Single-embryo RNA sequencing for continuous and sex-specific gene expression analysis on Drosophila.

Exploring early embryonic gene expression is challenging due to the rate of development and the limited material available. Here, we present a protocol for ordering Drosophila embryos along a developmental pseudo-time trajectory and determining the sex of the embryos using RNA-seq data. We describe steps for sample collection, RNA isolation, RNA-seq, and RNA-seq data processing. We then detail the establishment of a continuous transcriptome dataset for assessing gene expression throughout early development and in a sex-specific manner. For complete details on the use and execution of this protocol, please refer to Pérez-Mojica et al.1.

Prenatal benzene exposure in mice alters offspring hypothalamic development predisposing to metabolic disease in later life.

Maternal exposure to environmental contaminants during pregnancy poses a significant threat to a developing fetus, as these substances can easily cross the placenta and disrupt the neurodevelopment of offspring. Specifically, the hypothalamus is essential in the regulation of metabolism, notably during critical windows of development. An abnormal hormonal and inflammatory milieu during development can trigger persistent changes in the function of hypothalamic circuits, leading to long-lasting effects on the body's energy homeostasis and metabolism. We recently demonstrated that gestational exposure to clinically relevant levels of benzene induces severe metabolic dysregulation in the offspring. Given the central role of the hypothalamus in metabolic control, we hypothesized that prenatal exposure to benzene impacts hypothalamic development, contributing to the adverse metabolic effects in the offspring. C57BL/6JB dams were exposed to benzene at 50 ppm in the inhalation chambers exclusively during pregnancy (from E0.5 to E19). Transcriptomic analysis of the exposed offspring at postnatal day 21 (P21) revealed hypothalamic changes in genes related to metabolic regulation, inflammation, and neurodevelopment exclusively in males. Moreover, the hypothalamus of prenatally benzene-exposed male offspring displayed alterations in orexigenic and anorexigenic projections, impairments in leptin signaling, and increased microgliosis. Additional exposure to benzene during lactation did not promote further microgliosis or astrogliosis in the offspring, while the high-fat diet (HFD) challenge in adulthood exacerbated glucose metabolism and hypothalamic inflammation in benzene-exposed offspring of both sexes. These findings reveal the persistent adverse effects of prenatal benzene exposure on hypothalamic circuits and neuroinflammation, predisposing the offspring to long-lasting metabolic health conditions.

Continuous transcriptome analysis reveals novel patterns of early gene expression in Drosophila embryos.

The transformative events during early organismal development lay the foundation for body formation and long-term phenotype. The rapid progression of events and the limited material available present major barriers to studying these earliest stages of development. Herein, we report an operationally simple RNA sequencing approach for high-resolution, time-sensitive transcriptome analysis in early (≤3 h) Drosophila embryos. This method does not require embryo staging but relies on single-embryo RNA sequencing and transcriptome ordering along a developmental trajectory (pseudo-time). The resulting high-resolution, time-sensitive mRNA expression profiles reveal the exact onset of transcription and degradation for thousands of transcripts. Further, using sex-specific transcription signatures, embryos can be sexed directly, eliminating the need for Y chromosome genotyping and revealing patterns of sex-biased transcription from the beginning of zygotic transcription. Our data provide an unparalleled resolution of gene expression during early development and enhance the current understanding of early transcriptional processes.

Prenatal benzene exposure alters offspring hypothalamic development predisposing to metabolic disease in later life.

The hypothalamus is essential in the regulation of metabolism, notably during critical windows of development. An abnormal hormonal and inflammatory milieu during development can trigger persistent changes in the function of hypothalamic circuits, leading to long-lasting effects on the body’s energy homeostasis and metabolism. We recently demonstrated that gestational exposure to benzene at smoking levels induces severe metabolic dysregulation in the offspring. Given the central role of the hypothalamus in metabolic control, we hypothesized that prenatal exposure to benzene impacts hypothalamic development, contributing to the adverse metabolic effects in the offspring. C57BL/6JB dams were exposed to benzene in the inhalation chambers exclusively during pregnancy (from E0.5 to E19). The transcriptome analysis of the offspring hypothalamus at postnatal day 21 (P21) revealed changes in genes related to metabolic regulation, inflammation, and neurodevelopment exclusively in benzene-exposed male offspring. Moreover, the hypothalamus of prenatally benzene-exposed male offspring displayed alterations in orexigenic and anorexigenic projections, impairments in leptin signaling, and increased microgliosis. Additional exposure to benzene during lactation did not promote further microgliosis or astrogliosis in the offspring, while the high-fat diet (HFD) challenge in adulthood exacerbated glucose metabolism and hypothalamic inflammation in benzene-exposed offspring of both sexes. These findings reveal the persistent impact of prenatal benzene exposure on hypothalamic circuits and neuroinflammation, predisposing the offspring to long-lasting metabolic health conditions.

