Molecular analyses of zebrafish V0v spinal interneurons and identification of transcriptional regulators downstream of Evx1 and Evx2 in these cells.

BACKGROUND: V0v spinal interneurons are highly conserved, glutamatergic, commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However, we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. METHODS: To identify candidate members of V0v gene regulatory networks, we FAC-sorted wild-type and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression-profiled them using microarrays and single cell RNA-seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 double mutants and wild-type siblings. RESULTS: Our data reveal two molecularly distinct subtypes of zebrafish V0v spinal interneurons at 48 h and suggest that, by this stage of development, evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons, or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons, plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly, we show that Hmx2/3a, repress dI2 interneuron expression of skor1a and nefma, two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. CONCLUSIONS: This study identifies two molecularly distinct subsets of zebrafish V0v spinal interneurons, as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of these cells. Our data further suggest that in the absence of both Evx1 and Evx2, V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then, later, start to express markers of distinct types of inhibitory spinal interneurons, or motoneurons. Taken together, our findings significantly increase our knowledge of V0v and spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type.

Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in these Cells.

Background: V0v spinal interneurons are highly conserved, glutamatergic, commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However, we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks, we FAC-sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression-profiled them using microarrays and single cell RNA-seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 double mutants and wild-type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that, by this stage of development, evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons, or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons, plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly, we show that Hmx2/3a, repress dI2 interneuronal expression of skor1a and nefma, two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons, as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of these cells. Our data further suggest that in the absence of both Evx1 and Evx2, V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then, later, start to express markers of distinct types of inhibitory spinal interneurons, or motoneurons. Taken together, our findings significantly increase our knowledge of V0v and spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type.

Semicontinuous and batch ozonation combined with peroxymonosulfate for inactivation of microalgae in ballast water.

The Ballast Water Management Convention (BWMC) establishes limits regarding the permissible number of viable organisms in discharged ballast water. Ozone as a ballast water treatment is interesting because it can be generated in-situ and has strong oxidant power. Additionally, some oxidants can be formed in reaction with seawater, especially brominated compounds, that assist in inactivating microorganisms. The objective of this study is to assess the efficacy of semicontinuous and batch ozonation as well as their combination with peroxymonosulfate salt (PMS) as methods to be used to ensure compliance with regulation D2 of the BWMC using Tetraselmis suecica as a standard microorganism. Growth modeling method was employed to determine the inactivation achieved by the treatments. The results show that ozone is an effective treatment for accomplishing the D2 of the BWMC. Batch ozonation is more efficient than semicontinuous ozonation probably because of the brominated compounds formed during the ozone saturation of the water. The oxidants that are developed during the ozonation of seawater prolong the residual effect of the treatment throughout the days of storage with practically no presence of them in the ballast tanks at 72 h. The addition of the PMS increases the inactivation in the semicontinuous ozonation, but a threshold concentration of ozone is needed to observe the synergistic effect of both oxidants. No increase is associated with the combination of O3 and PMS in the case of batch ozonation.

Acute and long-term success of ventricular tachycardia ablation in patients with ischemic heart disease in a Mexican center.

Objective: . To report the results of ventricular tachycardia (VT) catheter ablation in ischemic heart disease (IHD), and to identify risk factors associated with recurrence in a Mexican center. Materials and methods: . We made a retrospective review of the cases of VT ablation performed in our center from 2015 to 2022. We analyzed the characteristics of the patients and those of the procedures separately and we determined factors associated with recurrence. Results: . Fifty procedures were performed in 38 patients (84% male; mean age 58.1 years). Acute success rate was 82%, with a 28% of recurrences. Female sex (OR 3.33, IC 95% 1.66-6.68, p=0.006), atrial fibrillation (OR 3.5, IC 95% 2.08-5.9, p=0.012), electrical storm (OR 2.4, IC 95% 1.06-5.41, p=0.045), functional class greater than II (OR 2.86, IC 95% 1.34-6.10, p=0.018) were risk factors for recurrence and the presence of clinical VT at the time of ablation (OR 0.29, IC 95% 0.12-0.70, p=0.004) and the use of more than 2 techniques for mapping (OR 0.64, IC 95% 0.48-0.86, p=0.013) were protective factors. Conclusions: . Ablation of ventricular tachycardia in ischemic heart disease has had good results in our center. The recurrence is similar to that reported by other authors and there are some factors associated with it.

