Knockout mice with pituitary malformations help identify human cases of hypopituitarism.

BACKGROUND: Congenital hypopituitarism (CH) and its associated syndromes, septo-optic dysplasia (SOD) and holoprosencephaly (HPE), are midline defects that cause significant morbidity for affected people. Variants in 67 genes are associated with CH, but a vast majority of CH cases lack a genetic diagnosis. Whole exome and whole genome sequencing of CH patients identifies sequence variants in genes known to cause CH, and in new candidate genes, but many of these are variants of uncertain significance (VUS). METHODS: The International Mouse Phenotyping Consortium (IMPC) is an effort to establish gene function by knocking-out all genes in the mouse genome and generating corresponding phenotype data. We used mouse embryonic imaging data generated by the Deciphering Mechanisms of Developmental Disorders (DMDD) project to screen 209 embryonic lethal and sub-viable knockout mouse lines for pituitary malformations. RESULTS: Of the 209 knockout mouse lines, we identified 51 that have embryonic pituitary malformations. These genes not only represent new candidates for CH, but also reveal new molecular pathways not previously associated with pituitary organogenesis. We used this list of candidate genes to mine whole exome sequencing data of a cohort of patients with CH, and we identified variants in two unrelated cases for two genes, MORC2 and SETD5, with CH and other syndromic features. CONCLUSIONS: The screening and analysis of IMPC phenotyping data provide proof-of-principle that recessive lethal mouse mutants generated by the knockout mouse project are an excellent source of candidate genes for congenital hypopituitarism in children.

Myomatrix arrays for high-definition muscle recording.

Neurons coordinate their activity to produce an astonishing variety of motor behaviors. Our present understanding of motor control has grown rapidly thanks to new methods for recording and analyzing populations of many individual neurons over time. In contrast, current methods for recording the nervous system's actual motor output - the activation of muscle fibers by motor neurons - typically cannot detect the individual electrical events produced by muscle fibers during natural behaviors and scale poorly across species and muscle groups. Here we present a novel class of electrode devices ('Myomatrix arrays') that record muscle activity at unprecedented resolution across muscles and behaviors. High-density, flexible electrode arrays allow for stable recordings from the muscle fibers activated by a single motor neuron, called a 'motor unit,' during natural behaviors in many species, including mice, rats, primates, songbirds, frogs, and insects. This technology therefore allows the nervous system's motor output to be monitored in unprecedented detail during complex behaviors across species and muscle morphologies. We anticipate that this technology will allow rapid advances in understanding the neural control of behavior and identifying pathologies of the motor system.

Myomatrix arrays for high-definition muscle recording.

Neurons coordinate their activity to produce an astonishing variety of motor behaviors. Our present understanding of motor control has grown rapidly thanks to new methods for recording and analyzing populations of many individual neurons over time. In contrast, current methods for recording the nervous system's actual motor output - the activation of muscle fibers by motor neurons - typically cannot detect the individual electrical events produced by muscle fibers during natural behaviors and scale poorly across species and muscle groups. Here we present a novel class of electrode devices ("Myomatrix arrays") that record muscle activity at unprecedented resolution across muscles and behaviors. High-density, flexible electrode arrays allow for stable recordings from the muscle fibers activated by a single motor neuron, called a "motor unit", during natural behaviors in many species, including mice, rats, primates, songbirds, frogs, and insects. This technology therefore allows the nervous system's motor output to be monitored in unprecedented detail during complex behaviors across species and muscle morphologies. We anticipate that this technology will allow rapid advances in understanding the neural control of behavior and in identifying pathologies of the motor system.

Anisotropic Surface Broadening and Core Depletion during the Evolution of a Strong-Field Induced Nanoplasma.

