Rebuttal to the article Pathological crystal structures.

A section in the Acta Crystallographica Section C article by Raymond & Girolami [Acta Cryst. (2023), C79, 445-455] stated that the product of the reaction of [(Cp*Rh)2(µ-OH)3]+ (Cp* is 1,2,3,4,5-pentamethylcyclopentadiene) with 1-methylthymine (1-MT) at pH 10 and 60 °C, to synthesize the anionic component [RhI(η1-N3-1-MT)2]-, was not an RhI complex, but rather an AgI complex, due to the use of silver triflate (AgOTf) to remove Cl- from [Cp*RhCl2]2 to synthesize [Cp*Rh(H2O)3](OTf)2, a water-soluble crystalline complex. We will clearly show that this premise, as stated, is invalid, while the authors have simply avoided several important facts, including that Cp*OH, a reductive elimination product, at pH 10 and 60 °C, was unequivocally identified, thus leading to the RhI anionic component [RhI(η1-N3-1-MT)2]-. More importantly, AgOH, from the reaction of NaOH at pH 10 with any potentially remaining AgOTf, after the AgCl was filtered off, would be insoluble in water. Furthermore, a control experiment with the inorganic complex Rh(OH)3, reacting with 1-methylthymine at pH 10, provided no product, and this bodes well for a similar fate with AgOTf and 1-methylthymine, i.e. at pH 10, AgOTf would again be converted to the water-insoluble AgOH; therefore, no reaction would occur! Finally, a 1H NMR spectroscopy experiment was carried out with synthesized and crystallized [Cp*Rh(H2O)3](OTf)2 in D2O at various pD values; at pD 8.65 no reaction took place, while at pD 13.6, and at 60 °C for 2 h, a reductive elimination reaction caused the precipitation of Cp*OH. The subsequent 1H NMR spectrum clearly demonstrated, in the absence of any AgI complexes, that the solution structure and the X-ray crystals in D2O were similar. A postulated mechanism for this novel anionic component structure, as published previously [Smith et al. (2014). Organometallics, 33, 2389-2404], will be presented, along with the experimental data, to insure the credibility of our results. We will also answer the comments in the response of Drs Raymond and Girolami to this rebuttal.

MANAGEMENT OF LARGE FULL-THICKNESS MACULAR HOLES: Long-Term Outcomes of Internal Limiting Membrane Flaps and Internal Limiting Membrane Peels.

BACKGROUND/PURPOSE: To determine and compare the efficacy of a surgical internal limiting membrane (ILM) flap technique with the traditional ILM peel on long-term visual and anatomical outcomes for large (>400 µm) full-thickness macular holes. METHODS: From October 2016 to July 2022, patients undergoing initial full-thickness macular hole repair with the ILM flap or ILM peel technique were reviewed. Final outcomes were recorded and based on size in microns: 401 to 800, 801 to 1,200, and >1,200. RESULTS: Patients treated with ILM flap (n = 52, 94.2% closure rate) or ILM peel (n = 407, 93.6% closure rate) were followed with a mean follow-up time of 15.0 ± 10.2 and 20.0 ± 13.4 months, respectively. Success rates for ILM flaps and ILM peels were compared for full-thickness macular holes of 401 to 800 (100%, 95.8%, P = 0.39), 801 to 1,200 (95%, 93%, P = 0.74), and >1,200 (86.7%, 86.7%, P = 1.0) µm. Mean best-recorded logarithm of the minimal angle of resolution visual acuity for ILM flaps and ILM peels, respectively, was 1.02 ± 0.46 and 0.87 ± 0.47 preoperatively, with follow-up acuity of 0.48 ± 0.32 (P < 0.03) and 0.39 ± 0.42 (P < 0.01) at Year 3. CONCLUSION: Both techniques provide a similar anatomical closure rate and functional improvement in vision. Comparisons should be cautiously made based on difference in preoperative hole size.

Adverse pregnancy outcomes in pregnant women with chronic kidney disease: A systematic review and meta-analysis.

