Characterizing the nuclear and cytoplasmic transcriptomes in developing and mature human cortex uncovers new insight into psychiatric disease gene regulation.

Transcriptome compartmentalization by the nuclear membrane provides both stochastic and functional buffering of transcript activity in the cytoplasm, and has recently been implicated in neurodegenerative disease processes. Although many mechanisms regulating transcript compartmentalization are also prevalent in brain development, the extent to which subcellular localization differs as the brain matures has yet to be addressed. To characterize the nuclear and cytoplasmic transcriptomes during brain development, we sequenced both RNA fractions from homogenate prenatal and adult human postmortem cortex using poly(A)+ and Ribo-Zero library preparation methods. We find that while many genes are differentially expressed by fraction and developmental expression changes are similarly detectable in nuclear and cytoplasmic RNA, the compartmented transcriptomes become more distinct as the brain matures, perhaps reflecting increased utilization of nuclear retention as a regulatory strategy in adult brain. We examined potential mechanisms of this developmental divergence including alternative splicing, RNA editing, nuclear pore composition, RNA-binding protein motif enrichment, and RNA secondary structure. Intron retention is associated with greater nuclear abundance in a subset of transcripts, as is enrichment for several splicing factor binding motifs. Finally, we examined disease association with fraction-regulated gene sets and found nuclear-enriched genes were also preferentially enriched in gene sets associated with neurodevelopmental psychiatric disorders. These results suggest that although gene-level expression is globally comparable between fractions, nuclear retention of transcripts may play an underappreciated role in developmental regulation of gene expression in brain, particularly in genes whose dysregulation is related to neuropsychiatric disorders.

Cortical cellular diversity and development in schizophrenia.

While a definitive understanding of schizophrenia etiology is far from current reality, an increasing body of evidence implicates perturbations in early development that alter the trajectory of brain maturation in this disorder, leading to abnormal function in early childhood and adulthood. This atypical development likely arises from an interaction of many brain cell types that follow distinct developmental paths. Because both cellular identity and development are governed by the transcriptome and epigenome, two levels of gene regulation that have the potential to reflect both genetic and environmental influences, mapping "omic" changes over development in diverse cells is a fruitful avenue for schizophrenia research. In this review, we provide a survey of human brain cellular composition and development, levels of genomic regulation that determine cellular identity and developmental trajectories, and what is known about how genomic regulation is dysregulated in specific cell types in schizophrenia. We also outline technical challenges and solutions to conducting cell type-specific functional genomic studies in human postmortem brain.

HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis.

During the early stages of infection, the HIV-1 capsid protects viral components from cytosolic sensors and nucleases such as cGAS and TREX, respectively, while allowing access to nucleotides for efficient reverse transcription. Here we show that each capsid hexamer has a size-selective pore bound by a ring of six arginine residues and a 'molecular iris' formed by the amino-terminal ß-hairpin. The arginine ring creates a strongly positively charged channel that recruits the four nucleotides with on-rates that approach diffusion limits. Progressive removal of pore arginines results in a dose-dependent and concomitant decrease in nucleotide affinity, reverse transcription and infectivity. This positively charged channel is universally conserved in lentiviral capsids despite the fact that it is strongly destabilizing without nucleotides to counteract charge repulsion. We also describe a channel inhibitor, hexacarboxybenzene, which competes for nucleotide binding and efficiently blocks encapsidated reverse transcription, demonstrating the tractability of the pore as a novel drug target.

Sticking It Straight: Pediatric Procedure Curriculum Initiative.