Strong-field ionization of nanoscale clusters provides excellent opportunities to study the complex correlated electronic and nuclear dynamics of near-solid density plasmas. Yet, monitoring ultrafast, nanoscopic dynamics in real-time is challenging, which often complicates a direct comparison between theory and experiment. Here, near-infrared laser-induced plasma dynamics in ∼600 nm diameter helium droplets are studied by femtosecond time-resolved x-ray coherent diffractive imaging. An anisotropic, ∼20 nm wide surface region, defined as the range where the density lies between 10% and 90% of the core value, is established within ∼100 fs, in qualitative agreement with theoretical predictions. At longer timescales, however, the width of this region remains largely constant while the radius of the dense plasma core shrinks at average rates of ≈71 nm/ps along and ≈33 nm/ps perpendicular to the laser polarization. These dynamics are not captured by previous plasma expansion models. The observations are phenomenologically described within a numerical simulation; details of the underlying physics, however, remain to be explored.

Characterization of Bacterial Community Dynamics of the Human Mouth Throughout Decomposition via Metagenomic, Metatranscriptomic, and Culturing Techniques.

The postmortem microbiome has recently moved to the forefront of forensic research, and many studies have focused on the idea that predictable fluctuations in decomposer communities could be used as a "microbial clock" to determine time of death. Commonly, the oral microbiome has been evaluated using 16S rRNA gene sequencing to assess the changes in community composition throughout decomposition. We sampled the hard palates of three human donors over time to identify the prominent members of the microbiome. This study combined 16S rRNA sequencing with whole metagenomic (MetaG) and metatranscriptomic (MetaT) sequencing and culturing methodologies in an attempt to broaden current knowledge about how these postmortem microbiota change and might function throughout decomposition. In all four methods, Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were the dominant phyla, but their distributions were insufficient in separating samples based on decomposition stage or time or by donor. Better resolution was observed at the level of genus, with fresher samples from decomposition clustering away from others via principal components analysis (PCA) of the sequencing data. Key genera in driving these trends included Rothia; Lysinibacillus, Lactobacillus, Staphylococcus, and other Firmicutes; and yeasts including Candida and Yarrowia. The majority of cultures (89%) matched to sequences obtained from at least one of the sequencing methods, while 11 cultures were found in the same samples using all three methods. These included Acinetobacter gerneri, Comamonas terrigena, Morganella morganii, Proteus vulgaris, Pseudomonas koreensis, Pseudomonas moraviensis, Raoutella terrigena, Stenotrophomonas maltophilia, Bacillus cereus, Kurthia zopfii, and Lactobacillus paracasei. MetaG and MetaT data also revealed many novel insects as likely visitors to the donors in this study, opening the door to investigating them as potential vectors of microorganisms during decomposition. The presence of cultures at specific time points in decomposition, including samples for which we have MetaT data, will yield future studies tying specific taxa to metabolic pathways involved in decomposition. Overall, we have shown that our 16S rRNA sequencing results from the human hard palate are consistent with other studies and have expanded on the range of taxa shown to be associated with human decomposition, including eukaryotes, based on additional sequencing technologies.

Disintegration of diminutive liquid helium jets in vacuum.

The phenomenon of liquid jets disintegrating into droplets has attracted the attention of researchers for more than 200 years. An overwhelming fraction of these studies considered classical viscous liquid jets issuing into ambient atmospheric gases, such as air. Here, we present an optical shadowgraphy study of the disintegration of a cryogenic liquid helium jet produced with a 5 µm diameter nozzle into vacuum. The physical properties of liquid helium, such as its density, surface tension, and viscosity, change dramatically as the jet flows through the nozzle and evaporatively cools in vacuum, eventually reaching the superfluid state. In this study, we demonstrate that, at different stagnation pressures and temperatures, droplet formation may involve spraying, capillary breakup, jet branching, and/or flashing and cavitation. The average droplet sizes produced in this work range from 3.4 × 1012 to 6.5 × 1012 helium atoms or 6.7-8.3 µm in diameter. This paper also reports on the distributions of sizes and shapes of the resulting droplets.

Angular Momentum in Rotating Superfluid Droplets.