Impact of the coronavirus disease-19 pandemic on electrophysiological procedures at a national referral center.

INTRODUCTION: The coronavirus disease (COVID-19) pandemic has generated serious repercussions on the health system, reducing the number of all cardiology procedures worldwide. OBJECTIVES: Describe the impact of the COVID-19 pandemic on the procedures performed by the electrophysiology department in a national referral center. METHODS: We made a retrospective review of our data base and we compared procedures made in the past 3 years since 2017-2019 with the procedures made in the 2020. We divided the procedures into two large groups: Cardiac Implantable Electronic Devices (CIED) related procedures and electrophysiological procedures (EP) which included conventional and complex ablations. RESULTS: There was a significant reduction in all the procedures, the average of procedures performed in the last 3 previous years was 467, while in 2020, we performed only 319 (p = 0.01); this represents a reduction of 33.4% in the total number of procedures. There was no statistical difference regarding the CIED related procedures, the average of procedures in the past 3 previous years was 174, and in 2020 we performed 190 procedures (p = 0.46). Regarding the EP, the average of the past 3 previous years was 293, while in 2020, we performed only 129 procedures (p < 0.01). The reduction in the EP was 55.97%. The most affected months were April, May, and June. CONCLUSIONS: The COVID-19 pandemic considerably affected the number of the procedures in our center, reducing it by 33.4%. The reduction of procedures fundamentally affected the ablations, with a reduction of 55.97%. The number of CIED related procedures was not affected.

Diagnostic challenges of Brugada Syndrome in pediatric patients.

Although most cases of Brugada syndrome have been described in adults, pediatric patients with the disease have been reported since the original article from Josep and Pedro Brugada. Herein is presented the case series of Brugada syndrome in pediatric population of the National Institute of Cardiology Ignacio Chavez. One boy and two adolescent males had palpitations as clinical presentation of the disease. Atrial arrhythmias were documented in two, in the third case there was a high clinical suspicion and quinidine abolished symptoms. The aim of this report is to highlight the importance of performing a detailed clinical history as well as the usefulness of high precordial leads for the diagnosis of this entity.

Porphyrin Supramolecular Arrays Formed by Weakly Interacting Meso-Functional Groups on Au(111).

The formation of a binary porphyrinic self-assembled system between meso-tetrakis(4-carboxyphenyl) porphyrin (TCPP) and meso-tetrakis(4-dimethyl amino) porphyrin (TDAP) was easily designed through non-covalent interactions in solution and adsorbed on a gold substrate. It was found that non-covalent interactions and geometrical conformations between porphyrins allow their self-assembly into a well-defined arrangement, which was confirmed by UV-Vis spectroscopy, electrochemistry, atomic force microscopy and density functional theory (DFT) studies.

Zebrafish transgenic constructs label specific neurons in Xenopus laevis spinal cord and identify frog V0v spinal neurons.

A correctly functioning spinal cord is crucial for locomotion and communication between body and brain but there are fundamental gaps in our knowledge of how spinal neuronal circuitry is established and functions. To understand the genetic program that regulates specification and functions of this circuitry, we need to connect neuronal molecular phenotypes with physiological analyses. Studies using Xenopus laevis tadpoles have increased our understanding of spinal cord neuronal physiology and function, particularly in locomotor circuitry. However, the X. laevis tetraploid genome and long generation time make it difficult to investigate how neurons are specified. The opacity of X. laevis embryos also makes it hard to connect functional classes of neurons and the genes that they express. We demonstrate here that Tol2 transgenic constructs using zebrafish enhancers that drive expression in specific zebrafish spinal neurons label equivalent neurons in X. laevis and that the incorporation of a Gal4:UAS amplification cassette enables cells to be observed in live X. laevis tadpoles. This technique should enable the molecular phenotypes, morphologies and physiologies of distinct X. laevis spinal neurons to be examined together in vivo. We have used an islet1 enhancer to label Rohon-Beard sensory neurons and evx enhancers to identify V0v neurons, for the first time, in X. laevis spinal cord. Our work demonstrates the homology of spinal cord circuitry in zebrafish and X. laevis, suggesting that future work could combine their relative strengths to elucidate a more complete picture of how vertebrate spinal cord neurons are specified, and function to generate behavior. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1007-1020, 2017.