OBJECTIVES: Literature demonstrates that pediatric residents are not graduating with procedural confidence and competency. This was confirmed with our own institution's Accreditation Council for Graduate Medical Education and internal surveys. Our primary objective was to improve procedural confidence among pediatric residents with the introduction of a mandatory longitudinal pediatric procedural curriculum, including simulation in combination with online modules. METHODS: We performed a quality improvement intervention to increase resident comfort level performing Accreditation Council for Graduate Medical Education-required procedures. This study involved pediatric residents, postgraduation year (PGY) 1-3, at an academic, tertiary care hospital. Between April 2015 and June 2017, the combination of online self-directed learning modules and hands-on simulation curriculum was implemented for pediatric residents. Surveys were administered at 1-year intervals to assess self-reported comfort level on 12 procedures using a Likert scale (1 for "strongly disagree" to 5 for "strongly agree, maximum score of 60 for all procedures). RESULTS: Forty (63%) of 63 participant presurveys and 45 (71%) of 63 postsurveys were available for analysis. The mean comfort level for all procedures demonstrated a statistically significant increase from 32.4 to 37.1, or 12.7% (P = 0.005). By PGY level, the score increased from 24.4 to 30.9 (21%) for PGY1, 34.4 to 37.5 (8.3%) for PGY2, and 38.6 to 42.8 (9.8%) for PGY3 (P < 0.005). Overall, pediatric residents rated the simulation experience very favorably. CONCLUSIONS: A mandatory longitudinal procedure curriculum improved procedural comfort level among pediatric residents. Iterative curriculum designs found the most productive combination to be deliberate practice within mastery learning simulation sessions with required precourse online modules.

Esophageal Bougienage for Management of Lodged Esophageal Coins: Safe, Effective, Efficient, and Underused.

OBJECTIVES: The aims of the study are to evaluate outcomes of esophageal bougienage for management of lodged esophageal coins and to assess the extent of bougienage usage and barriers among emergency providers. METHODS: We performed a retrospective chart review of pediatric patients with single lodged esophageal coins presenting to our single academic pediatric emergency department between November 2012 and December 2018. We compared procedural outcomes, complications, length of stay (LOS), and cost between those managed with bougienage and with endoscopy. We further surveyed emergency physicians to assess the extent of bougienage usage and barriers to utilization across different institutions. RESULTS: We identified 205 patients with single lodged esophageal coins presenting during our study window. One hundred forty-seven patients ultimately underwent bougienage with 97% success and no major complications. Fifty-six patients were managed by endoscopy with 100% success and one major complication. Bougienage had significantly lower LOS (median 2.18 vs 11.92 hours, P < 0.001) and hospital charges (median $3533 vs $12,679, P < 0.001) compared with endoscopy. We received 242 completed surveys representing 38 states from primarily academic pediatric emergency physicians. The majority of respondents (90%) used specialist consult with only 4.5% performing bougienage. A total of 36.4% of respondents had never heard of the procedure and only 16.1% had ever performed it. Barriers to usage included lack of provider training (95.6%), perceived risk of complications (94.4%), and perceived lack of success (80.5%). CONCLUSIONS: Bougienage is safe and effective with significant LOS and cost benefits compared with endoscopy. Despite these advantages, the procedure is underused, because of lack of provider education and concerns regarding safety, efficacy, and both family and specialist preference, which are not supported by current literature. These data support the need for broader education regarding the bougienage technique, as well as larger prospective studies of its safety and outcomes.

Pediatric Ovarian Torsion: Should You Go With the Flow?