Docosahexaenoic acid and oleic acid induce altered DNA methylation of individual CpG loci in Jurkat T cells.

Docosahexaenoic acid (DHA, 22:6n-3) and oleic acid (18:1n-9) can alter the DNA methylation of individual CpG loci in vivo and in vitro, although the targeting mechanism is unknown. We tested the hypothesis that the targeting of altered methylation is associated with putative transcription factor response elements (pTREs) proximal to modified loci. Jurkat cells were treated with 22:6n-3 or 18:1n-9 (both 15 µM) for eight days and DNA methylation measured using the MethylationEPIC 850K array. 1596 CpG loci were altered significantly (508 hypermethylated) by 22:6n-3 and 563 CpG loci (294 hypermethylated) by 18:1n-9. 78 loci were modified by both fatty acids. Induced differential methylation was not modified by the PPARα antagonist GW6471. DNA sequences proximal to differentially methylated CpG loci were enriched in zinc-finger pTREs. These findings suggest that zinc-finger-containing transcription factors may be involved in targeting altered DNA methylation modifying processes induced by fatty acids to individual CpG loci.

Polyunsaturated Fatty Acid Biosynthesis Involving Δ8 Desaturation and Differential DNA Methylation of FADS2 Regulates Proliferation of Human Peripheral Blood Mononuclear Cells.

Polyunsaturated fatty acids (PUFAs) are important for immune function. Limited evidence indicates that immune cell activation involves endogenous PUFA synthesis, but this has not been characterised. To address this, we measured metabolism of 18:3n-3 in quiescent and activated peripheral blood mononuclear cells (PBMCs), and in Jurkat T cell leukaemia. PBMCs from men and women (n = 34) were incubated with [1-13C]18:3n-3 with or without Concanavalin A (Con. A). 18:3n-3 conversion was undetectable in unstimulated PBMCs, but up-regulated when stimulated. The main products were 20:3n-3 and 20:4n-3, while 18:4n-3 was undetectable, suggesting initial elongation and Δ8 desaturation. PUFA synthesis was 17.4-fold greater in Jurkat cells than PBMCs. The major products of 18:3n-3 conversion in Jurkat cells were 20:4n-3, 20:5n-3, and 22:5n-3. 13C Enrichment of 18:4n-3 and 20:3n-3 suggests parallel initial elongation and Δ6 desaturation. The FADS2 inhibitor SC26196 reduced PBMC, but not Jurkat cell, proliferation suggesting PUFA synthesis is involved in regulating mitosis in PBMCs. Con. A stimulation increased FADS2, FADS1, ELOVL5 and ELOVL4 mRNA expression in PBMCs. A single transcript corresponding to the major isoform of FADS2, FADS20001, was detected in PBMCs and Jurkat cells. PBMC activation induced hypermethylation of a 470bp region in the FADS2 5'-regulatory sequence. This region was hypomethylated in Jurkat cells compared to quiescent PBMCs. These findings show that PUFA synthesis involving initial elongation and Δ8 desaturation is involved in regulating PBMC proliferation and is regulated via transcription possibly by altered DNA methylation. These processes were dysregulated in Jurkat cells. This has implications for understanding the regulation of mitosis in normal and transformed lymphocytes.

Associations between whole peripheral blood fatty acids and DNA methylation in humans.

Fatty acids (FA) modify DNA methylation in vitro, but limited information is available on whether corresponding associations exist in vivo and reflect any short-term effect of the diet. Associations between global DNA methylation and FAs were sought in blood from lactating infants (LI; n = 49) and adult males (AMM; n = 12) equally distributed across the three conventional BMI classes. AMM provided multiple samples at 2-hour intervals during 8 hours after either a single Western diet-representative meal (post-prandial samples) or no meal (fasting samples). Lipid/glucose profile, HDAC4 promoter and PDK4 5'UTR methylation were determined in AMM. Multiple regression analysis revealed that global (in LI) and both global and PDK4-specific DNA methylation (in AMM) were positively associated with eicosapentaenoic and arachidonic acid. HDAC4 methylation was inversely associated with arachidonic acid post-prandially in AMM. Global DNA methylation did not show any defined within-day pattern that would suggest a short-term response to the diet. Nonetheless, global DNA methylation was higher in normal weight subjects both post-prandially and in fasting and coincided with higher polyunsaturated relative to monounsaturated and saturated FAs. We show for the first time strong associations of DNA methylation with specific FAs in two human cohorts of distinct age, diet and postnatal development stage.

