did or did not subsequently experience POP. Differences in community structure (alpha diversity measurements; P < 0.05), composition (P < 0.05), and OTUs (Q < 0.05) were observed in gestation week 12 sows scored PS1 compared to week 15 sows scored PS1 or PS3, suggesting that sow vaginal microbiota shifts during late gestation differently as POP risk changes. Collectively, these data demonstrate that sows with greater POP risk have unique vaginal microflora, for which a better understanding could aid in the development of mitigation strategies.

The Aryl hydrocarbon receptor mediates reproductive toxicity of polychlorinated biphenyl congener 126 in rats.

Polychlorinated biphenyls (PCBs) are endocrine disrupting chemicals with documented, though mechanistically ill-defined, reproductive toxicity. The toxicity of dioxin-like PCBs, such as PCB126, is mediated via the aryl hydrocarbon receptor (AHR) in non-ovarian tissues. The goal of this study was to examine the uterine and ovarian effects of PCB126 and test the hypothesis that the AHR is required for PCB126-induced reproductive toxicity. Female Holzman-Sprague Dawley wild type (n = 14; WT) and Ahr knock out (n = 11; AHR-/-) rats received a single intraperitoneal injection of either corn oil vehicle (5 ml/kg: WT_O and AHR-/-_O) or PCB126 (1.63 mg/kg in corn oil: WT_PCB and AHR-/-_PCB) at four weeks of age. The estrous cycle was synchronized and ovary and uterus were collected 28 days after exposure. In WT rats, PCB126 exposure reduced (P < 0.05) body and ovary weight, uterine gland number, uterine area, progesterone, 17ß-estradiol and anti-Müllerian hormone level, secondary and antral follicle and corpora lutea number but follicle stimulating hormone level increased (P < 0.05). In AHR-/- rats, PCB126 exposure increased (P ≤ 0.05) circulating luteinizing hormone level. Ovarian or uterine mRNA abundance of biotransformation, and inflammation genes were altered (P < 0.05) in WT rats due to PCB126 exposure. In AHR-/- rats, the transcriptional effects of PCB126 were restricted to reductions (P < 0.05) in three inflammatory genes. These findings support a functional role for AHR in the female reproductive tract, illustrate AHR's requirement in PCB126-induced reprotoxicity, and highlight the potential risk of dioxin-like compounds on female reproduction.

Characterization of the effects of heat stress on autophagy induction in the pig oocyte.

BACKGROUND: Heat stress (HS) occurs when body heat accumulation exceeds heat dissipation and is associated with swine seasonal infertility. HS contributes to compromised oocyte integrity and reduced embryo development. Autophagy is a potential mechanism for the oocyte to mitigate the detrimental effects of HS by recycling damaged cellular components. METHODS: To characterize the effect of HS on autophagy in oocyte maturation, we utilized an in vitro maturation (IVM) system where oocytes underwent thermal neutral (TN) conditions throughout the entire maturation period (TN/TN), HS conditions during the first half of IVM (HS/TN), or HS conditions during the second half of IVM (TN/HS). RESULTS: To determine the effect of HS on autophagy induction within the oocyte, we compared the relative abundance and localization of autophagy-related proteins. Heat stress treatment affected the abundance of two well described markers of autophagy induction: autophagy related gene 12 (ATG12) in complex with ATG5 and the cleaved form of microtubule-associated protein 1 light chain 3 beta (LC3B-II). The HS/TN IVM treatment increased the abundance of the ATG12-ATG5 complex and exacerbated the loss of LC3B-II in oocytes. The B-cell lymphoma 2 like 1 protein (BCL2L1) can inhibit autophagy or apoptosis through its interaction with either beclin1 (BECN1) or BCL2 associated X, apoptosis regulator (BAX), respectively. We detected colocalization of BCL2L1 with BAX but not BCL2L1 with BECN1, suggesting that apoptosis is inhibited under the HS/TN treatment but not autophagy. Interestingly, low doses of the autophagy inducer, rapamycin, increased oocyte maturation. CONCLUSIONS: Our results here suggest that HS increases autophagy induction in the oocyte during IVM, and that artificial induction of autophagy increases the maturation rate of oocytes during IVM. These data support autophagy as a potential mechanism activated in the oocyte during HS to recycle damaged cellular components and maintain developmental competence.

Mother-child transmission of epigenetic information by tunable polymorphic imprinting.

Genomic imprinting mediated by DNA methylation restricts gene expression to a single allele determined by parental origin and is not generally considered to be under genetic or environmental influence. Here, we focused on a differentially methylated region (DMR) of approximately 1.9 kb that includes a 101-bp noncoding RNA gene (nc886/VTRNA2-1), which is maternally imprinted in ∼75% of humans. This is unlike other imprinted genes, which demonstrate monoallelic methylation in 100% of individuals. The DMR includes a CTCF binding site on the centromeric side defining the DMR boundary and is flanked by a CTCF binding site on the telomeric side. The centromeric CTCF binding site contains an A/C polymorphism (rs2346018); the C allele is associated with less imprinting. The frequency of imprinting of the nc886 DMR in infants was linked to at least two nongenetic factors, maternal age at delivery and season of conception. In a separate cohort, nc886 imprinting was associated with lower body mass index in children at 5 y of age. Thus, we propose that the imprinting status of the nc886 DMR is "tunable" in that it is associated with maternal haplotype and prenatal environment. This provides a potential mechanism for transmitting information, with phenotypic consequences, from mother to child.

