The Hox transcription factor Ultrabithorax binds RNA and regulates co-transcriptional splicing through an interplay with RNA polymerase II.

Transcription factors (TFs) play a pivotal role in cell fate decision by coordinating gene expression programs. Although most TFs act at the DNA layer, few TFs bind RNA and modulate splicing. Yet, the mechanistic cues underlying TFs activity in splicing remain elusive. Focusing on the Drosophila Hox TF Ultrabithorax (Ubx), our work shed light on a novel layer of Ubx function at the RNA level. Transcriptome and genome-wide binding profiles in embryonic mesoderm and Drosophila cells indicate that Ubx regulates mRNA expression and splicing to promote distinct outcomes in defined cellular contexts. Our results demonstrate a new RNA-binding ability of Ubx. We find that the N51 amino acid of the DNA-binding Homeodomain is non-essential for RNA interaction in vitro, but is required for RNA interaction in vivo and Ubx splicing activity. Moreover, mutation of the N51 amino acid weakens the interaction between Ubx and active RNA Polymerase II (Pol II). Our results reveal that Ubx regulates elongation-coupled splicing, which could be coordinated by a dynamic interplay with active Pol II on chromatin. Overall, our work uncovered a novel role of the Hox TFs at the mRNA regulatory layer. This could be an essential function for other classes of TFs to control cell diversity.

Single-cell RNA sequencing of motoneurons identifies regulators of synaptic wiring in Drosophila embryos.

The correct wiring of neuronal circuits is one of the most complex processes in development, since axons form highly specific connections out of a vast number of possibilities. Circuit structure is genetically determined in vertebrates and invertebrates, but the mechanisms guiding each axon to precisely innervate a unique pre-specified target cell are poorly understood. We investigated Drosophila embryonic motoneurons using single-cell genomics, imaging, and genetics. We show that a cell-specific combination of homeodomain transcription factors and downstream immunoglobulin domain proteins is expressed in individual cells and plays an important role in determining cell-specific connections between differentiated motoneurons and target muscles. We provide genetic evidence for a functional role of five homeodomain transcription factors and four immunoglobulins in the neuromuscular wiring. Knockdown and ectopic expression of these homeodomain transcription factors induces cell-specific synaptic wiring defects that are partly phenocopied by genetic modulations of their immunoglobulin targets. Taken together, our data suggest that homeodomain transcription factor and immunoglobulin molecule expression could be directly linked and function as a crucial determinant of neuronal circuit structure.

Multiple two-step oscillation regimes produced by the alto saxophone.

A saxophone mouthpiece fitted with sensors is used to observe the oscillation of a saxophone reed, as well as the internal acoustic pressure, allowing to identify qualitatively different oscillating regimes. In addition to the standard two-step regime, where the reed channel successively opens and closes once during an oscillation cycle, the experimental results show regimes featuring two closures of the reed channel per cycle, as well as inverted regimes, where the reed closure episode is longer than the open episode. These regimes are well-known on bowed string instruments and some were already described on the Uilleann pipes. A simple saxophone model using measured input impedance is studied with the harmonic balance method, and is shown to reproduce the same two-step regimes. The experiment shows qualitative agreement with the simulation: in both cases, the various regimes appear in the same order as the blowing pressure is increased. Similar results are obtained with other values of the reed opening control parameter, as well as another fingering.

Percutaneous Ultrasonic Tenotomy for Achilles Tendinopathy Is a Surgical Procedure With Similar Complications.

Percutaneous ultrasonic tenotomy is a relatively new treatment option for multiple types of tendinopathy. However, a paucity of high-level data is available on its use for chronic Achilles tendinosis. The present case series details the complications associated with the use of this technique. When considering percutaneous ultrasonic tenotomy, the surgeon should be cognizant that it is a surgical procedure with complications similar to those of other Achilles tendon surgeries.

Analysis of Bub3 and Nup75 in the Drosophila male germline lineage.

