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Lameness in pigs is one of the major reasons for culling and early losses in pigs. This can be linked to osteoporosis due to pathologic alterations in bone mineral density (BMD) or bone mineral content (BMC) and may also be linked to the sex. Dealing with the ban on piglet castration without anaesthesia in Germany 2021, we have three male “sex” types: entire boars (EB), immunocastrated boars (IB), and surgically castrated boars (SB). The hypothesis of the present study is that BMC or BMD varies between different male sex types. If sex has an effect on bone mineralization (BMC or BMD) and if this affects leg health, it could result in more lameness and problems during fattening in the negatively affected sex type. The present study evaluated bone mineralization (in terms of BMD and BMC) and body composition traits using dual-energy X-ray absorptiometry (DXA) three times during growth at 30, 50, and 90 kg live body weight. Nine body regions were analysed for bone mineral traits and compared for different male sex types and the fattening season. Significant differences were found regarding BMD (and BMC) among EB, IB, and SB for whole-body BMD (BMC). Additionally significant differences were found in the front and lower hind limbs, where SB showed a significantly higher BMD compared to EB, with IB in between. Additionally regional differences were detected among the groups. Further studies are needed to evaluate the effect of these differences in bone mineralization on leg health.

Cleavage of amyloid precursor protein (APP) by β-secretase BACE1 initiates the production and accumulation of neurotoxic amyloid-β peptides, which is widely considered an essential pathogenic mechanism in Alzheimer's disease (AD). Here, we report that BACE1 is essential for normal auditory function. Compared with wild-type littermates, BACE1−/−mice of either sex exhibit significant hearing deficits, as indicated by increased thresholds and reduced amplitudes in auditory brainstem responses (ABRs) and decreased distortion product otoacoustic emissions (DPOAEs). Immunohistochemistry revealed aberrant synaptic organization in the cochlea and hypomyelination of auditory nerve fibers as predominant neuropathological substrates of hearing loss in BACE1−/−mice. In particular, we found that fibers of spiral ganglion neurons (SGN) close to the organ of Corti are disorganized and abnormally swollen. BACE1 deficiency also engenders organization defects in the postsynaptic compartment of SGN fibers with ectopic overexpression of PSD95 far outside the synaptic region. During postnatal development, auditory fiber myelination in BACE1−/−mice lags behind dramatically and remains incomplete into adulthood. We relate the marked hypomyelination to the impaired processing of Neuregulin-1 when BACE1 is absent. To determine whether the cochlea of adult wild-type mice is susceptible to AD treatment-like suppression of BACE1, we administered the established BACE1 inhibitor NB-360 for 6 weeks. The drug suppressed BACE1 activity in the brain, but did not impair hearing performance and, upon neuropathological examination, did not produce the characteristic cochlear abnormalities of BACE1−/−mice. Together, these data strongly suggest that the hearing loss of BACE1 knock-out mice represents a developmental phenotype.SIGNIFICANCE STATEMENTGiven its crucial role in the pathogenesis of Alzheimer's disease (AD), BACE1 is a prime pharmacological target for AD prevention and therapy. However, the safe and long-term administration of BACE1-inhibitors as envisioned in AD requires a comprehensive understanding of the various physiological functions of BACE1. Here, we report that BACE1 is essential for the processing of auditory signals in the inner ear, as BACE1-deficient mice exhibit significant hearing loss. We relate this deficit to impaired myelination and aberrant synapse formation in the cochlea, which manifest during postnatal development. By contrast, prolonged pharmacological suppression of BACE1 activity in adult wild-type mice did not reproduce the hearing deficit or the cochlear abnormalities of BACE1 null mice.