PTPRN2 and PLCß1 promote metastatic breast cancer cell migration through PI(4,5)P2-dependent actin remodeling.

Altered abundance of phosphatidyl inositides (PIs) is a feature of cancer. Various PIs mark the identity of diverse membranes in normal and malignant cells. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) resides predominantly in the plasma membrane, where it regulates cellular processes by recruiting, activating, or inhibiting proteins at the plasma membrane. We find that PTPRN2 and PLCß1 enzymatically reduce plasma membrane PI(4,5)P2 levels in metastatic breast cancer cells through two independent mechanisms. These genes are upregulated in highly metastatic breast cancer cells, and their increased expression associates with human metastatic relapse. Reduction in plasma membrane PI(4,5)P2 abundance by these enzymes releases the PI(4,5)P2-binding protein cofilin from its inactive membrane-associated state into the cytoplasm where it mediates actin turnover dynamics, thereby enhancing cellular migration and metastatic capacity. Our findings reveal an enzymatic network that regulates metastatic cell migration through lipid-dependent sequestration of an actin-remodeling factor.

Inhibition of germinal vesicle breakdown using IBMX increases microRNA-21 in the porcine oocyte.

BACKGROUND: Germinal vesicle breakdown (GVBD) occurs during oocyte meiotic maturation, a period when transcriptional processes are virtually inactive. Thus, the maturing oocyte is reliant on processes such as post-transcriptional gene regulation (PTGR) to regulate the mRNA and protein repertoire. MicroRNA (miRNA) are a class of functional small RNA that target mRNA to affect their abundance and translational efficiency. Of particular importance is miRNA-21 (MIR21) due to its role in regulating programmed cell death 4 (PDCD4). The objective of this study was to characterize the abundance and regulation of MIR21 in relation to GVBD. METHODS: Oocytes were collected from aspirated porcine tertiary follicles. Relative abundance of mature MIR21 was quantified at 0, 8, 16, 24, 32, and 42 h of in vitro (IVM) with or without treatment with 3-isobutyl-1-methylxanthine (IBMX). RESULTS: IBMX increased abundance of MIR21 at 24 h approximately 30-fold compared to control oocytes (P < 0.05), and the induced increase in MIR21 abundance at 24 h was concomitant with premature depletion of PDCD4 protein abundance. To characterize the effect of artificially increasing MIR21 on oocyte competence without inhibiting GVBD, a MIR21 mimic, scrambled microRNA negative control, or nuclease free water was micro-injected into denuded oocytes at 21 h of IVM. The maturation rate of oocytes injected with synthetic MIR21 (63.0 ± 7.5%) was higher than oocytes injected with negative controls (P < 0.05). CONCLUSIONS: Inhibition of nuclear meiotic maturation via IBMX significantly increased MIR21 and decreased its target, PDCD4. Injection of a MIR21 mimic increased oocyte maturation rate. Our results indicate MIR21 is active and important during meiotic maturation of the oocyte.

Genetic and genomic characterization of vulva size traits in Yorkshire and Landrace gilts.

BACKGROUND: Reproductive performance is critical for efficient swine production. Recent results indicated that vulva size (VS) may be predictive of reproductive performance in sows. Study objectives were to estimate genetic parameters, identify genomic regions associated, and estimate genomic prediction accuracies (GPA) for VS traits. RESULTS: Heritability estimates of VS traits, vulva area (VA), height (VH), and width (VW) measurements, were moderately to highly heritable in Yorkshire, with 0.46 ± 0.10, 0.55 ± 0.10, 0.31 ± 0.09, respectively, whereas these estimates were low to moderate in Landrace, with 0.16 ± 0.09, 0.24 ± 0.11, and 0.08 ± 0.06, respectively. Genetic correlations within VS traits were very high for both breeds, with the lowest of 0.67 ± 0.29 for VH and VW for Landrace. Genome-wide association studies (GWAS) for Landrace, reveled genomic region associated with VS traits on Sus scrofa chromosome (SSC) 2 (154-157 Mb), 7 (107-110 Mb), 8 (4-6 Mb), and 10 (8-19 Mb). For Yorkshire, genomic regions on SSC 1 (87-91 and 282-287 Mb) and 5 (67 Mb) were identified. All regions explained at least 3.4% of the genetic variance. Accuracies of genomic prediction were moderate in Landrace, ranging from 0.30 (VH) to 0.61 (VA), and lower for Yorkshire, with 0.07 (VW) to 0.11 (VH). Between-breed and multi-breed genomic prediction accuracies were low. CONCLUSIONS: Our findings suggest that VS traits are heritable in Landrace and Yorkshire gilts. Genomic analyses show that major QTL control these traits, and they differ between breed. Genomic information can be used to increase genetic gains for these traits in gilts. Additional research must be done to validate the GWAS and genomic prediction results reported in our study.