OBJECTIVES: Ovarian torsion (OT) is an emergency that mandates early detection and surgical detorsion to avoid catastrophic consequences of further adnexal injury. Prompt ultrasound is critical for accurate diagnosis. Traditionally, evaluation of arterial and venous flow was used as a diagnostic tool for OT, but recent radiologic research has indicated that ovarian size and size discrepancy between sides is a better diagnostic criterion. This study seeks to determine whether ovarian size discrepancy or vascular flow to the ovary is more accurate in the diagnosis of OT in the pediatric emergency population and to better describe symptoms that distinguish OT from other abdominal and pelvic pathology. METHODS: This was a retrospective, cross-sectional study evaluating all female pediatric patients, aged 1 to 18 years, who underwent a pelvic ultrasound to evaluate for OT over a 2-year period in our pediatric emergency department. Patients suitable for inclusion were identified via Nuance mPowerTM, a search engine that provides clinical analytics based on radiology reports generated within our institution. RESULTS: We reviewed the medical records of 193 female patients aged 1 to 18 years, all of whom had a pelvic ultrasound (with or without Doppler) to evaluate for OT during the study period. In comparing ovarian size on ultrasound, patients with OT had a significantly larger magnitude of difference in ovarian volume than patients without torsion (5.57× [interquartile range, 3-12.5] vs 1.56× [interquartile range, 1.24-2.25; P < 0.001]). Ovarian torsion was associated with a 33-fold increased risk of lack of arterial flow (relative risk, 33.33) and with a 9-fold increased risk of lack of venous flow (relative risk, 9.27), when compared with those patients without OT. Patients with OT were significantly more likely to have emesis and peritoneal signs on examination, as well as previous history of OT (P = 0.01, 0.02, and 0.002, respectively) than those without OT. All patients with OT reported abdominal pain. CONCLUSIONS: We found that a large size discrepancy between ovaries is indicative of OT. Our data also suggest that presence of Doppler flow on ultrasound cannot be used to exclude OT but that lack of Doppler flow on ultrasound is a significant diagnostic marker. As previous studies have also found, clinical symptoms of OT are nonspecific and do not offer any certainty in differentiating OT from other pathologies.

Schizophrenia risk variants influence multiple classes of transcripts of sorting nexin 19 (SNX19).

Genome-wide association studies (GWAS) have identified many genomic loci associated with risk for schizophrenia, but unambiguous identification of the relationship between disease-associated variants and specific genes, and in particular their effect on risk conferring transcripts, has proven difficult. To better understand the specific molecular mechanism(s) at the schizophrenia locus in 11q25, we undertook cis expression quantitative trait loci (cis-eQTL) mapping for this 2 megabase genomic region using postmortem human brain samples. To comprehensively assess the effects of genetic risk upon local expression, we evaluated multiple transcript features: genes, exons, and exon-exon junctions in multiple brain regions-dorsolateral prefrontal cortex (DLPFC), hippocampus, and caudate. Genetic risk variants strongly associated with expression of SNX19 transcript features that tag multiple rare classes of SNX19 transcripts, whereas they only weakly affected expression of an exon-exon junction that tags the majority of abundant transcripts. The most prominent class of SNX19 risk-associated transcripts is predicted to be overexpressed, defined by an exon-exon splice junction between exons 8 and 10 (junc8.10) and that is predicted to encode proteins that lack the characteristic nexin C terminal domain. Risk alleles were also associated with either increased or decreased expression of multiple additional classes of transcripts. With RACE, molecular cloning, and long read sequencing, we found a number of novel SNX19 transcripts that further define the set of potential etiological transcripts. We explored epigenetic regulation of SNX19 expression and found that DNA methylation at CpG sites near the primary transcription start site and within exon 2 partially mediate the effects of risk variants on risk-associated expression. ATAC sequencing revealed that some of the most strongly risk-associated SNPs are located within a region of open chromatin, suggesting a nearby regulatory element is involved. These findings indicate a potentially complex molecular etiology, in which risk alleles for schizophrenia generate epigenetic alterations and dysregulation of multiple classes of SNX19 transcripts.

ACEP SimBox: A Pediatric Simulation-Based Training Innovation.

Thirty million pediatric visits (<18 years old) occur across 5,000 US emergency departments (EDs) each year, with most of these cases presenting to community EDs. Simulation-based training is an effective method to improve and sustain EDs' readiness to triage and stabilize critically ill infants and children, but large simulation centers are mostly concentrated at academic hospitals. The use of pediatric simulation-based training has been limited in the community ED setting due to the high cost of equipment and limited access to content experts in pediatric critical care. We designed an innovative "off-the-shelf" simulation-based training resource, "American College of Emergency Physicians (ACEP) SimBox," that provides a free low-technology manikin along with teaching aids and train-the-trainer materials to community EDs to run a simulation drill in their own workspaces with local educators. The goal was to develop an "off-the-shelf," free, open-access, simulation-based resource to improve the readiness of community EDs to triage, resuscitate, and transfer critically ill infants as measured by presimulation and postsimulation surveys measuring opinions regarding the scenario, session experience, and most valuable aspect of the session. Between January 2018 and December 2019, 179 ACEP SimBoxes were shipped across the United States, reaching 36 of 50 states. Facilitators and participants who completed the postsimulation survey evaluated the session as a valuable use of their time. All facilitator respondents reported that the low-technology manikins, paired with their institution-specific equipment, were sufficient for learning, thus reducing costs. All participant respondents reported an increased commitment to pediatric readiness for their ED after completing the simulation session. This innovation resulted in the implementation of a unique simulation-based training intervention across many community EDs in the United States. The ACEP SimBox innovation demonstrates that an easy to use and unique simulation-based training tool can be developed, distributed, and implemented across many community EDs in the United States to help improve community ED pediatric readiness.