The angular momentum of rotating superfluid droplets originates from quantized vortices and capillary waves, the interplay between which remains to be uncovered. Here, the rotation of isolated submicrometer superfluid ^{4}He droplets is studied by ultrafast x-ray diffraction using a free electron laser. The diffraction patterns provide simultaneous access to the morphology of the droplets and the vortex arrays they host. In capsule-shaped droplets, vortices form a distorted triangular lattice, whereas they arrange along elliptical contours in ellipsoidal droplets. The combined action of vortices and capillary waves results in droplet shapes close to those of classical droplets rotating with the same angular velocity. The findings are corroborated by density functional theory calculations describing the velocity fields and shape deformations of a rotating superfluid cylinder.

Preformulation study of NSC-726796.

A stability-indicating high-performance liquid chromatography method to quantify 2-(2,4-difluorophenyl)-4,5,6,7-tetrafluoroisoindoline-1,3-dione (NSC-726796) and its three main degradation products was developed. This method was used to investigate its degradation kinetics and mechanism. The reaction follows first-order kinetics and appears to be base catalyzed with the maximum stability at pH 1. The products were identified as 2-(2,4-difluorophenylcarbamoyl)-3,4,5,6-tetrafluorobenzoic acid (NSC-749820), 2,4-difluoroaniline, and tetrafluorophthalic acid. The parent drug, NSC-726796, was also found to react with methanol and ethanol. NSC-726796 demonstrates antiangiogenic activity, however, when its degradant NSC749820 does not show antiangiogenic activity.

Cascade® Autologous System Platelet-Rich Fibrin Matrix in the Treatment of Chronic Leg Ulcers.

PROBLEM: Lower extremity ulcers (venous, diabetic) are often unresponsive to standard treatment. Various systemic and local cellular, vascular, and anatomical factors can result in nonhealing wounds that are refractory to normal healing processes and standard care. SOLUTION: Several published wound care guidelines strongly suggest that if an ulcer does not respond to standard good wound care within 4 weeks, then advanced wound therapies should be considered. These advanced therapies include wound bed preparation agents (negative wound pressure therapy, hyperbaric oxygen), recombinant growth factors, or bioengineered cell therapies. NEW TECHNOLOGY: The Cascade® system produces platelet-rich fibrin matrix (PRFM), a novel autologous sterile biologic, produced at the bedside from a small volume (18 mL) of the patient's own blood by using Vacutainer® separation technology optimized for fibrin and platelet isolation. Prepared as an easy to apply, suturable membrane, without the use of exogenous thrombin, PRFM consists of a dense cross-linked fibrin lattice containing intact, viable platelets with their full complement of platelet-derived growth factors. INDICATIONS FOR USE: From the FDA 510(k) clearance: The Cascade system "is designed to be used for the safe and rapid preparation of autologous platelet-rich plasma from a small sample of blood at the patient point of care." PRFM has been used to successfully treat severe venous leg ulcer (VLU), neuropathic diabetic foot ulcer (DFU), mixed arterial and Charcot-deformity associated foot ulcers. CAUTIONS: When treating venous or DFUs, the Cascade system should be used together with standard wound care practice (therapeutic compression for VLU and weight off-loading, debridement, and infection control for DFU) in patients with an adequate blood supply to the lower limb.

Stability of 5-fluoro-2'-deoxycytidine and tetrahydrouridine in combination.

In vivo, the DNA methyltransferase inhibitor, 5-fluoro-2'-deoxycytidine (FdCyd, NSC-48006), is rapidly converted to its unwanted metabolites. Tetrahydrouridine (THU, NSC-112907), a cytidine deaminase inhibitor can block the first metabolic step in FdCyd catabolism. Clinical studies have shown that co-administration with THU can inhibit the metabolism of FdCyd. The National Cancer Institute is particularly interested in a 1:5 FdCyd/THU formulation. The purpose of this study was to investigate the in vitro pH stability of FdCyd and THU individually and in combination. A stability-indicating high-performance liquid chromatography method for the quantification of both compounds and their degradants was developed using a ZIC(R)-HILIC column. The effect of THU and FdCyd on the in vitro degradation of each other was studied as a function of pH from 1.0 to 7.4 in aqueous solutions at 37 degrees C. The degradation of FdCyd appears to be first-order and acid-catalyzed. THU equilibrates with at least one of its degradants. The combination of FdCyd and THU in solution does not affect the stability of either compound. The stability and compatibility of FdCyd and THU in the solid state at increased relative humidity and at various temperatures are also evaluated.

