SLC41A1 is essential for magnesium homeostasis in vivo.

Solute carrier family 41 member A1 (SLC41A1) has been suggested to mediate magnesium (Mg2+) transport by several in vitro studies. However, the physiological function of SLC41A1 remains to be elucidated. In this study, cellular Mg2+ transport assays combined with zebrafish slc41a1 knockdown experiments were performed to disclose SLC41A1 function and its physiological relevance. The gene slc41a1 is ubiquitously expressed in zebrafish tissues and is regulated by water and dietary Mg2+ availability. Knockdown of slc41a1 in zebrafish larvae grown in a Mg2+-free medium resulted in a unique phenotype characterized by a decrease in zebrafish Mg content. This decrease shows that SLC41A1 is required to maintain Mg2+ balance and its dysfunction results in renal Mg2+ wasting in zebrafish larvae. Importantly, the Mg content of the larvae is rescued when mouse SLC41A1 is expressed in slc41a1-knockdown zebrafish. Conversely, expression of mammalian SLC41A1-p.Asp262Ala, harboring a mutation in the ion-conducting SLC41A1 pore, did not reverse the renal Mg2+ wasting. 25Mg2+ transport assays in human embryonic kidney 293 (HEK293) cells overexpressing SLC41A1 demonstrated that SLC41A1 mediates cellular Mg2+ extrusion independently of sodium (Na+). In contrast, SLC41A1-p.Asp262Ala expressing HEK293 cells displayed similar Mg2+ extrusion activities than control (mock) cells. In polarized Madin-Darby canine kidney cells, SLC41A1 localized to the basolateral cell membrane. Our results demonstrate that SLC41A1 facilitates renal Mg2+ reabsorption in the zebrafish model. Furthermore, our data suggest that SLC41A1 mediates both Mg2+ uptake and extrusion.

Escape Mutations in NS4B Render Dengue Virus Insensitive to the Antiviral Activity of the Paracetamol Metabolite AM404.

Despite the enormous disease burden associated with dengue virus infections, a licensed antiviral drug is lacking. Here, we show that the paracetamol (acetaminophen) metabolite AM404 inhibits dengue virus replication. Moreover, we find that mutations in NS4B that were previously found to confer resistance to the antiviral compounds NITD-618 and SDM25N also render dengue virus insensitive to AM404. Our work provides further support for NS4B as a direct or indirect target for antiviral drug development.

Identification of a new dengue virus inhibitor that targets the viral NS4B protein and restricts genomic RNA replication.

Dengue virus (DENV) is an important human arthropod-borne virus with a major impact on public health. Nevertheless, a licensed vaccine or specific treatment is still lacking. We therefore screened the NIH Clinical Collection (NCC), a library of drug-like small molecules, for inhibitors of DENV replication using a cell line that contains a stably replicating DENV serotype 2 (DENV2) subgenomic replicon. The most potent DENV inhibitor in the NCC was δ opioid receptor antagonist SDM25N. This compound showed antiviral activity against wild-type DENV2 in both Hela and BHK-21 cells, but not in the C6/36 cell line derived from the mosquito Aedes albopictus. The structurally related compound naltrindole also inhibited DENV replication, albeit less potently. Using a transient subgenomic replicon, we demonstrate that SDM25N restricts genomic RNA replication rather than translation of the viral genome. We identified a single amino acid substitution (F164L) in the NS4B protein that confers resistance to SDM25N. Remarkably, an NS4B amino acid substitution (P104L), which was previously shown to confer resistance to the DENV inhibitor NITD-618, also provided resistance to SDM25N. In conclusion, we have identified a new DENV inhibitor, SDM25N, which restricts genomic RNA replication by - directly or indirectly - targeting the viral NS4B protein.

Fistula after 2-flap palatoplasty: a 20-year review.