Relative importance of attribute preferences for radial vs. femoral arterial access: A crowdsourcing study of healthy online-recruited volunteers.

INTRODUCTION: Percutaneous coronary intervention (PCI) is typically performed with vascular access provided by the radial or femoral artery. However, little is known about how patients value aspects of these different vascular access approaches. METHODS: Conjoint analysis is a survey-based statistical technique used in market research that helps determine how individuals value different attributes that make up a particular product or services. We utilized conjoint analysis to assess the relative importance of four attributes associated with PCI: access site, risk of bleeding, hospital stay, and radiation exposure. Participants were healthy individuals recruited by Amazon Mechanical Turk (MTURK). After completing a conjoint analysis survey, the software Conjoint.ly was used to calculate the relative importance for these four different attributes of PCI. RESULTS: The relative importance of hospital stay, radiation exposure, bleeding risk, and procedure site was 32.7% (95% CI 29.5-35.8), 27.3% (95% CI 24.8-29.8), 24.4% (95% CI 22.3-26.5), and 15.7% (95% CI 13.6-17.8), respectively. The difference between these groups was statistically significant (P-value < 0.00001). The difference between duration of hospital stay and radiation exposure was statistically significant (P-value < 0.00433). CONCLUSION: Patients undergoing PCI place largest relative value on duration of hospital stay. Access site appears the least valued attribute. These findings carry implications to guide further research on access site choices and the consent process in the context of shared decision-making.

Heat stress decreases metabolic flexibility in skeletal muscle of growing pigs.

Heat-stressed pigs experience metabolic alterations, including altered insulin profiles, reduced lipid mobilization, and compromised intestinal integrity. This is bioenergetically distinct from thermal neutral pigs on a similar nutritional plane. To delineate differences in substrate preferences between direct and indirect (via reduced feed intake) heat stress effects, skeletal muscle fuel metabolism was assessed. Pigs (35.3 ± 0.8 kg) were randomly assigned to three treatments: thermal neutral fed ad libitum (TN; 21°C, n = 8), heat stress fed ad libitum (HS; 35°C, n = 8), and TN, pair-fed/HS intake (PF; n = 8) for 7 days. Body temperature (TB) and feed intake (FI) were recorded daily. Longissimus dorsi muscle was biopsied for metabolic assays on days -2, 3, and 7 relative to initiation of environmental treatments. Heat stress increased TB and decreased FI ( P < 0.05). Heat stress inhibited incomplete fatty acid oxidation and glucose oxidation ( P < 0.05). Metabolic flexibility decreased in HS pigs compared with TN and PF controls ( P < 0.05). Both phosphofructokinase and pyruvate dehydrogenase (PDH) activities increased in PF ( P < 0.05); however, TN and HS did not differ. Heat stress inhibited citrate synthase and ß-hydroxyacyl-CoA dehydrogenase (ß-HAD) activities ( P < 0.05). Heat stress did not alter PDH phosphorylation or carnitine palmitoyltransferase 1 abundance but reduced acetyl-CoA carboxylase 1 (ACC1) protein abundance ( P < 0.05). In conclusion, HS decreased skeletal muscle fatty acid oxidation and metabolic flexibility, likely involving ß-HAD and ACC regulation.

Using CRISPR-Cas9 to Generate Gene-Corrected Autologous iPSCs for the Treatment of Inherited Retinal Degeneration.

Patient-derived induced pluripotent stem cells (iPSCs) hold great promise for autologous cell replacement. However, for many inherited diseases, treatment will likely require genetic repair pre-transplantation. Genome editing technologies are useful for this application. The purpose of this study was to develop CRISPR-Cas9-mediated genome editing strategies to target and correct the three most common types of disease-causing variants in patient-derived iPSCs: (1) exonic, (2) deep intronic, and (3) dominant gain of function. We developed a homology-directed repair strategy targeting a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) and demonstrated restoration of the retinal transcript and protein in patient cells. We generated a CRISPR-Cas9-mediated non-homologous end joining (NHEJ) approach to excise a major contributor to Leber congenital amaurosis, the IVS26 cryptic-splice mutation in CEP290, and demonstrated correction of the transcript and protein in patient iPSCs. Lastly, we designed allele-specific CRISPR guides that selectively target the mutant Pro23His rhodopsin (RHO) allele, which, following delivery to both patient iPSCs in vitro and pig retina in vivo, created a frameshift and premature stop that would prevent transcription of the disease-causing variant. The strategies developed in this study will prove useful for correcting a wide range of genetic variants in genes that cause inherited retinal degeneration.