BACKGROUND: Chronic kidney disease (CKD) is associated with an increased risk of adverse pregnancy outcomes, but the risk at different stages of CKD (defined by estimated glomerular filtration rate, eGFR) compared with women without CKD has not been quantified in large cohorts. OBJECTIVES: To quantify the association between CKD and adverse pregnancy outcomes according to CKD definition, CKD stage and presence or absence of diabetes. SEARCH STRATEGY: A systematic search of EMBASE and MEDLINE from inception to 5 January 2023. SELECTION CRITERIA: English-language randomised controlled trials as well as cohort and case-control studies investigating adverse pregnancy outcomes in pregnant women with CKD. DATA COLLECTION AND ANALYSIS: Two reviewers conducted independent data extractions. A random-effects model was used to estimate risk. MAIN RESULTS: We included 19 studies with 3 251 902 women. Defining CKD using eGFR or serum creatinine produced results with greater effect size but wider confidence intervals. Compared with CKD stages 1-2, women with CKD stages 3-5 have a greater risk, but also greater imprecision in the risk estimate, of the following outcomes: pre-eclampsia (OR 55.18, 95% CI 2.63-1157.68, vs OR 24.74, 95% CI 1.75-348.70), preterm birth (OR 20.24, 95% CI 2.85-143.75, vs OR 8.18, 95% CI 1.54-43.46) and neonatal intensive care unit admission (OR 19.32, 95% CI 3.07-121.68, vs OR 9.77, 95% CI 2.49-38.39). Women with diabetic kidney disease, compared with women without diabetic kidney disease, have higher risks of maternal mortality, small-for-gestational-age neonates, pre-eclampsia and gestational hypertension. CONCLUSIONS: There is heterogeneity in the definition of CKD in pregnancy. Future studies should consider ways to standardise its definition and measurement in pregnancy.

Composition of thrombi in zebrafish: similarities and distinctions with mammals.

BACKGROUND: Blood clots are primarily composed of red blood cells (RBCs), platelets/thrombocytes, and fibrin. Despite the similarities observed between mammals and zebrafish, the composition of fish thrombi is not as well known. OBJECTIVES: To analyze the formation of zebrafish blood clots ex vivo and arterial and venous thrombi in vivo. METHODS: Transgenic zebrafish lines and laser-mediated endothelial injury were used to determine the relative ratio of RBCs and thrombocytes in clots. Scanning electron and confocal microscopy provided high-resolution images of the structure of adult and larval clots. Adult and larval thrombocyte spreading on fibrinogen was evaluated ex vivo. RESULTS: RBCs were present in arterial and venous thrombi, making up the majority of cells in both circulations. However, bloodless mutant fish demonstrated that fibrin clots can form in vivo in the absence of blood cells. Scanning electron and confocal microscopy showed that larval and adult zebrafish thrombi and mammalian thrombi look surprisingly similar externally and internally, even though the former have nucleated RBCs and thrombocytes. Although adult thrombocytes spread on fibrinogen, we found that larval cells do not fully activate without the addition of plasma from adult fish, suggesting a developmental deficiency of a plasma activating factor. Finally, mutants lacking αIIbß3 demonstrated that this integrin mediates thrombocyte spreading on fibrinogen. CONCLUSION: Our data showed strong conservation of arterial and venous and clot/thrombus formation across species, including developmental regulation of thrombocyte function. This correlation supports the possibility that mammals also do not absolutely require circulating cells to form fibrin clots in vivo.

Demographic and Socioeconomic Factors in Prospective Retina-Focused Clinical Trial Screening and Enrollment.

Historically marginalized populations are disproportionately affected by many diseases that commonly affect the retina, yet they have been traditionally underrepresented in prospective clinical trials. This study explores whether this disparity affects the clinical trial enrollment process in the retina field and aims to inform future trial recruitment and enrollment. Age, gender, race, ethnicity, preferred language, insurance status, social security number (SSN) status, and median household income (estimated using street address and zip code) for patients referred to at least one prospective, retina-focused clinical trial at a large, urban, retina-based practice were retrospectively extracted using electronic medical records. Data were collected for the 12-month period from 1 January 2022, through 31 December 2022. Recruitment status was categorized as Enrolled, Declined, Communication (defined as patients who were not contacted, were contacted with no response, were waiting for a follow-up, or were scheduled for screening following a clinical trial referral.), and Did Not Qualify (DNQ). Univariable and multivariable analyses were used to determine significant relationships between the Enrolled and Declined groups. Among the 1477 patients, the mean age was 68.5 years old, 647 (43.9%) were male, 900 (61.7%) were White, 139 (9.5%) were Black, and 275 (18.7%) were Hispanic. The distribution of recruitment status was: 635 (43.0%) Enrolled, 232 (15.7%) Declined, 290 (19.6%) Communication, and 320 (21.7%) DNQ. In comparing socioeconomic factors between the Enrolled and Declined groups, significant odds ratios were observed for age (p < 0.02, odds ratio (OR) = 0.98, 95% confidence interval (CI) [0.97, 1.00]), and between patients who preferred English versus Spanish (p = 0.004, OR = 0.35, 95% CI [0.17, 0.72]. Significant differences between the Enrolled and Declined groups were also observed for age (p < 0.05), ethnicity (p = 0.01), preferred language (p < 0.05), insurance status (p = 0.001), and SSN status (p < 0.001). These factors may contribute to patient participation in retina-focused clinical trials. An awareness of these demographic and socioeconomic disparities may be valuable to consider when attempting to make clinical trial enrollment an equitable process for all patients, and strategies may be useful to help address these challenges.