Idiopathic Lead Migration: Concept and Variants of an Uncommon Cause of Cardiac Implantable Electronic Device Dysfunction.

OBJECTIVES: This cumulative case study was performed to properly address the possible mechanisms, forms, and consequences of "twiddler's," "reel," and "ratchet" syndromes. BACKGROUND: Twiddler's, reel, and ratchet syndromes are rare entities responsible for lead displacement of cardiac implantable electronic devices (CIED). METHODS: From 2007 to 2012, 1,472 CIED were implanted at our center. Eighty-nine cases were reviewed for failure of pacing circuit integrity. Only 9 met the inclusion criteria for idiopathic lead migration (ILM) and were grouped as ILM (twiddler) or ILM (reel). For a pooled analysis of cases, a review of the literature from 1990 to 2012 was performed, and the authors identified 78 cases from 64 publications. RESULTS: The study population consisted of 87 cases (45 women; median age, 66 years; 46 with ILM [twiddler] and 41 with ILM [reel]). Migration affected only 1 lead in 65% of 46 devices with more than 1 lead. None of the previously reported risk factors-manual manipulation of the device, elderly age, obesity, oversized pocket, and psychiatric history-correlated with the risk of ILM. CONCLUSIONS: Neither manual manipulation of the device nor the other traditional risk factors reported in the literature for ILM syndrome correlated with the risk of ILM.

Peroxisome proliferator-activated receptor-ß/δ modulates mast cell phenotype.

The peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) is known to have multiple anti-inflammatory effects, typically observed in endothelial cells, macrophages, T cells and B cells. Despite the fact that mast cells are important mediators of inflammation, to date, the role of PPARß/δ in mast cells has not been examined. Hence, the present study examined the hypothesis that PPARß/δ modulates mast cell phenotype. Bone-marrow-derived mast cells (BMMCs) and peritoneal mast cells from Pparß/δ+/+ mice expressed higher levels of high-affinity IgE receptor (FcεRI) compared with Pparß/δ-/- mice. BMMCs from Pparß/δ+/+ mice also exhibited dense granules, associated with higher expression of enzymes and proteases compared with Pparß/δ-/- mice. Resting BMMCs from Pparß/δ+/+ mice secreted lower levels of inflammatory cytokines, associated with the altered activation of phospholipase Cγ1 and extracellular signal-regulated kinases compared with Pparß/δ-/- mice. Moreover, the production of cytokines by mast cells induced by various stimuli was highly dependent on PPARß/δ expression. This study demonstrates that PPARß/δ is an important regulator of mast cell phenotype.

Lmx1b is required for the glutamatergic fates of a subset of spinal cord neurons.

BACKGROUND: Alterations in neurotransmitter phenotypes of specific neurons can cause imbalances in excitation and inhibition in the central nervous system (CNS), leading to diseases. Therefore, the correct specification and maintenance of neurotransmitter phenotypes is vital. As with other neuronal properties, neurotransmitter phenotypes are often specified and maintained by particular transcription factors. However, the specific molecular mechanisms and transcription factors that regulate neurotransmitter phenotypes remain largely unknown. METHODS: In this paper we use single mutant, double mutant and transgenic zebrafish embryos to elucidate the functions of Lmx1ba and Lmx1bb in the regulation of spinal cord interneuron neurotransmitter phenotypes. RESULTS: We demonstrate that lmx1ba and lmx1bb are both expressed in zebrafish spinal cord and that lmx1bb is expressed by both V0v cells and dI5 cells. Our functional analyses demonstrate that these transcription factors are not required for neurotransmitter fate specification at early stages of development, but that in embryos with at least two lmx1ba and/or lmx1bb mutant alleles there is a reduced number of excitatory (glutamatergic) spinal interneurons at later stages of development. In contrast, there is no change in the numbers of V0v or dI5 cells. These data suggest that lmx1b-expressing spinal neurons still form normally, but at least a subset of them lose, or do not form, their normal excitatory fates. As the reduction in glutamatergic cells is only seen at later stages of development, Lmx1b is probably required either for the maintenance of glutamatergic fates or to specify glutamatergic phenotypes of a subset of later forming neurons. Using double labeling experiments, we also show that at least some of the cells that lose their normal glutamatergic phenotype are V0v cells. Finally, we also establish that Evx1 and Evx2, two transcription factors that are required for V0v cells to acquire their excitatory neurotransmitter phenotype, are also required for lmx1ba and lmx1bb expression in these cells, suggesting that Lmx1ba and Lmx1bb act downstream of Evx1 and Evx2 in V0v cells. CONCLUSIONS: Lmx1ba and Lmx1bb function at least partially redundantly in the spinal cord and three functional lmx1b alleles are required in zebrafish for correct numbers of excitatory spinal interneurons at later developmental stages. Taken together, our data significantly enhance our understanding of how spinal cord neurotransmitter fates are regulated.