Development of a new lingual range-of-motion assessment scale: normative data in surgically treated oral cancer patients.

Surgical resection in oral cancer patients can result in altered speech, swallowing, and patient perception of quality of life (QOL). Oral surgery can result in reduced lingual range of motion (ROM). However, few studies have quantified the degree of lingual restriction after surgery. This pilot study describes a new measurement system to define tongue ROM in surgically treated tongue cancer patients. This measurement system was validated by comparing results in these treated surgical patients versus healthy individuals. This scale was further validated by correlating ROM with performance status, oral outcomes, and patient-rated QOL. Thirty-six patients who underwent oral tongue surgery and 31 healthy individuals were included. Tongue ROM was assessed using a novel ROM assessment system. This novel system was examined in these patients versus healthy subjects. This measurement tool was further validated by correlating tongue ROM in treated patients with performance status, oral outcomes, and patient-rated QOL. Tongue ROM was found to be significantly lower in the surgically treated patients than in the healthy individuals (p = 0.0001). Tongue ROM correlated with performance status, oral outcomes, and all QOL measures. This new tongue ROM measurement system defined tongue deficits in surgically treated oral cancer patients. This tool was validated by comparing results to those in healthy individuals, as well as by correlating tongue ROM to performance status, oral outcomes, and QOL. This measurement tool can be used to define baseline and postsurgery tongue ROM in oral cancer patients, as well as track change over time with recovery and therapy. Future studies should examine use of this measurement tool with other populations demonstrating tongue deficits.

Relationship between aggressive behavior, depressed mood, and other disruptive behavior in Puerto Rican children diagnosed with attention deficit and disruptive behaviors disorders

OBJECTIVE: The present study was directed at examining the relationship between aggressive behavior, depressed mood, other disruptive behaviors in children diagnosed with ADHD or ODD disorders in Puerto Rico. METHODS: One hundred seventy six (176) students (127 males and 49 females) from 12 public elementary schools in the San Juan Area of Puerto Rico participated in the study. The participants were divided into a group of ADHD children who exhibited aggressive behavior, a group of ADHD children that did not show aggressive behavior, and a normal group. Several self-report measures were administered to the children and teachers. RESULTS: Our results indicate that the best predictor of aggressive behavior was the hyperactivity and impulsiveness for both ADHD males and females. In addition, depressed mood in both males and females was also a significant predictor of aggressive behavior in Puerto Rican ADHD children. However, in females the social problems variable was also found to be a significant grouping variable. CONCLUSION: The first conclusion of these results is that inattentiveness does not appear to be a relevant factor in ADHD Puerto Rican children who exhibit aggressive behavior. Second, we need to be cognizant to the fact that Puerto Rican ADHD children do exhibit high co-morbidity for aggressive behavior, depressed mood, and social problems. Thus, our diagnostic and treatment approaches with ADHD Puerto Rican children need to include an assessment of the social environment of the child and its effect on his emotional state, in particular his or her mood.

Repair of posterior coronary lacerations.

Posterior lacerations of the coronary arteries that occur during arteriotomy should be repaired if significant. We describe a modification of the standard technique to repair posterior coronary artery lacerations. This technique avoids distortion and narrowing of the coronary artery.

Fat augmentation for glottic insufficiency.

OBJECTIVES: Fat lipoinjection augmentation for glottic insufficiency has been used in patients with vocal fold paralysis. Relatively little information is available on the effectiveness of fat injection in patients with vocal atrophy, intubation trauma, and post-hemilaryngectomy defects. STUDY DESIGN: This paper retrospectively compares the efficiency of fat injection in patients with vocal cord paralysis (n = 9), vocal scar (n = 13), and vocal atrophy (n = 11). METHODS: The perceptual acoustic, phonatory function, and videolaryngostroboscopic data were evaluated before and after fat augmentation in 33 patients. RESULTS: Mean follow-up time was 9.7 months. Nineteen patients had excellent results. Three patients had no change. Five patients had late failure. Six patients were lost to follow-up. Phonatory function showed significant improvement in jitter, shimmer, noise-to-harmonic ratio, maximal phonation time, grade, asthenia, and breathiness (P < .05). Videolaryngostroboscopic rating showed significant improvement in right linearity of the vocal fold edge, amplitude of vocal fold vibration, excursion of the mucosal wave, vibratory behavior, and phase symmetry (P < .05). Anterior defects did better than posterior defects. Small vocal fold defects did better than large defects. CONCLUSIONS: Fat injection is a good autogenous implant and may be considered as an option in management of patients with vocal fold scar, defect, or atrophy. Reabsorption of fat is a problem, but the procedure may be repeated.