The XRN1-regulated RNA helicase activity of YTHDC2 ensures mouse fertility independently of m6A recognition.

The functional consequence of N6-methyladenosine (m6A) RNA modification is mediated by "reader" proteins of the YTH family. YTH domain-containing 2 (YTHDC2) is essential for mammalian fertility, but its molecular function is poorly understood. Here, we identify U-rich motifs as binding sites of YTHDC2 on 3' UTRs of mouse testicular RNA targets. Although its YTH domain is an m6A-binder in vitro, the YTH point mutant mice are fertile. Significantly, the loss of its 3'→5' RNA helicase activity causes mouse infertility, with the catalytic-dead mutation being dominant negative. Biochemical studies reveal that the weak helicase activity of YTHDC2 is enhanced by its interaction with the 5'→3' exoribonuclease XRN1. Single-cell transcriptomics indicate that Ythdc2 mutant mitotic germ cells transition into meiosis but accumulate a transcriptome with mixed mitotic/meiotic identity that fail to progress further into meiosis. Finally, our demonstration that ythdc2 mutant zebrafish are infertile highlights its conserved role in animal germ cell development.

Venous Thrombosis and Thrombocyte Activity in Zebrafish Models of Quantitative and Qualitative Fibrinogen Disorders.

Venous thrombosis occurs in patients with quantitative and qualitative fibrinogen disorders. Injury-induced thrombosis in zebrafish larvae has been used to model human coagulopathies. We aimed to determine whether zebrafish models of afibrinogenemia and dysfibrinogenemia have different thrombotic phenotypes. Laser injuries were used to induce venous thrombosis and the time-to-occlusion (TTO) and the binding and aggregation of fluorescent Tg(itga2b:EGFP) thrombocytes measured. The fga-/- larvae failed to support occlusive venous thrombosis and showed reduced thrombocyte binding and aggregation at injury sites. The fga+/- larvae were largely unaffected. When genome editing zebrafish to produce fibrinogen Aα R28C, equivalent to the human Aα R35C dysfibrinogenemia mutation, we detected in-frame skipping of exon 2 in the fga mRNA, thereby encoding AαΔ19-56. This mutation is similar to Fibrinogen Montpellier II which causes hypodysfibrinogenemia. Aα+/Δ19-56 fish had prolonged TTO and reduced thrombocyte activity, a dominant effect of the mutation. Finally, we used transgenic expression of fga R28C cDNA in fga knock-down or fga-/- mutants to model thrombosis in dysfibrinogenemia. Aα R28C expression had similar effects on TTO and thrombocyte activity as Aα+/Δ19-56. We conclude that thrombosis assays in larval zebrafish can distinguish between quantitative and qualitative fibrinogen disorder models and may assist in anticipating a thrombotic phenotype of novel fibrinogen mutations.

Transcatheter aortic valve replacement after chest radiation: A propensity-matched analysis.