Dazed and Confused: Altered Mental Status in an Adolescent Male.

BACKGROUND: Absence status epilepticus (ASE) is a form of generalized nonconvulsive status epilepticus. ASE is characterized by impairment in consciousness, which can vary widely, making the diagnosis more difficult. The typical patient with ASE will be confused yet responsive and in a "trance-like state" with delayed speech, clumsy gait, and the ability to perform simple tasks after prompting. With treatment, typical ASE has an excellent prognosis and does not appear to be associated with significant neuronal damage. CASE PRESENTATION: An 11-year-old boy with history of febrile seizures presented to the emergency department (ED) with altered mental status without trauma or ingestion. His vital signs and physical examination were normal, with the exception of appearing intoxicated with sparse verbalization and inappropriate emotional responses. All laboratory results and imaging were unremarkable. While in the ED, his neurologic examination trended toward normal, returning almost to baseline. He was admitted to the hospital for video electroencephalogram, which revealed status epilepticus. After benzodiazepine therapy, epileptic electrical activity ceased and the patient's symptoms resolved. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: ASE is a rare condition that is uncommonly described in the pediatric population. These patients are frequently misdiagnosed on initial presentation as their alteration in mental status can be easily confused with ingestion, trauma, psychiatric illness, or infectious etiologies. Overturning the long-standing emergency dogma of "if they're talking to you, it's not a seizure" is undoubtedly difficult, but both pediatric and adult providers should be aware of this clinical entity.

Point-of-Care Ultrasound Facilitates Management of Ruptured Ectopic Pregnancy.

ABSTRACT: Abdominal pain in the pregnant adolescent presents a diagnostic dilemma with potential life-threatening etiologies. We present a case where point-of-care ultrasound was used to facilitate diagnosis and expedite lifesaving management of a ruptured ectopic pregnancy. We further review the technique and literature for first-trimester transabdominal point-of-care ultrasound.

HIV-1 evades innate immune recognition through specific cofactor recruitment.

Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.

Anterior insula activity regulates the associated behaviors of high fat food binge intake and cue reactivity in male rats.

Binge eating episodes are characterized by uncontrollable, excessive intake of food and are associated with binge eating disorder and some subtypes of obesity. One factor thought to contribute to binge episodes is a high level of reactivity to food-associated cues (i.e., cue reactivity). The insula is a neural node poised to regulate both binge eating and cue reactivity because of its prominent role in interpretation of internal and external cues. This work established a positive association between high fat food (HFF) binge intake and cue reactivity in male rats. Furthermore, we demonstrated that activation of the anterior insula suppressed both HFF binge intake and cue reactivity, without altering homeostatic intake of food. We further show that attenuation of HFF binge intake and cue reactivity is not due to decreased food-reward efficacy or deficits in motivation. Together, these data establish a key role for the anterior insula in the control of binge eating related-behaviors and support novel avenues for the treatment of binge eating.

Observational review of paediatric intraosseous needle placement in the paediatric emergency department.