Oronasal fistula formation is a recalcitrant complication following palatoplasty, resulting in nasal emission during speech and deglutition. We review our series to identify factors associated with fistula incidence. A retrospective review of all children with nonsyndromic cleft palate who underwent 2-flap palatoplasty by the senior author from July 1983 to August 2004, was performed. Patient demographics, cleft characteristics, and operative techniques were recorded for each patient. The incidence rates of fistula, pharyngeal flap, and reoperation were used as primary outcomes. Statistical comparisons of frequencies were performed using Fisher exact test. Comparisons of means were performed using chi2 analysis. A total of 332 consecutive children met inclusion criteria. Mean age at palatoplasty was 10.8 months, and mean follow-up was 74.1 months. Eight children (2.4%) were found to have fistulae postoperatively, ranging in size from 2 to 15 mm. Four palatal fistulas occurred in the soft palate, 2 at the junction of the hard and soft palate, 1 in the hard palate, and 1 at the incisive foramen. Symptomatic nasal emission requiring reoperation occurred in 5 children. Two of these 5 children required a second operation to achieve fistula closure. Forty pharyngeal flaps were required for correction of velopharyngeal incompetence (12.0%). Two-flap palatoplasty remains a highly successful technique for closure of a variety of palatal clefts, with low fistula incidence. Surgical technique and experience are factors associated with low fistula incidence.

Revascularization of a specific angiosome for limb salvage: does the target artery matter?

Ischemic wounds of the lower extremity can fail to heal despite successful revascularization. The foot can be divided into six anatomic regions (angiosomes) fed by distinct source arteries arising from the posterior tibial (three), anterior tibial (one), and peroneal (two) arteries. This study investigated whether bypass to the artery directly feeding the ischemic angiosome had an impact on wound healing and limb salvage. Retrospective analysis was performed for 52 nonhealing lower extremity wounds (48 patients) requiring tibial bypass over a 2-year period. Preoperative arteriograms were reviewed to determine arterial anatomy relative to each wound's specific angiosome and bypass anatomy. Patients were divided into two groups; direct revascularization (DR, bypass to the artery directly feeding the ischemic angiosome) or indirect revascularization (IR, bypass unrelated to the ischemic angiosome). Wound outcome was analyzed with regard to the endpoints of complete healing, amputation, or death unrelated to the wound. Time to healing was also noted for healed wounds. Based on preoperative arteriography, 51% (n = 27) of the wounds received DR to the ischemic angiosome, while 49% (n = 25) underwent IR. There were no statistically significant differences in the comorbidities of the two groups. Revascularization was via tibial bypass using the saphenous vein (n = 34, 65%) or polytetrafluoroethylene with a distal vein patch (n = 18, 35%). Bypasses were performed to the anterior tibial (n = 22, 42%), posterior tibial (n = 17, 33%), or peroneal (n = 13, 25%) arteries based on the surgeon's judgment. One bypass failed in the perioperative period and was excluded from the analysis. The remaining bypasses were patent at the time of wound analysis. Due to a 17% mortality rate during follow-up, 43 wounds were available for endpoint analysis. This analysis demonstrated that 77% of wounds (n = 33) progressed to complete healing and 23% of wounds (n = 10) failed to heal with resultant amputation. In the DR group, there was 91% healing with a 9% amputation rate. In the IR group, there was 62% healing with a 38% amputation rate (p = 0.03). In those wounds that did heal, total time to healing was not significantly different--DR 162.4 days versus IR 159.8 days (p = 0.95). Revascularization plays a crucial role in the treatment of ischemic lower extremity wounds. We believe that direct revascularization of the angiosome specific to the anatomy of the wound leads to a higher rate of healing and limb salvage. Although many factors must be considered in choosing the target artery for revascularization, consideration should be given to revascularization of the artery directly feeding the ischemic angiosome.

Alterations in CNS activity induced by botulinum toxin treatment in spasmodic dysphonia: an H215O PET study.

Speech-related changes in regional cerebral blood flow (rCBF) were measured using H(2)(15)O positron-emission tomography in 9 adults with adductor spasmodic dysphonia (ADSD) before and after botulinum toxin (BTX) injection and 10 age- and gender-matched volunteers without neurological disorders. Scans were acquired at rest and during production of continuous narrative speech and whispered speech. Speech was recorded during scan acquisition for offline quantification of voice breaks, pitch breaks, and percentage aperiodicity to assess correlations between treatment-related changes in rCBF and clinical improvement. Results demonstrated that speech-related responses in heteromodal sensory areas were significantly reduced in persons with ADSD, compared with volunteers, before the administration of BTX. Three to 4 weeks after BTX injection, speech-related responses were significantly augmented in these regions and in left hemisphere motor areas commonly associated with oral-laryngeal motor control. This pattern of responses was most strongly correlated with the objective measures of clinical improvement (decreases in the frequency of voice breaks, pitch breaks, and percentage aperiodicity). These data suggest a pathophysiological model for ADSD in which BTX treatment results in more efficient cortical processing of sensory information, making this information available to motor areas that use it to more effectively regulate laryngeal movements.