BACKGROUND: Chest radiation therapy (CRT) for malignant thoracic neoplasms is associated with development of valvular heart disease years later. As previous radiation exposure can complicate surgical treatment, transcatheter aortic valve replacement (TAVR) has emerged as an alternative. However, outcomes data are lacking for TAVR patients with a history of CRT. METHODS: We conducted a retrospective study of all patients who underwent a TAVR procedure at a single institution between September 2012 and November 2018. Among 1341 total patients, 50 had previous CRT. These were propensity-matched in a 1:2 ratio to 100 patients without history of CRT. Thirty-day adverse events were analyzed with generalized estimating equation models. Overall mortality was analyzed with stratified Cox regression modelling. RESULTS: Median clinical follow-up was 24 months (interquartile range [IQR], 12-44 months). There was no difference between CRT and non-CRT patients in overall mortality (hazard ratio [HR] 0.84 [0.37-1.90], P = 0.67), 30-day mortality (HR 3.1 [0.49-20.03], P = 0.23), or 30-day readmission rate (HR 1.0 [0.43-2.31], P = 1). There were no differences in the rates of most adverse events, but patients with CRT history had higher rates of postprocedural respiratory failure (HR 3.63 [1.32-10.02], P = 0.01) and permanent pacemaker implantation (HR 2.84 [1.15-7.01], P = 0.02). CONCLUSIONS: For patients with aortic valve stenosis and previous CRT, TAVR is safe and effective, with outcomes similar to those in the general aortic stenosis population. Patients with history of CRT are more likely to have postprocedural respiratory failure and to require permanent pacemaker implantation.

Methods to Investigate miRNA Function: Focus on Platelet Reactivity.

MicroRNAs (miRNAs) are small noncoding RNAs modulating protein production. They are key players in regulation of cell function and are considered as biomarkers in several diseases. The identification of the proteins they regulate, and their impact on cell physiology, may delineate their role as diagnostic or prognostic markers and identify new therapeutic strategies. During the last 3 decades, development of a large panel of techniques has given rise to multiple models dedicated to the study of miRNAs. Since plasma samples are easily accessible, circulating miRNAs can be studied in clinical trials. To quantify miRNAs in numerous plasma samples, the choice of extraction and purification techniques, as well as normalization procedures, are important for comparisons of miRNA levels in populations and over time. Recent advances in bioinformatics provide tools to identify putative miRNAs targets that can then be validated with dedicated assays. In vitro and in vivo approaches aim to functionally validate candidate miRNAs from correlations and to understand their impact on cellular processes. This review describes the advantages and pitfalls of the available techniques for translational research to study miRNAs with a focus on their role in regulating platelet reactivity.

Chemical Modulators of Fibrinogen Production and Their Impact on Venous Thrombosis.

Thrombosis is a leading cause of morbidity and mortality. Fibrinogen, the soluble substrate for fibrin-based clotting, has a central role in haemostasis and thrombosis and its plasma concentration correlates with cardiovascular disease event risk and a prothrombotic state in experimental models. We aimed to identify chemical entities capable of changing fibrinogen production and test their impact on experimental thrombosis. A total of 1,280 bioactive compounds were screened for their ability to alter fibrinogen production by hepatocyte-derived cancer cells and a selected panel was tested in zebrafish larvae. Anthralin and all-trans retinoic acid (RA) were identified as fibrinogen-lowering and fibrinogen-increasing moieties, respectively. In zebrafish larvae, anthralin prolonged laser-induced venous- occlusion times and reduced thrombocyte accumulation at injury sites. RA had opposite effects. Treatment with RA, a nuclear receptor ligand, increased fibrinogen mRNA levels. Using an antisense morpholino oligonucleotide to deplete zebrafish fibrinogen, we correlated a shortening of laser-induced venous thrombosis times with RA treatment and fibrinogen protein levels. Anthralin had little effect on fibrinogen mRNA in zebrafish larvae, despite leading to lower detectable fibrinogen. Therefore, we made a proteomic scan of anthralin-treated cells and larvae. A reduced representation of proteins linked to the canonical secretory pathway was detected, suggesting that anthralin affects protein secretion. In summary, we found that chemical modulation of fibrinogen levels correlates with measured effects on experimental venous thrombosis and could be investigated as a therapeutic avenue for thrombosis prevention.

A genetic modifier of venous thrombosis in zebrafish reveals a functional role for fibrinogen AαE in early hemostasis.