Evx1 and Evx2 specify excitatory neurotransmitter fates and suppress inhibitory fates through a Pax2-independent mechanism.

BACKGROUND: For neurons to function correctly in neuronal circuitry they must utilize appropriate neurotransmitters. However, even though neurotransmitter specificity is one of the most important and defining properties of a neuron we still do not fully understand how neurotransmitter fates are specified during development. Most neuronal properties are determined by the transcription factors that neurons express as they start to differentiate. While we know a few transcription factors that specify the neurotransmitter fates of particular neurons, there are still many spinal neurons for which the transcription factors specifying this critical phenotype are unknown. Strikingly, all of the transcription factors that have been identified so far as specifying inhibitory fates in the spinal cord act through Pax2. Even Tlx1 and Tlx3, which specify the excitatory fates of dI3 and dI5 spinal neurons work at least in part by down-regulating Pax2. METHODS: In this paper we use single and double mutant zebrafish embryos to identify the spinal cord functions of Evx1 and Evx2. RESULTS: We demonstrate that Evx1 and Evx2 are expressed by spinal cord V0v cells and we show that these cells develop into excitatory (glutamatergic) Commissural Ascending (CoSA) interneurons. In the absence of both Evx1 and Evx2, V0v cells still form and develop a CoSA morphology. However, they lose their excitatory fate and instead express markers of a glycinergic fate. Interestingly, they do not express Pax2, suggesting that they are acquiring their inhibitory fate through a novel Pax2-independent mechanism. CONCLUSIONS: Evx1 and Evx2 are required, partially redundantly, for spinal cord V0v cells to become excitatory (glutamatergic) interneurons. These results significantly increase our understanding of the mechanisms of neuronal specification and the genetic networks involved in these processes.

Activation of peroxisome proliferator-activated receptor-ß/δ (PPAR-ß/δ) inhibits human breast cancer cell line tumorigenicity.

The effect of activation and overexpression of the nuclear receptor PPAR-ß/δ in human MDA-MB-231 (estrogen receptor-negative; ER(-)) and MCF7 (estrogen-receptor-positive; ER(+)) breast cancer cell lines was examined. Target gene induction by ligand activation of PPAR-ß/δ was increased by overexpression of PPAR-ß/δ compared with controls. Overexpression of PPAR-ß/δ caused a decrease in cell proliferation in MCF7 and MDA-MB-231 cells compared with controls, whereas ligand activation of PPAR-ß/δ further inhibited proliferation of MCF7 but not MDA-MB-231 cells. Overexpression and/or ligand activation of PPAR-ß/δ in MDA-MB-231 or MCF7 cells had no effect on experimental apoptosis. Decreased clonogenicity was observed in both MDA-MB-231 and MCF7 overexpressing PPAR-ß/δ in response to ligand activation of PPAR-ß/δ as compared with controls. Ectopic xenografts developed from MDA-MB-231 and MCF7 cells overexpressing PPAR-ß/δ were significantly smaller, and ligand activation of PPAR-ß/δ caused an even greater reduction in tumor volume as compared with controls. Interestingly, the decrease in MDA-MB-231 tumor size after overexpressing PPAR-ß/δ and ligand activation of PPAR-ß/δ correlated with increased necrosis. These data show that ligand activation and/or overexpression of PPAR-ß/δ in two human breast cancer cell lines inhibits relative breast cancer tumorigenicity and provide further support for the development of ligands for PPAR-ß/δ to specifically inhibit breast carcinogenesis. These new cell-based models will be invaluable tools for delineating the role of PPAR-ß/δ in breast cancer and evaluating the effects of PPAR-ß/δ agonists.