Extensive communication at the stem cell-niche interface and asymmetric stem cell division is key for the homeostasis of the Drosophila male germline stem cell system. To improve our understanding of these processes, we analysed the function of the mitotic checkpoint complex (MCC) component Bub3 and the nucleoporin Nup75, a component of the nuclear pore complex realizing the transport of signalling effector molecules to the nucleus, in the Drosophila testis. By lineage-specific interference, we found that the two genes control germline development and maintenance. Bub3 is continuously required in the germline, as its loss results in the beginning in an over-proliferation of early germ cells and later on in loss of the germline. The absence of the germline lineage in such testes has dramatic cell non-autonomous consequences, as cells co-expressing markers of hub and somatic cyst cell fates accumulate and populate in extreme cases the whole testis. Our analysis of Nups showed that some of them are critical for lineage maintenance, as their depletion results in the loss of the affected lineage. In contrast, Nup75 plays a role in controlling proliferation of early germ cells but not differentiating spermatogonia and seems to be involved in keeping hub cells quiescent. In sum, our analysis shows that Bub3 and Nup75 are required for male germline development and maintenance.

Long-term reorganization of respiratory pathways after partial cervical spinal cord injury.

High cervical spinal cord injury (SCI) interrupts bulbospinal respiratory pathways innervating phrenic motoneurons, and induces an inactivation of phrenic nerves (PN) and diaphragm. We have previously shown that the ipsilateral (ipsi) PN was inactivated following a lateral C2 SCI, but was spontaneously partially reactivated 7 days post-SCI. This phrenic reactivation depended on contralateral (contra) descending pathways, located laterally, that cross the spinal midline. We analysed here whether long-term post-lesional changes may occur in the respiratory network. We showed that ipsi PN reactivation was greater at 3 months compared with 7 days post-SCI, and that it was enhanced after acute contra phrenicotomy (Phx), which also induced a substantial reactivation of the ipsi diaphragm (not detected at 7 days post-SCI). At 3 months post-SCI (compared with 7 days post-SCI), ipsi PN activity was only moderately affected by ipsi Phx or by gallamine treatment, a nicotinic neuromuscular blocking agent, indicating that it was less dependent on ipsi sensory phrenic afferents. After an additional acute contra SCI (C1) performed laterally, ipsi PN activity was abolished in rats 7 days post-SCI, but persisted in rats 3 months post-SCI. This activity thus depended on new functional descending pathways located medially rather than laterally. These may not involve newly recruited neurons as retrograde labelling showed that ipsi phrenic motoneurons were innervated by a similar number of medullary respiratory neurons after a short and long post-lesional time. These results show that after a long post-lesional time, phrenic reactivation is reinforced by an anatomo-functional reorganization of spinal respiratory pathways.

Metatarsus Primus Elevatus Resolution After First Metatarsophalangeal Joint Arthroplasty.

BACKGROUND: Hallux limitus (HL) is the second-most common pathology associated with the first metatarsophalangeal joint. A common etiology believed to be associated with HL is metatarsus primus elevatus (MPE), although causation has been unsubstantiated by evidence. Historically, correction of MPE is surgically addressed with an osteotomy. However, some believe MPE is a secondary manifestation of HL due to retrograde pressure and lack of dorsiflexion at the first metatarsophalangeal joint. This study sought to determine whether MPE resolves spontaneously after first metatarsophalangeal joint arthroplasty and reinstitution of normal joint dorsiflexion. METHODS: Twenty-seven weightbearing lateral radiographs from patients with HL were reviewed before and after nonimplant first metatarsophalangeal joint arthroplasty. Radiographs were taken preoperatively and at postoperative visits 1 (mean, 2 weeks) and 2 (mean, 10 weeks). Measurements included first to second metatarsal elevation, Seiberg Index, first to fifth metatarsal distance, sagittal plane first to second metatarsal angle, Meary's angle, metatarsal declination angle, and hallux equinus angle. RESULTS: Statistically significant improvement was seen at both postoperative visits in all of the previously mentioned measurements except first to fifth metatarsal distance, which was reduced but was not statistically significant. CONCLUSIONS: Metatarsus primus elevatus was reduced significantly after first metatarsophalangeal joint arthroplasty. Resolution occurred rapidly and was maintained at the final postoperative visit. This could be due to MPE being a manifestation of HL and not a cause. Based on the results of this study, osteotomies may be unnecessary to surgically address MPE because it can spontaneously correct after reinstitution of first metatarsophalangeal joint motion.

Use of Dehydrated Human Amnion/Chorion Membrane Allografts in More Than 100 Patients with Six Major Types of Refractory Nonhealing Wounds.