Autologous platelet-rich fibrin matrix as cell therapy in the healing of chronic lower-extremity ulcers.

A novel autologous platelet-rich fibrin matrix membrane (PRFM) was assessed for the ability to facilitate healing in patients with chronic lower-extremity ulcers. Preliminary data are presented from a prospective trial (n=21). Twelve patients were identified with 17 venous leg ulcers (VLU) and nine bearing 13 nonvenous lower-extremity ulcers. Before enrollment, the patients were evaluated for vascular status and received appropriate surgical intervention to optimize arterial and venous circulatory status. None of the ulcers had responded to a variety of standard treatments from 4 months to 53 years. Initial ulcer size ranged from 0.7 to 65 cm(2) (mean, 11.2 cm(2)). Each PRFM-treated patient received up to three applications of either a 35 or 50 mm fenestrated membrane, depending on initial ulcer size. The primary endpoints were percent and rate of complete closure as measured by digital photography, computerized planimetery, and clinical examination. Patients were followed weekly for 12 weeks with a follow-up visit at 16 weeks. At each 4-week interval, the extent of healing was assessed, and those patients with >50% reduction in wound area were allowed to continue to complete closure. Patients with <50% closure received repeated applications. Complete closure was achieved in 66.7% of the VLU patients (64.7% of treated ulcers) in 7.1 weeks (median, 6 weeks) with an average of two applications per patient. Forty-four percent complete closure was seen with non-VLU patients (31% of treated ulcers). From the results of this small-scale pilot study, PRFM shows significant potential for closing of chronic leg ulcers.

Best-practice algorithms for the use of a bilayered living cell therapy (Apligraf) in the treatment of lower-extremity ulcers.

Tissue-engineered skin substitutes such as Apligraf have emerged over the past 20 years as among the most carefully studied and efficacious of the advanced wound modalities. These products have been proven as effective enhancements to general wound care, promoting wound closure particularly in instances where conventional wound care fails. Marketed for hard-to-heal wounds since 1998, Apligraf has become part of standard wound care in many wound centers across the United States. Despite this situation, few general wound care guidelines incorporate advanced and active wound-healing technologies, such as tissue-engineered skin products. Because of this deficiency, appropriate patient selection and proper use of these product remain largely unaddressed within the general wound care community. Here, we describe the development of guidelines surrounding optimal use of the bilayered living cell therapy, Apligraf, in the treatment of the two types of lower extremity ulcers for which the product is FDA approved: venous leg ulcer and diabetic foot ulcer. The guidelines detailed in this article focus on the identification and selection of patients who are at risk for failure of standard wound care therapy and thus appropriate for Apligraf treatment. The intended audience for these guidelines is the general wound care practitioner, for whom the developed treatment algorithms and accompanying figure legends should provide practical, user-friendly direction simplifying both patient selection and appropriate use of Apligraf within the context of good wound-healing practice.

Telling tales in school: a queer response to the heterosexist narrative structure of higher education.

If we look at general education programs, we find that implicit in them is a narrative structure. Drawing on the work of Judith Roof, I argue that this structure can promote a heterosexist logic which has among its implications the marginalization of gay and lesbian studies. I further argue, however, that this need not be the case. By appealing to Paul Ricoeur's account of narrative and to Immanuel Levinas's description of one's obligation to respond to the face of the Other, I articulate a mode of engaging the narratives at play in general education programs that mitigates their potential for marginalization. More pointedly, I argue that truly vital and ethically sound programs must work against marginalization, which means, among other things, that they must promote the cultivation of gay and lesbian and queer studies.

Detection of fatty acids from intact microorganisms by molecular beam static secondary ion mass spectrometry.