Reuse of pacemakers: comparison of short and long-term performance.

BACKGROUND: In developing economies, there are patients in whom pacemaker implantation is delayed because they cannot afford one. Reused devices have been a solution. To address concerns about safety, a cohort of consecutive patients implanted with a reused pacemaker was compared with a control group. METHODS AND RESULTS: A cohort of 603 consecutive patients from 2000 to 2010 was studied in an ambispective noninferiority study. The study group patients (n=307) received resterilized pacemakers, and the control group patients (n=296) received a new pacemaker. A combined end point of 3 major outcomes-unexpected battery depletion, infection, and device dysfunction-was analyzed. A total of 85 pacemakers had to be explanted, 31 in the control group (10.5%) and 54 in the study group (17.6%; relative risk, 1.68; 95% confidence interval, 1.1-2.5; P=0.02). Forty-three reached the primary end point, 16 in the control group (5.5%) and 27 in the study group (7.2%; relative risk, 1.3; 95% confidence interval, 0.70-2.45; P=0.794). In terms of individual outcomes, 5 new pacemakers (1.7%) and 11 resterilized pacemakers (3.6%) had unexpected battery depletion (relative risk, 2.12; 95% confidence interval, 0.75-6; P=0.116); 3.7% new pacemakers and 3.2% reused pacemakers had a procedure-related infection (relative risk, 0.87; 95% confidence interval, 0.38-2.03; P=0.46); and 1 pacemaker in the study group malfunctioned. CONCLUSIONS: Pacemaker reuse is feasible and safe and is a viable option for patient with bradyarrhythmias. Other than the expected shorter battery life, reuse of pacemaker generators is not inferior to the use of new devices.

Evx1 is required for joint formation in zebrafish fin dermoskeleton.

The transcription factor Evx1 is expressed in the joints between individual lepidotrichia (bony ray) segments and at the distal tips of the lepidotrichia in developing zebrafish fins. It is also expressed in the apical growth zone in regenerating fins. However, so far there is no functional evidence that addresses whether Evx1 is required for any aspect of fin development or regeneration. In this study, we use a novel mutation in evx1 to address this. We find that Evx1 is not required for either fin outgrowth or regeneration. All of the fins form normally in evx1 mutants, and there are no significant changes in fin length. In contrast, Evx1 is required for lepidotrichia joint formation during both fin development and regeneration. This is a very specific phenotype as both lepidotrichia hemisegment separations and lepidotrichia bifurcations still form normally in evx1 mutant fins, as do joints in the more proximal endoskeletal radials.

Modulation of gastrointestinal inflammation and colorectal tumorigenesis by peroxisome proliferator-activated receptor-ß/δ (PPARß/δ).

Critical physiological roles of peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) include the regulation glucose and lipid homeostasis, cellular differentiation, and modulation of inflammation. The potential for targeting PPARß/δ for the prevention and treatment of metabolic diseases or cancer, is compromised because of major inconsistencies in the literature. This is due primarily to uncertainty regarding the effect of PPARß/δ and its activation on cell proliferation, apoptosis and cell survival. This review summarizes both the confirmed and conflicting mechanisms that have been described for PPARß/δ and the potential for targeting this nuclear receptor for the prevention and treatment of colon cancer.

Evidence for ligand-mediated selective modulation of aryl hydrocarbon receptor activity.