BACKGROUND: Biochemical properties of the amniotic membrane help modulate inflammation and enhance soft-tissue healing. In controlled trials, the efficacy of dehydrated human amnion/chorion membrane (dHACM) allografts has been established. Our purpose is to describe our experience with using dHACM to treat nonhealing wounds of various etiologies. METHODS: We conducted a retrospective review of deidentified data from 117 consecutive patients treated in an outpatient clinic with dHACM allografts with wounds of various etiologies over 2 years. The decision to use advanced wound-care treatments is based on rate of healing observed after initiation of standard wound care and patient risk factors. Eligibility for treatments such as amniotic membrane allografts includes wounds without 50% reduction after 4 weeks, or earlier in patients deemed to be at high risk for nonhealing or with a history of chronic wounds. In micronized or sheet formulation, dHACM is applied to the wound weekly after sharp/mechanical debridement as necessary, and wound-care practices appropriate for wound type and location are continued. RESULTS: Thirty-four percent of allograft recipients had diabetic foot ulcers, 25% had venous leg ulcers, 20% had surgical wounds, 14% had pressure ulcers, 6% had ischemic wounds, and 2% had traumatic wounds. Complete healing occurred in 91.1% of treated patients, with a mean ± SD number of weekly applications per healed wound of 5.1 ± 4.2. CONCLUSIONS: In addition to wounds of diabetic origin, dHACM can significantly expedite healing in refractory wounds of varying etiologies.

Predictors of vitamin D deficiency in inflammatory bowel disease and health: A Mississippi perspective.

AIM: To identify the predictors of vitamin D deficiency in patients with and without inflammatory bowel disease (IBD). METHODS: Patients with ulcerative colitis (UC) or Crohn's disease (CD) related diagnostic codes who received medical care at University of Mississippi Medical Center between July 2012 and 2015 were identified. After thorough chart review, we identified patients with biopsy proven IBD who had also been tested for serum 25-hydroxyvitamin D [25(OH)D] concentration. We compared these patients to a previously studied cohort of healthy controls who also had vitamin D concentration checked. Logistic regression analysis was performed to determine the association between vitamin d deficiency and UC, CD, race, age, gender and body mass index (BMI). RESULTS: We identified 237 patients with confirmed IBD. Of these, only 211 had a serum 25(OH)D concentrations available in the medical record. The group of healthy controls consisted of 98 individuals with available serum 25(OH)D concentration. 43% of IBD patients were African American (AA). Patients with CD were more likely to have vitamin D concentration checked. Bivariate analysis showed that AA (51% vs 21%, P = 0.00001), subjects with BMI >30 kg/m2 (39% vs 23% P = 0.01) and CD (40% vs 26%, P = 0.04) were more likely to be vitamin D deficient than vitamin D sufficient. Those with Age > 65 were more likely to be vitamin D sufficient (46% vs 15%, P = 0.04). Multiple regression showed that only BMI > 30 kg/m2 and AA race are associated with vitamin D deficiency. CONCLUSION: BMI > 30 kg/m2 and AA race are predictive of vitamin D deficiency. Gender, age and diagnosis of IBD are not predictive of vitamin D deficiency.

Kidney biopsy for renal tubular acidosis: when tissue diagnosis makes a difference.

Renal tubular acidosis (RTA) is a disorder with variable presentations and oftentimes a nebulous underlying primary diagnosis. We describe a rare cause of RTA as an unusual complication of proton pump inhibitor (PPI) therapy. We report a case of a 33-year-old male with history of hypertension, acid reflux, allergic rhinitis, and low testosterone admitted with complaints of fatigue, weight loss, and unexplained acidosis for ~ 2 months. His medications prior to admission included losartan, omeprazole, potassium chloride, sildenafil, and testosterone propionate injections. His physical exam was unremarkable with a blood pressure of 120/80 mmHg. Initial lab work showed a nonanion gap metabolic acidosis with serum bicarbonate level of 16 mM/L and potassium 3 mM/L. Urine studies showed urine pH of 6.5 and a positive urine anion gap. The serum creatinine was within normal range(1.13 mg/dL). He required massive doses of bicarbonate and potassium supplementation with minimal improvement of serum chemistries achieved. The cause of apparent distal RTA remained elusive despite extensive blood, urine, and imaging testing. Ultimately a renal biopsy was obtained showing mild to moderate tubule-interstitial inflammation with 5% fibrosis. PPI therapy (omeprazole) was stopped, and he was started on prednisone 60 mg per day. Two weeks later, his RTA findings resolved, and he no longer required bicarbonate and potassium supplementation. Our case highlights the importance of recognizing a unique complication of RTA following PPI therapy. It also underscores the possible need for considering a kidney biopsy in the setting of nondiagnostic laboratory work up to uncover the underlying etiology of RTA and suspected allergic interstitial nephritis (AIN).