Plasma fibrinogen molecules comprise 2 copies of Aα, Bß, and γ chains folded into a hexameric protein. A minor fibrinogen isoform with an extended Aα chain (AαE) is more abundant in newborn human blood than in adults. Larval zebrafish produce predominantly AαE-containing fibrinogen, but its functional significance is unclear. In 3-day-old zebrafish, when hemostasis is reliant on fibrinogen and erythrocyte-rich clotting but is largely thrombocyte-independent, we measured the time to occlusion (TTO) in a laser-induced venous thrombosis assay in 3 zebrafish strains (AB, TU, and AB × TL hybrids). AB larvae showed delayed TTO compared with the TU and AB × TL strains. Mating AB with TU or TL produced larvae with a TU-like TTO. In contrast to TU, AB larvae failed to produce fibrinogen AαE, due to a mutation in the AαE-specific coding region of fibrinogen α-chain gene (fga). We investigated whether the lack of AαE explained the delayed AB TTO. Transgenic expression of AαE, but not Aα, shortened the AB TTO to that of TU. AαE rescued venous occlusion in fibrinogen mutants or larvae with morpholino-targeted fibrinogen α-chain messenger RNA, but Aα was less effective. In 5-day-old larvae, circulating thrombocytes contribute to hemostasis, as visualized in Tg(itga2b:EGFP) transgenics. Laser-induced venous thrombocyte adhesion and aggregation is reduced in fibrinogen mutants, but transgenic expression of Aα or AαE restored similar thrombocyte accumulation at the injury site. Our data demonstrate a genetic modifier of venous thrombosis and a role for fibrinogen AαE in early developmental blood coagulation, and suggest a link between differentially expressed fibrinogen isoforms and the cell types available for clotting.

Metabolite Profiling of Javanese Ginger Zingiber purpureum and Identification of Antiseizure Metabolites via a Low-Cost Open-Source Zebrafish Bioassay-Guided Isolation.

The rhizomes of Zingiber purpureum, "Bangle", were investigated for its antiseizure properties using a streamlined and cost-effective zebrafish screening strategy and a mouse epilepsy assay. Its hexane extract demonstrated strong antiseizure activity in zebrafish epilepsy assay and was, therefore, selected for bioactivity-guided fractionation. Twelve compounds (1-12) were isolated, and two bioactive phenylbutenoids, trans- (11) and cis-banglene (12), reduced up to 70% of pentylenetetrazole (PTZ)-induced seizures. These compounds showed moderate activity against PTZ-induced seizures in a mouse epilepsy assay. To understand the specificity of Z. purpureum active compounds, its chemical profile was compared to that of Z. officinale. Their composition was assessed by differential metabolite profiling visualized by a molecular network, which revealed only vanillin derivatives and terpenoids as common metabolites and gave a comprehensive view of Z. purpureum composition. This study demonstrates the efficacy of a streamlined zebrafish epilepsy assay, which is therefore suitable for routine screening in phytochemistry laboratories.

MicroRNA-126 is a regulator of platelet-supported thrombin generation.

Circulating microRNA (miRNA) expression profiles correlate with platelet reactivity. MiR-126 is a promising candidates in this regard. We generated a transgenic zebrafish line with thrombocyte-specific overexpression of miR-126. Laser injury of the posterior cardinal vein of 5 day-old larvae was performed with or without antithrombotic pre-treatment. Platelet-like structures (PLS) derived from human megakaryocytes transfected with miR-126 were also evaluated for procoagulant activity. Finally, we studied the correlation between miR-126 level and thrombin generation markers in a cohort of stable cardiovascular patients. Control zebrafish developed small thrombocyte-rich thrombi at the site of vessel injury, without vessel occlusion. The miR-126 transgenic line developed an occluding thrombus in 75% (95% CI: 51-91%) of larvae. Pre-treatment with the direct thrombin inhibitor argatroban, but not aspirin, prevented vessel occlusion in the transgenic line (0% occlusion, 95%CI: 0-18%). Upon activation, human PLS showed an increased procoagulant profile after miR-126 transfection compared to control. Finally, the plasma levels of miR-126, but not a control platelet-derived miRNA, correlated with markers of in vivo thrombin generation in a cohort of 185 cardiovascular patients. Our results from three complementary approaches support a key role for miR-126 in platelet-supported thrombin generation and open new avenues in the tailoring of antithrombotic treatment.

Loss to Follow-Up Among Patients With Proliferative Diabetic Retinopathy in Clinical Practice.