The concept of selective receptor modulators has been established for the nuclear steroid hormone receptors. Such selective modulators have been used therapeutically with great success in the treatment of cancer. However, this concept has not been examined with regard to the aryl hydrocarbon receptor (AHR) because of the latent toxicity commonly associated with AHR activation. AHR-mediated toxicity is primarily derived from AHR binding to its dioxin response element (DRE) and driving expression of CYP1 family members, which have the capacity to metabolize procarcinogens to genotoxic carcinogens. Recent evidence using a non-DRE binding AHR mutant has established the DRE-independent suppression of inflammatory markers by the AHR. We wished to determine whether such DRE-independent repression with wild-type AHR could be dissociated from canonical DRE-dependent transactivation in a ligand-dependent manner and, in doing so, prove the concept of a selective AHR modulator (SAhRM). Here, we identify the selective estrogen receptor (ER) modulator Way-169916 as a dually selective modulator, binding both ER and AHR. Inflammatory gene expression associated with the cytokine-inducible acute-phase response (e.g., SAA1 and CRP) are diminished by Way-169916 in an AHR-dependent manner. Furthermore, activation of AHR by Way-169916 fails to stimulate canonical DRE-driven AHR-mediated CYP1A1 expression, thus eliminating the potential for AHR-mediated genotoxic stress. Such anti-inflammatory activity in the absence of DRE-mediated expression fulfills the major criteria of an SAhRM, which suggests that selective modulation of AHR is possible and renders the AHR a therapeutically viable drug target for the amelioration of inflammatory disease.

Conservation of gene linkage in dispersed vertebrate NK homeobox clusters.

Nk homeobox genes are important regulators of many different developmental processes including muscle, heart, central nervous system and sensory organ development. They are thought to have arisen as part of the ANTP megacluster, which also gave rise to Hox and ParaHox genes, and at least some NK genes remain tightly linked in all animals examined so far. The protostome-deuterostome ancestor probably contained a cluster of nine Nk genes: (Msx)-(Nk4/tinman)-(Nk3/bagpipe)-(Lbx/ladybird)-(Tlx/c15)-(Nk7)-(Nk6/hgtx)-(Nk1/slouch)-(Nk5/Hmx). Of these genes, only NKX2.6-NKX3.1, LBX1-TLX1 and LBX2-TLX2 remain tightly linked in humans. However, it is currently unclear whether this is unique to the human genome as we do not know which of these Nk genes are clustered in other vertebrates. This makes it difficult to assess whether the remaining linkages are due to selective pressures or because chance rearrangements have "missed" certain genes. In this paper, we identify all of the paralogs of these ancestrally clustered NK genes in several distinct vertebrates. We demonstrate that tight linkages of Lbx1-Tlx1, Lbx2-Tlx2 and Nkx3.1-Nkx2.6 have been widely maintained in both the ray-finned and lobe-finned fish lineages. Moreover, the recently duplicated Hmx2-Hmx3 genes are also tightly linked. Finally, we show that Lbx1-Tlx1 and Hmx2-Hmx3 are flanked by highly conserved noncoding elements, suggesting that shared regulatory regions may have resulted in evolutionary pressure to maintain these linkages. Consistent with this, these pairs of genes have overlapping expression domains. In contrast, Lbx2-Tlx2 and Nkx3.1-Nkx2.6, which do not seem to be coexpressed, are also not associated with conserved noncoding sequences, suggesting that an alternative mechanism may be responsible for the continued clustering of these genes.

Intracranial portion of the trochlear nerve and dorsal oblique muscle composition in dog: a structural and ultrastructural study.

In the present investigation the right intracranial portion of the trochlear nerves and dorsal oblique muscle of the right ocular globe were removed from six adult dogs and analyzed by light and electron microscopy. Unmyelinated fibers were observed in the analyzed nerves. The number, diameter, area, and density of myelinated fibers were determined, as were corresponding axon area and diameter and myelin sheath thickness. Frequency histograms of myelin sheath thickness and fiber size show a bimodal distribution with a similar proportion of large and small fibers. Muscle samples were taken from the central portion of the muscle belly, subsequently frozen, cut, and stained with m-ATPase at pH 4.6. Fibers were classified as Type 1 or Type 2 according to their reaction to the m-ATPase and detailed morphologic and morphometric studies were made. The muscles showed two clearly distinct layers, a central layer and a peripheral layer, chiefly composed of Type 2 fibers. The fibers in the central layer were larger in size than those in the peripheral layer.