Parental cannabis use has been associated with adverse neurodevelopmental outcomes in offspring, but how such phenotypes are transmitted is largely unknown. Using reduced representation bisulphite sequencing (RRBS), we recently demonstrated that cannabis use is associated with widespread DNA methylation changes in human and rat sperm. Discs-Large Associated Protein 2 (DLGAP2), involved in synapse organization, neuronal signaling, and strongly implicated in autism, exhibited significant hypomethylation (p

Cannabis use alters sperm DNA methylation, but the potential reversibility of these changes is unknown. Semen samples from cannabis users and non-user controls were collected at baseline and again following a 77-day period of cannabis abstinence (one spermatogenic cycle). Users and controls did not significantly differ by demographics or semen analyses. Whole-genome bisulfite sequencing identified 163 CpG sites with significantly different DNA methylation in sperm between groups (P

Background: The human auditory nerve contains 30,000 nerve fibers (NFs) that relay complex speech information to the brain with spectacular acuity. How speech is coded and influenced by various conditions is not known. It is also uncertain whether human nerve signaling involves exclusive proteins and gene manifestations compared with that of other species. Such information is difficult to determine due to the vulnerable, “esoteric,” and encapsulated human ear surrounded by the hardest bone in the body. We collected human inner ear material for nanoscale visualization combining transmission electron microscopy (TEM), super-resolution structured illumination microscopy (SR-SIM), and RNA-scope analysis for the first time. Our aim was to gain information about the molecular instruments in human auditory nerve processing and deviations, and ways to perform electric modeling of prosthetic devices.Material and Methods: Human tissue was collected during trans-cochlear procedures to remove petro-clival meningioma after ethical permission. Cochlear neurons were processed for electron microscopy, confocal microscopy (CM), SR-SIM, and high-sensitive in situ hybridization for labeling single mRNA transcripts to detect ion channel and transporter proteins associated with nerve signal initiation and conductance.Results: Transport proteins and RNA transcripts were localized at the subcellular level. Hemi-nodal proteins were identified beneath the inner hair cells (IHCs). Voltage-gated ion channels (VGICs) were expressed in the spiral ganglion (SG) and axonal initial segments (AISs). Nodes of Ranvier (NR) expressed Nav1.6 proteins, and encoding genes critical for inter-cellular coupling were disclosed.Discussion: Our results suggest that initial spike generators are located beneath the IHCs in humans. The first NRs appear at different places. Additional spike generators and transcellular communication may boost, sharpen, and synchronize afferent signals by cell clusters at different frequency bands. These instruments may be essential for the filtering of complex sounds and may be challenged by various pathological conditions.

Background: The human cochlea was earlier believed to lack capacity to mount specific immune responses. Recent studies established that the human cochlea holds macrophages. The cells appear to surveil, dispose of, and restore wasted cells to maintain tissue integrity. Macrophage activities are believed to be the central elements in immune responses and could swiftly defuse invading microbes that enter via adjacent infection-prone areas. This review updates recent human studies in light of the current literature and adds information about chemokine gene expression.Materials and Methods: We analyzed surgically obtained human tissue using immunohistochemistry, confocal microscopy, and multichannel super-resolution structured illumination microscopy. The samples were considered representative of steady-state conditions. Antibodies against the ionized calcium-binding adaptor molecule 1 were used to identify the macrophages. CD68 and CD11b, and the major histocompatibility complex type II (MHCII) and CD4 and CD8 were analyzed. The RNAscope technique was used for fractalkine gene localization.Results: Many macrophages were found around blood vessels in the stria vascularis but not CD4 and CD8 lymphocytes. Amoeboid macrophages were identified in the spiral ganglion with surveilling “antennae” projecting against targeted cells. Synapse-like contacts were seen on spiral ganglion cell bodies richly expressing single CXC3CL gene transcripts. Branching neurite-like processes extended along central and peripheral axons. Active macrophages were occasionally found near degenerating hair cells. Some macrophage-interacting T lymphocytes were observed between the scala tympani wall and Rosenthal's canal. CD4 and CD8 cells were not found in the organ of Corti.Conclusions: The results indicate that the human cochlea is equipped with macrophages and potentially lymphocytes, suggesting both an innate and adaptive immune capacity. A rich expression of fractalkine gene transcripts in spiral ganglion neurons suggest an essential role for auditory nerve protection, as has been demonstrated experimentally. The findings provide further information on the important role of the immune machinery present in the human inner ear and its potential to carry adverse immune reactions, including cytotoxic and foreign body responses. The results can be used to form a rationale for therapies aiming to modulate these immune activities.