PURPOSE: To determine the proportion of patients with proliferative diabetic retinopathy (PDR) who were counted as loss to follow-up (LTFU) patients and to investigate predictive factors. DESIGN: Retrospective cohort study. METHODS: Information was collected for 4,423 patients with PDR between April 30, 2012, and April 30, 2017. Two definitions of LTFU were used. Complete LTFU referred to the population who never returned to care within the study period. Interval LTFU referred to the population who did not adhere to clinical recommendations and missed scheduled appointments, resulting in intervals longer than 6 months or 1 year between 2 appointments. Age, average gross income, and insurance were assessed as potential predictors of interval LTFU. RESULTS: Among 4,423 patients with PDR, 2,407 (54.4%) and 2,320 (52.4%) were complete LTFU at 6 months and 1 year, respectively; 782 (17.7%) and 468 (10.6%) patients were interval LTFU for 6 months and 1 year, respectively. Age and average gross income were not found to be significant predictors of interval LTFU. Compared to self-pay, government and private insurance patients were more likely to be interval LTFU at 6 months (government, P = .035; private, P = .005). Private insurance patients were also more likely to be interval LTFU at 1 year (P = .003). CONCLUSIONS: The identified complete LTFU rates were notably high and warrant further study. More than 1 of 6 patients were interval LTFU for at least 6 months, and 1 of 10 patients was interval LTFU for more than 1 year. Insurance status was significant in determining interval LTFU status. Consistent with other analyses, these results indicate that compliance with clinical appointments among patients with PDR is a substantial clinical challenge.

Fibrin(ogen) in human disease: both friend and foe.

Fibrinogen is an abundant protein synthesized in the liver, present in human blood plasma at concentrations ranging from 1.5-4 g/L in healthy individuals with a normal half-life of 3-5 days. With fibrin, produced by thrombin-mediated cleavage, fibrinogen plays important roles in many physiological processes. Indeed, the formation of a stable blood clot, containing polymerized and cross-linked fibrin, is crucial to prevent blood loss and drive wound healing upon vascular injury. A balance between clotting, notably the conversion of fibrinogen to fibrin, and fibrinolysis, the proteolytic degradation of the fibrin mesh, is essential. Disruption of this equilibrium can cause disease in distinct manners. While some pathological conditions are the consequence of altered levels of fibrinogen, others are related to structural properties of the molecule. The source of fibrinogen expression and the localization of fibrin(ogen) protein also have clinical implications. Low levels of fibrinogen expression have been detected in extra-hepatic tissues, including carcinomas, potentially contributing to disease. Fibrin(ogen) deposits at aberrant sites including the central nervous system or kidney, can also be pathological. In this review, we discuss disorders in which fibrinogen and fibrin are implicated, highlighting mechanisms that may contribute to disease.

KIAA1109 Variants Are Associated with a Severe Disorder of Brain Development and Arthrogryposis.

Whole-exome and targeted sequencing of 13 individuals from 10 unrelated families with overlapping clinical manifestations identified loss-of-function and missense variants in KIAA1109 allowing delineation of an autosomal-recessive multi-system syndrome, which we suggest to name Alkuraya-Kucinskas syndrome (MIM 617822). Shared phenotypic features representing the cardinal characteristics of this syndrome combine brain atrophy with clubfoot and arthrogryposis. Affected individuals present with cerebral parenchymal underdevelopment, ranging from major cerebral parenchymal thinning with lissencephalic aspect to moderate parenchymal rarefaction, severe to mild ventriculomegaly, cerebellar hypoplasia with brainstem dysgenesis, and cardiac and ophthalmologic anomalies, such as microphthalmia and cataract. Severe loss-of-function cases were incompatible with life, whereas those individuals with milder missense variants presented with severe global developmental delay, syndactyly of 2nd and 3rd toes, and severe muscle hypotonia resulting in incapacity to stand without support. Consistent with a causative role for KIAA1109 loss-of-function/hypomorphic variants in this syndrome, knockdowns of the zebrafish orthologous gene resulted in embryos with hydrocephaly and abnormally curved notochords and overall body shape, whereas published knockouts of the fruit fly and mouse orthologous genes resulted in lethality or severe neurological defects reminiscent of the probands' features.

Inactivation of AMMECR1 is associated with growth, bone, and heart alterations.

We report five individuals with loss-of-function of the X-linked AMMECR1: a girl with a balanced X-autosome translocation and inactivation of the normal X-chromosome; two boys with maternally inherited and de novo nonsense variants; and two half-brothers with maternally inherited microdeletion variants. They present with short stature, cardiac and skeletal abnormalities, and hearing loss. Variants of unknown significance in AMMECR1 in four male patients from two families with partially overlapping phenotypes were previously reported. AMMECR1 is coexpressed with genes implicated in cell cycle regulation, five of which were previously associated with growth and bone alterations. Our knockdown of the zebrafish orthologous gene resulted in phenotypes reminiscent of patients' features. The increased transcript and encoded protein levels of AMMECR1L, an AMMECR1 paralog, in the t(X;9) patient's cells indicate a possible partial compensatory mechanism. AMMECR1 and AMMECR1L proteins dimerize and localize to the nucleus as suggested by their nucleic acid-binding RAGNYA folds. Our results suggest that AMMECR1 is potentially involved in cell cycle control and linked to a new syndrome with growth, bone, heart, and kidney alterations with or without elliptocytosis.