Videolaryngostroboscopy following vertical partial laryngectomy.

Phonation after partial laryngeal ablative surgery has not often been examined. Videolaryngostroboscopic recordings made after vertical partial laryngectomy (VPL) were retrospectively reviewed and correlated with patient historical and operative factors. Among VPL patients (n = 42), the most common site of vibration during phonation was the contralateral false vocal fold (17/42 patients or 40.5%), followed by the contralateral arytenoid mucosa (10/42 or 23.8%) and the contralateral true vocal fold (8/42 patients or 19.0%). There was no overall difference in vocal quality judgment with respect to site of vibration (ANOVA, p = .373). Vocal quality scores were similar with use of the pyriform mucosal flap versus other reconstructive methods (Student's t-test, p = .568). This study highlights the fact that reconstruction of a new vibratory source after VPL is important for voice production. Because VPL patients infrequently demonstrated true vocal fold vibration, alternative sites (ie, false vocal fold, arytenoid mucosa) must be considered as new phonatory sources after VPL.

Intraluminal milrinone for dilation of the radial artery graft.

There is renewed interest in the use of the radial artery as a conduit for coronary artery bypass surgery. The radial artery is, however, a very muscular artery, prone to vasospasm. Milrinone, a potent vasodilator, has demonstrated vasodilatory properties superior to those of papaverine. In this report, we describe our technique of radial artery harvesting and the adjunctive use of intraluminal milrinone as a vasodilator in the preparation of this conduit for coronary artery bypass grafting. We have used these techniques in 25 patients who have undergone coronary artery bypass grafting using the radial artery. No hand ischemic complications have been observed in this group. Intraluminal milrinone appears to dilate and relax the radial artery, rendering this large conduit spasm free and very easy to use. We recommend the skeletonization technique for radial artery harvesting and the use of intraluminal milrinone as a radial artery vasodilator in routine myocardial revascularization.

Left ventricular volume reduction and reconstruction in ischemic cardiomyopathy.

BACKGROUND: Ischemic cardiomyopathy can be the result of large or small myocardial infarctions or due to myocardial hibernation. Patients with an end-systolic volume index >100 mL¿m2 do not benefit from revascularization alone and require an operation that reduces ventricular volume. Various approaches to reduce ventricular volume have been described. We applied several of these techniques in patients with end-stage ischemic cardiomyopathy. METHODS: Forty eight patients with end-stage ischemic cardiomyopathy (Class III-IV) underwent left ventricular volume reduction operations with coronary revascularization and mitral valve repair or Alfieri valvoplasty. Fourteen patients underwent interpapillary resections, 22 anterior resections, 4 posterior resections, 2 anterior and posterior resections, and 6 patients reduction of left ventricular volume with endocavitary patches. RESULTS: All the techniques used improved left ventricular function. Analysis of mortality revealed that extensive resections (interpapillary, anterior, and posterior resection) had a 43% mortality. However, a limited resection or a ventricular reconstruction with an endocavitary patch had only a 12.5% mortality. When we changed our approach to a more conservative one, mortality was reduced from 26% the first 12 months to 13% in the last 15 months of the study. CONCLUSIONS: Ischemic cardiomyopathy has a poor prognosis if the end-systolic volume index exceeds 100 mL/m2. Various procedures exist to reduce left ventricular volume. Extensive ventricular resections improve ventricular function, but have a high mortality. This led us to use other methods of ventricular volume reduction such as more conservative resections combined with left ventricular reconstructions or ventricular volume reduction with endocavitary patches. Mortality was reduced significantly by this approach. The patients that survived have remained Class I-II in a follow-up that extends up to 30 months. Surgical therapy of Class III-IV ischemic cardiomyopathy is feasible, but aggressive ventricular resections have a high mortality. We advocate a more reconstructive approach with limited or no ventricular resection.

Surgical treatment of aortic dissections: initial experience with the adventitial inversion technique.