AIM: Intraosseous (IO) access is a life-saving option during resuscitations in the paediatric emergency department (PED). This study aimed to compare success rates and time to placement for Manual IO versus EZ-IO needles in PED patients ≤8 and >8 kg. METHODS: This was a retrospective cross-sectional descriptive study of IO use in a single-centre tertiary PED from 2006 to 2014. Cases were identified through diagnosis codes for IO infusion, cardiopulmonary resuscitation and cardiac arrest and admissions to the intensive care unit. Categorical measures were compared with Z-test for comparison of two proportions and continuous with Student's t-tests. RESULTS: Of 1748 charts screened, 50 had an IO attempted. In patients ≤8 kg, Manual IO had success rate of 55% (17/31) versus 47% (8/17) for EZ-IO (P = 0.61). In patients >8 kg, Manual had success rate of 100% (2/2) versus 93% (14/15) for EZ-IO (P = 0.71). Manual performance was no different for ≤8 kg than >8 kg (P = 0.21), but EZ-IO was less successful for ≤8 kg than >8 kg (P = 0.005). In patients ≤8 kg, Manual IO had a shorter time to placement at 4.5 min versus 12.8 for EZ-IO (P = 0.02). CONCLUSION: We observed no difference in performance between Manual and EZ-IO devices in children ≤8 kg, but the Manual IO were placed more quickly. We observed lower success rates with EZ-IO devices in children ≤8 kg compared to >8 kg. Future investigations should focus specifically on training for IO placement in children ≤8 kg.

Papillary thyroid microcarcinoma: decision-making, extent of surgery, and outcomes.

BACKGROUND: The optimal extent of surgery for patients with papillary thyroid microcarcinoma (PTMC), tumors ≤1 cm, is controversial because survival is excellent regardless of approach. The objective of this study was to investigate patient and surgeon decision-making about the extent of surgery for PTMC. MATERIALS AND METHODS: We conducted a retrospective review of thyroid cancer patients operated on at a single institution from 2008-2016. To examine decision-making about the extent of surgery, we performed a discourse analysis on all available documentation looking for patient or surgeon reasons. RESULTS: Of the 853 thyroid cancer patients, 125 (14.7%) had a PTMC as their largest tumor. Overall, 27.2% of the PTMC patients underwent a thyroid lobectomy, whereas 72.8% had a total thyroidectomy (TT). Of those patients diagnosed with PTMC preoperatively (19/125), a significantly higher proportion underwent a TT (94.7% versus 68.9%, P = 0.02). In all cases, documentation indicated that these preoperatively diagnosed patients followed the surgeon's recommendation regarding the extent of surgery. Reasons surgeons cited for recommending a TT included patient and disease factors (34.6%), belief that TT was the standard treatment (21.7%), ease of follow-up (8.7%), and referring provider preference (4.3%). Of the 19 patients diagnosed preoperatively, four (21.1%) patients had a complication, one (5.3%) of which was permanent and potentially avoidable with less extensive surgery. CONCLUSIONS: These data suggest that surgeons drive decision-making about the extent of thyroidectomy in patients with preoperatively diagnosed PTMC. With recent guidelines recommending thyroid lobectomy, closer examination of decision-making is needed to ensure that patients make well-informed, preference-based decisions.

Activation of human γδ T cells by cytosolic interactions of BTN3A1 with soluble phosphoantigens and the cytoskeletal adaptor periplakin.

The three butyrophilin BTN3A molecules, BTN3A1, BTN3A2, and BTN3A3, are members of the B7/butyrophilin-like group of Ig superfamily receptors, which modulate the function of T cells. BTN3A1 controls activation of human Vγ9/Vδ2 T cells by direct or indirect presentation of self and nonself phosphoantigens (pAg). We show that the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate binds to the intracellular B30.2 domain of BTN3A1 with an affinity of 1.1 µM, whereas the endogenous pAg isopentenyl pyrophosphate binds with an affinity of 627 µM. Coculture experiments using knockdown cell lines showed that in addition to BTN3A1, BTN3A2 and BTN3A3 transmit activation signals to human γδ T cells in response to (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and the aminobisphosphonate drug zoledronate that causes intracellular accumulation of isopentenyl pyrophosphate. The plakin family member periplakin, identified in yeast two-hybrid assays, interacted with a membrane-proximal di-leucine motif, located proximal to the B30.2 domain in the BTN3A1 cytoplasmic tail. Periplakin did not interact with BTN3A2 or BTN3A3, which do not contain the di-leucine motif. Re-expression into a BTN3A1 knockdown line of wild-type BTN3A1, but not of a variant lacking the periplakin binding motif, BTN3A1Δexon5, restored γδ T cell responses, demonstrating a functional role for periplakin interaction. These data, together with the widespread expression in epithelial cells, tumor tissues, and macrophages detected using BTN3A antiserum, are consistent with complex functions for BTN3A molecules in tissue immune surveillance and infection, linking the cell cytoskeleton to γδ T cell activation by indirectly presenting pAg to the Vγ9/Vδ2 TCR.