The effect of genetic variation on promoter usage and enhancer activity.

The identification of genetic variants affecting gene expression, namely expression quantitative trait loci (eQTLs), has contributed to the understanding of mechanisms underlying human traits and diseases. The majority of these variants map in non-coding regulatory regions of the genome and their identification remains challenging. Here, we use natural genetic variation and CAGE transcriptomes from 154 EBV-transformed lymphoblastoid cell lines, derived from unrelated individuals, to map 5376 and 110 regulatory variants associated with promoter usage (puQTLs) and enhancer activity (eaQTLs), respectively. We characterize five categories of genes associated with puQTLs, distinguishing single from multi-promoter genes. Among multi-promoter genes, we find puQTL effects either specific to a single promoter or to multiple promoters with variable effect orientations. Regulatory variants associated with opposite effects on different mRNA isoforms suggest compensatory mechanisms occurring between alternative promoters. Our analyses identify differential promoter usage and modulation of enhancer activity as molecular mechanisms underlying eQTLs related to regulatory elements.

Effect of Wearing a Telemetry Jacket on Behavioral and Physiologic Parameters of Dogs in the Open-Field Test.

Safety pharmacology studies in dogs often integrate behavioral assessments made using video recording with physiologic measurements collected by telemetry. However, whether merely wearing the telemetry vest affects canine behavior and other parameters has not been evaluated. This pilot study assessed the effect of a telemetry vest on behavioral and physiologic responses to an environmental stressor, the sounds of a thunderstorm, in Labrador retrievers. Dogs were assigned to one of 2 experimental groups (Vest and No-Vest, n = 8 dogs per group) by using a matched pairs design, with a previously determined, sound-associated anxiety score as the blocking variable. Dogs were individually retested with the same standardized sound stimulus (thunderstorm) in an open-field arena, and their behavioral responses were video recorded. Video analysis of locomotor activity and anxiety-related behavior and manual determination of heart rate and body temperature were performed; results were compared between groups. Vest wearing did not affect total locomotor activity or rectal body temperature but significantly decreased heart rate by 8% and overall mean anxiety score by 34% during open-field test sessions. Our results suggest that the use of telemetry vests in dogs influences the measurement of physiologic parameters and behaviors that are assessed in safety pharmacology studies.

Oral Transmucosal Detomidine Gel in New Zealand White Rabbits (Oryctolagus cuniculus).

Handling and restraining rabbits for routine procedures may be impossible without prior sedation, result in unnecessary stress or injury to the rabbit or handler, and increase experimental variability. Parenteral administration of sedatives can cause stress also, as well as localized pain and tissue damage, especially in fractious animals. Detomidine hydrochloride, an α2-adrenergic receptor agonist, is commercially available in an oral transmucosal (OTM) gel formulation that is FDA-approved for sedation and restraint in horses. This study investigated the efficacy and safety of detomidine gel as an alternative to injectable sedation in rabbits. Eight adult male New Zealand White rabbits each received 0.6, 1.2, or 1.8 mg/kg OTM detomidine gel. Physiologic parameters and sedation scores (SS) were assessed at 10-min intervals from before administration until 100 min afterward. Histopathology of cardiac tissue was scored through 12 d after dosing. Gel administration increased the SS in all rabbits, but none of the animals developed clinically effective sedation (SS of 10 or greater, based on 5 reflex responses on a 3- or 4-point scale). The SS did not differ among dosage groups, and the time-dose interaction was not statistically significant. Heart rate decreased rapidly in all rabbits, with no difference among dosage groups, and there was no effect of time or dosage on peripheral capillary oxygen saturation. Minimal to mild degenerative changes were seen in the myocardium of all treated rabbits, but myocyte necrosis, inflammation, fibrosis, and mural thrombi-reported previously in rabbits that had received parenteral detomidine-did not occur. OTM detomidine gel was safely and easily administered to rabbits, but the duration and level of sedation were unpredictable. The use of OTM detomidine as a sole agent to facilitate handling and restraint of rabbits does not offer advantages over existing parenteral regimens.