We report the use of a surface analysis approach, static secondary ion mass spectrometry (SIMS) equipped with a molecular (ReO(4)(-)) ion primary beam, to analyze the surface of intact microbial cells. SIMS spectra of 28 microorganisms were compared to fatty acid profiles determined by gas chromatographic analysis of transesterfied fatty acids extracted from the same organisms. The results indicate that surface bombardment using the molecular primary beam cleaved the ester linkage characteristic of bacteria at the glycerophosphate backbone of the phospholipid components of the cell membrane. This cleavage enables direct detection of the fatty acid conjugate base of intact microorganisms by static SIMS. The limit of detection for this approach is approximately 10(7) bacterial cells/cm(2). Multivariate statistical methods were applied in a graded approach to the SIMS microbial data. The results showed that the full data set could initially be statistically grouped based upon major differences in biochemical composition of the cell wall. The gram-positive bacteria were further statistically analyzed, followed by final analysis of a specific bacterial genus that was successfully grouped by species. Additionally, the use of SIMS to detect microbes on mineral surfaces is demonstrated by an analysis of Shewanella oneidensis on crushed hematite. The results of this study provide evidence for the potential of static SIMS to rapidly detect bacterial species based on ion fragments originating from cell membrane lipids directly from sample surfaces.

Detection of Euryarchaeota and Crenarchaeota in an oxic basalt aquifer.

Groundwater from an oxic, fractured basalt aquifer was examined for the presence of Archaea. DNA was extracted from cells concentrated from groundwater collected from five wells penetrating the eastern Snake River Plain Aquifer (Idaho, USA). Polymerase chain reaction (PCR) amplification of 16S rDNA was performed with Archaea-specific primers using both nested (ca. 200-bp product) and direct (ca. 600-bp product) PCR approaches. Estimates of the archaeal diversity were made by separating PCR products from all five wells by denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis of partial 16S rDNA sequences from two wells was performed following cloning procedures. Archaea were detected in all wells and the number of DGGE bands per well ranged from two to nine and varied according to PCR approach. There were 30 unique clonal 16S rDNA partial sequences (ca. 600 bp) within a total of 100 clones that were screened from two wells. Twenty-two of the 16S rDNA fragments recovered from the aquifer were related to the Crenarchaeota and Euryarchaeota kingdoms (one large clade of clones in the former and six smaller clades in the latter), with sequences ranging from 23.7 to 95.4% similar to those found in other investigations. The presence of potentially thermophilic or methanogenic Archaea in this fully oxic aquifer may be related to deep thermal sources or elevated dissolved methane concentrations. Many sequences were similar to those that represent non-thermophilic Crenarchaeota of which there are no known cultured members and therefore no putative function.

Comparison of extracellular enzyme activities and community composition of attached and free-living bacteria in porous medium columns.

Free-living and surface-associated microbial communities in sand-packed columns perfused with groundwater were compared by examination of compositional and functional characteristics. The composition of the microbial communities was assessed by bulk DNA extraction, PCR amplification of 16S ribosomal DNA fragments, separation of these fragments by denaturing gradient gel electrophoresis, and sequence analysis. Community function was assessed by measurement of beta-glucosidase and aminopeptidase extracellular enzyme activities. Free-living populations in the aqueous phase exhibited a greater diversity of phylotypes than populations associated with the solid phase. The attached bacterial community displayed significantly greater beta-glucosidase and aminopeptidase enzyme activities per volume of porous medium than those of the free-living community. On a per-cell basis, the attached community had a significantly higher cell-specific aminopeptidase enzyme activity (1.07 x 10(-7) nmol cell(-1) h(-1)) than the free-living community (5.02 x 10(-8) nmol cell(-1) h(-1)). Conversely, the free-living community had a significantly higher cell-specific beta-glucosidase activity (1.92 x 10(-6) nmol cell(-1) h(-1)) than the surface-associated community (6.08 x 10(-7) nmol cell(-1) h(-1)). The compositional and functional differences observed between these two communities may reflect different roles for these distinct but interacting communities in the decomposition of natural organic matter or biodegradation of xenobiotics in aquifers.