BACKGROUND: The adventitial inversion technique obliterates the false lumen and converts a dissected aorta into a conduit with tough adventitia on the inside and outside. Dacron grafts can be anastomosed to the aorta with fine sutures, which hold without tears. METHODS: From August 1995 to March 1997, we treated 6 patients with acute dissecting aneurysms. Three aneurysms were type I (A) involving the entire aorta, two type II (A) involving the ascending aorta, and one type III (B) involving the thoracoabdominal aorta. Circulatory arrest was used in 3 patients, 1 with type I aneurysm (A), 1 type II (A), and 1 type III (B). RESULTS: All Dacron-aorta anastomoses held sutures well and did not bleed intraoperatively or postoperatively. One patient (type II [A]) died of intraoperative low cardiac output. In patients with type I (A) aneurysms, the false lumen was obliterated, but 1 patient required resection of a 6-cm abdominal aortic aneurysm. CONCLUSIONS: The adventitial inversion technique is a safe technique for the treatment of acute dissecting aneurysms, which facilitates operation and solves the problem of intraoperative or postoperative bleeding due to tissue friability.

Management of bilateral glottic tumors.

OBJECTIVE: To compare the rates of cure and complication and the time to decannulation and deglutition in patients undergoing resection of bilateral glottic tumors. DESIGN: A 22-year, nonrandomized, prospective, retrospective analysis. SETTING: Two academic tertiary care referral centers. PARTICIPANTS: Seventy-two patients with bilateral glottic carcinoma were treated using bilateral hemilaryngectomy. Depending on the size of the tumor and the extent of thyroid cartilage resection, patients underwent 1 of 3 methods of reconstruction: group 1, placement of an anterior commissure stent (34 patients); group 2, epiglottic laryngoplasty (15 patients); and group 3, staged posterior thyroid alar transposition laryngoplasty (23 patients). INTERVENTION: Resection and reconstruction of 72 larynges with bilateral glottic tumors using the bilateral hemilaryngectomy procedures. MAIN OUTCOME MEASURES: Acceptable rates of cure and complication, intervals to decannulation and deglutition, and quality of speech. RESULTS: High rates of tumor control and cure, low rates of recurrence and complication, acceptable time to decannulation and deglutition, and adequate quality and intelligibility of speech. CONCLUSIONS: Bilateral vocal cord carcinoma can be treated surgically with a high degree of tumor control and cure. The use of all 3 methods maintained laryngeal function with regard to tracheal decannulation, oral alimentation, and speech intelligibility.

[Re-emergence of dengue in Panama]. / Reemergencia del dengue en Panama.

The dengue surveillance system in Panama during the 1994 and 1995 epidemics shows the following results: In 1994 we registered 793 dengue cases and a year after 3,083 dengue cases, with three dengue haemorrhagic cases (one death). To estimate the accumulated incidence rates in 1994-1995 period, we consolidated the cases. The highest risk of dengue and the highest dengue haemorrhagic propension were observed in: West Panama (709.49 per 100,000 persons), Los Santos (405.74 per 100,000 persons), Herrera (337.62 per 100,000), Metropolitana (126.98 per 100,000) and San Miguelito (125.10 per 100,000 persons).

Reemergencia del dengue en Panama / Re-emergence of dengue in Panama

The dengue surveillance system in Panama during the 1994 and 1995 epidemics shows the following results: In 1994 we registered 793 dengue cases and a year after 3,083 dengue cases, with three dengue haemorrhagic cases (one death). To estimate the accumulated incidence rates in 1994-1995 period, we consolidated the cases. The highest risk of dengue and the highest dengue haemorrhagic propension were observed in: West Panama (709.49 per 100,000 persons), Los Santos (405.74 per 100,000 persons), Herrera (337.62 per 100,000), Metropolitana (126.98 per 100,000) and San Miguelito (125.10 per 100,000 persons)

The use of 25% glutaraldehyde solution to strengthen the aorta of patients with annuloaortic ectasia, ascending and aortic arch aneurysms.

Aortic friability is a problem of great concern and frustration for those performing operations on patients with ascending and arch aortic aneurysms, and particularly, on patients with annuloaortic ectasia. We have used 25% glutaraldehyde to successfully strengthen the distal aorta of three such patients. After the application, the aorta wall held sutures without tearing. Postoperative bleeding was minimal in each case. No neurological events were noticed, and all of the patients were discharged home within 2 weeks. The application of this tanning solution to such a friable aortic tissue should be considered as a helpful alternative to reduce postoperative bleeding, which is the most common complication after this type of surgery.