Biophysical studies of the interaction of squalamine and other cationic amphiphilic molecules with bacterial and eukaryotic membranes: importance of the distribution coefficient in membrane selectivity.

The interaction of squalamine (SQ) with eukaryotic and prokaryotic membranes was studied and compared with the interaction of two other cationic amphipathic antimicrobials (CAAs), i.e. the antibiotic polymyxin B (PMB) and the detergent hexadecyltrimethylammonium bromide (CTAB). Whole cell experiments showed that the three CAA have in common the ability to interact with lipopolysaccharide-containing membranes through a divalent cation sensitive process. Differences were found regarding their kinetics of membrane permeabilisation and their selectivity for bacteria, with a preferential permeabilisation of bacteria by PMB>SQ and no selectivity for CTAB. Experiments with lipid monolayers and bilayers showed that this selectivity did not correlate with a preferential interaction of the CAAs with lipids but rather relies on differences in their ability to penetrate lipid bilayers and to cause electrically active lesions. Incidentally, our results also suggest that the distribution coefficient of CAAs could be used to predict their selectivity for bacteria.

The insertion and transport of anandamide in synthetic lipid membranes are both cholesterol-dependent.

BACKGROUND: Anandamide is a lipid neurotransmitter which belongs to a class of molecules termed the endocannabinoids involved in multiple physiological functions. Anandamide is readily taken up into cells, but there is considerable controversy as to the nature of this transport process (passive diffusion through the lipid bilayer vs. involvement of putative proteic transporters). This issue is of major importance since anandamide transport through the plasma membrane is crucial for its biological activity and intracellular degradation. The aim of the present study was to evaluate the involvement of cholesterol in membrane uptake and transport of anandamide. METHODOLOGY/PRINCIPAL FINDINGS: Molecular modeling simulations suggested that anandamide can adopt a shape that is remarkably complementary to cholesterol. Physicochemical studies showed that in the nanomolar concentration range, anandamide strongly interacted with cholesterol monolayers at the air-water interface. The specificity of this interaction was assessed by: i) the lack of activity of structurally related unsaturated fatty acids (oleic acid and arachidonic acid at 50 nM) on cholesterol monolayers, and ii) the weak insertion of anandamide into phosphatidylcholine or sphingomyelin monolayers. In agreement with these data, the presence of cholesterol in reconstituted planar lipid bilayers triggered the stable insertion of anandamide detected as an increase in bilayer capacitance. Kinetics transport studies showed that pure phosphatidylcholine bilayers were weakly permeable to anandamide. The incorporation of cholesterol in phosphatidylcholine bilayers dose-dependently stimulated the translocation of anandamide. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that cholesterol stimulates both the insertion of anandamide into synthetic lipid monolayers and bilayers, and its transport across bilayer membranes. In this respect, we suggest that besides putative anandamide protein-transporters, cholesterol could be an important component of the anandamide transport machinery. Finally, this study provides a mechanistic explanation for the key regulatory activity played by membrane cholesterol in the responsiveness of cells to anandamide.

Isolation and characterization of microsatellite loci for mountain mullet (Agonostomus monticola).

We report on the isolation of 15 polymorphic microsatellite loci from mountain mullet (Agonostomus monticola). In the two populations sampled, loci exhibited two to 21 alleles and observed heterozygosity values ranged from 0.222 to 1.000. All loci conformed to Hardy-Weinberg equilibrium expectations, and none exhibited linkage disequilibrium. Although A. monticola is an important subsistence fishery in parts of its range, little is known about its ecology and many populations appear to be experiencing declines. These microsatellite loci should prove useful in the study of population structure of A. monticola and aid in other potential conservation efforts such as the management of hatchery broodstock.