Accuracy of intuition in clinical decision-making among novice clinicians.

AIMS: To assess the reliance on intuitive and analytical approaches during clinical decision-making among novice clinicians and whether that reliance is associated with accurate decision-making. BACKGROUND: Nurse educators and managers tend to emphasize analysis over intuition during clinical decision-making though nurses typically report some reliance on intuition in their practice. We hypothesized that under certain conditions, reliance on intuition would support accurate decision-making, even among novices. DESIGN: This study utilized an experimental design with clinical complication (familiar vs. novel) and decision phase (cue acquisition, diagnosis and action) as within-subjects' factors, and simulation role (observer, family, auxiliary nurse and primary nurse) as between-subjects' factor. METHODS: We examined clinical decision-making accuracy among final semester pre-licensure nursing students in a simulation experience. Students recorded their reasoning about emerging clinical complications with their patient during two distinct points in the simulation; one point involved a familiar complication and the other a relatively novel complication. All data were collected during Spring 2015. RESULTS: Although most participants relied more heavily on analysis than on intuition, use of intuition during the familiar complication was associated with more accurate decision-making, particularly in guiding attention to relevant cues. With the novel complication, use of intuition appeared to hamper decision-making, particularly for those in an observer role. CONCLUSION: Novice clinicians should be supported by educators and nurse managers to note when their intuitions are likely to be valid. Our findings emphasize the integrated nature of intuition and analysis in clinical decision-making.

HIV-1 Resistance to the Capsid-Targeting Inhibitor PF74 Results in Altered Dependence on Host Factors Required for Virus Nuclear Entry.

UNLABELLED: During HIV-1 infection of cells, the viral capsid plays critical roles in reverse transcription and nuclear entry of the virus. The capsid-targeting small molecule PF74 inhibits HIV-1 at early stages of infection. HIV-1 resistance to PF74 is complex, requiring multiple amino acid substitutions in the viral CA protein. Here we report the identification and analysis of a novel PF74-resistant mutant encoding amino acid changes in both domains of CA, three of which are near the pocket where PF74 binds. Interestingly, the mutant virus retained partial PF74 binding, and its replication was stimulated by the compound. The mutant capsid structure was not significantly perturbed by binding of PF74; rather, the mutations inhibited capsid interactions with CPSF6 and Nup153 and altered HIV-1 dependence on these host factors and on TNPO3. Moreover, the replication of the mutant virus was markedly impaired in activated primary CD4(+) T cells and macrophages. Our results suggest that HIV-1 escapes a capsid-targeting small molecule inhibitor by altering the virus's dependence on host factors normally required for entry into the nucleus. They further imply that clinical resistance to inhibitors targeting the PF74 binding pocket is likely to be strongly limited by functional constraints on HIV-1 evolution. IMPORTANCE: The HIV-1 capsid plays critical roles in early steps of infection and is an attractive target for therapy. Here we show that selection for resistance to a capsid-targeting small molecule inhibitor can result in viral dependence on the compound. The mutant virus was debilitated in primary T cells and macrophages--cellular targets of infection in vivo. The mutations also altered the virus's dependence on cellular factors that are normally required for HIV-1 entry into the nucleus. This work provides new information regarding mechanisms of HIV-1 resistance that should be useful in efforts to develop clinically useful drugs targeting the HIV-1 capsid.

Host cofactors and pharmacologic ligands share an essential interface in HIV-1 capsid that is lost upon disassembly.

The HIV-1 capsid is involved in all infectious steps from reverse transcription to integration site selection, and is the target of multiple host cell and pharmacologic ligands. However, structural studies have been limited to capsid monomers (CA), and the mechanistic basis for how these ligands influence infection is not well understood. Here we show that a multi-subunit interface formed exclusively within CA hexamers mediates binding to linear epitopes within cellular cofactors NUP153 and CPSF6, and is competed for by the antiretroviral compounds PF74 and BI-2. Each ligand is anchored via a shared phenylalanine-glycine (FG) motif to a pocket within the N-terminal domain of one monomer, and all but BI-2 also make essential interactions across the N-terminal domain: C-terminal domain (NTD:CTD) interface to a second monomer. Dissociation of hexamer into CA monomers prevents high affinity interaction with CPSF6 and PF74, and abolishes binding to NUP153. The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation. NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency. These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly.

Hyperglycaemic hyperosmolar syndrome in children: Patient characteristics, diagnostic delays and associated complications.

AIMS: The aim of this study was to describe the demographical and clinical characteristics, diagnostic difficulties, and morbidity and mortality in children with hyperglycaemic hyperosmolar syndrome (HHS). METHODS: Retrospective cross-section descriptive study of children (<18 years of age) at an urban, tertiary, academic Children's Hospital diagnosed with HHS from January 2002 to December 2011. RESULTS: Six patients met inclusion criteria for the diagnosis of HHS. Age ranged from 6 to 16 years with 4 (67%) patients younger than 13 years. Four (67%) patients were women and 5 (83%) were African-American. Body mass index (BMI)-for-age percentile was >97% in four (67%) patients. Three (60%) of five patients seen as outpatients were misdiagnosed, two cases were seen twice before an accurate diagnosis of HHS was made. There was one (17%) death. Complications included three patients with acute renal failure, one patient with seizures, and one patient with rhabdomyolysis and compartment syndrome leading to below the knee amputation. Malignant hyperthermia and ventricular arrhythmias occurred in the patient who expired. Three of the five patients who had autoantibody tests had positive results and were diagnosed with type 1 diabetes mellitus. CONCLUSIONS: Characteristics of children with HSS are variable for age, gender and BMI-for-age percentile and not predominately limited to obese male adolescent African-American. Delay in diagnosis is common and morbidity and mortality in paediatric HHS are significant. The subsequent diagnosis of patients presenting with HHS includes type 1 and 2 diabetes mellitus.

CIP4 coordinates with phospholipids and actin-associated proteins to localize to the protruding edge and produce actin ribs and veils.

Cdc42-interacting protein 4 (CIP4), a member of the F-BAR family of proteins, plays important roles in a variety of cellular events by regulating both membrane and actin dynamics. In many cell types, CIP4 functions in vesicle formation, endocytosis and membrane tubulation. However, recent data indicate that CIP4 is also involved in protrusion in some cell types, including cancer cells (lamellipodia and invadopodia) and neurons (ribbed lamellipodia and veils). In neurons, CIP4 localizes specifically to extending protrusions and functions to limit neurite outgrowth early in development. The mechanism by which CIP4 localizes to the protruding edge membrane and induces lamellipodial/veil protrusion and actin rib formation is not known. Here, we show that CIP4 localization to the protruding edge of neurons is dependent on both the phospholipid content of the plasma membrane and the underlying organization of actin filaments. Inhibiting phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production decreases CIP4 at the membrane. CIP4 localization to the protruding edge is also dependent on Rac1/WAVE1, rather than Cdc42/N-WASP. Capping actin filaments with low concentrations of cytochalasin D or by overexpressing capping protein dramatically decreases CIP4 at the protruding edge, whereas inactivating Arp2/3 drives CIP4 to the protruding edge. We also demonstrate that CIP4 dynamically colocalizes with Ena/VASP and DAAM1, two proteins known to induce unbranched actin filament arrays and play important roles in neuronal development. Together, this is the first study to show that the localization of an F-BAR protein depends on both actin filament architecture and phospholipids at the protruding edge of developing neurons.