Neuronal diversity in the cochlea is largely determined by ion channels. Among voltage‐gated channels, hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels open with hyperpolarization and depolarize the cell until the resting membrane potential. The functions for hearing are not well elucidated and knowledge about localization is controversial. We created a detailed map of subcellular location and co‐expression of all four HCN subunits across different mammalian species including CBA/J, C57Bl/6N, Ly5.1 mice, guinea pigs, cats, and human subjects. We correlated age‐related hearing deterioration in CBA/J and C57Bl/6N with expression levels of HCN1, −2, and −4 in individual auditory neurons from the same cohort. Spatiotemporal expression during murine postnatal development exposed HCN2 and HCN4 involvement in a critical phase of hair cell innervation. The huge diversity of subunit composition, but lack of relevant heteromeric pairing along the perisomatic membrane and axon initial segments, highlighted an active role for auditory neurons. Neuron clusters were found to be the hot spots of HCN1, −2, and −4 immunostaining. HCN channels were also located in afferent and efferent fibers of the sensory epithelium. Age‐related changes on HCN subtype expression were not uniform among mice and could not be directly correlated with audiometric data. The oldest mice groups revealed HCN channel up‐ or downregulation, depending on the mouse strain. The unexpected involvement of HCN channels in outer hair cell function where HCN3 overlaps prestin location emphasized the importance for auditory function. A better understanding may open up new possibilities to tune neuronal responses evoked through electrical stimulation by cochlear implants., All HCN channel subtypes were located at subcellular level in human and compared with other mammals such as guinea pig, cat, and three mouse strains. Changes in HCN expression levels were quantified in aging mice and murine postnatal development. The widespread expression and distinct localization emphasized the importance of HCN channels for sound coding and electrical stimulation with cochlear implants

Background Male obesity has profound effects on morbidity and mortality, but relatively little is known about the impact of obesity on gametes and the potential for adverse effects of male obesity to be passed to the next generation. DNA methylation contributes to gene regulation and is erased and re-established during gametogenesis. Throughout post-pubertal spermatogenesis, there are continual needs to both maintain established methylation and complete DNA methylation programming, even during epididymal maturation. This dynamic epigenetic landscape may confer increased vulnerability to environmental influences, including the obesogenic environment, that could disrupt reprogramming fidelity. Here we conducted an exploratory analysis that showed that overweight/obesity (n = 20) is associated with differences in mature spermatozoa DNA methylation profiles relative to controls with normal BMI (n = 47). Results We identified 3264 CpG sites in human sperm that are significantly associated with BMI (p Conclusions Male obesity is associated with altered sperm DNA methylation profiles that appear to affect reprogramming fidelity in a subset of sperm, suggestive of an influence on the spermatogonia. Further work is required to determine the potential heritability of these DNA methylation alterations. If heritable, these changes have the potential to impede normal development.

Men consume the most nicotine and cannabis products but impacts on sperm epigenetics are poorly characterized. Evidence suggests that preconception exposure to these drugs alters offspring neurodevelopment. Epigenetics may in part facilitate heritability. We therefore compared effects of exposure to tetrahydrocannabinol (THC) and nicotine on DNA methylation in rat sperm at genes involved in neurodevelopment. Reduced representation bisulfite sequencing data from sperm of rats exposed to THC via oral gavage showed that seven neurodevelopmentally active genes were significantly differentially methylated versus controls. Pyrosequencing data revealed majority overlap in differential methylation in sperm from rats exposed to THC via injection as well as those exposed to nicotine. Neurodevelopmental genes including autism candidates are vulnerable to environmental exposures and common features may mediate this vulnerability. We discovered that autism candidate genes are significantly enriched for bivalent chromatin structure, suggesting this configuration may increase vulnerability of genes in sperm to disrupted methylation.

As marijuana legalization is increasing, research regarding possible long-term risks for users and their offspring is needed. Little data exists on effects of paternal tetrahydrocannabinol (THC) exposure prior to reproduction. This study determined if chronic THC exposure alters sperm DNA methylation (DNAm) and if such effects are intergenerationally transmitted. Adult male rats underwent oral gavage with THC or vehicle control. Differentially methylated (DM) loci in motile sperm were identified using reduced representation bisulfite sequencing (RRBS). Another cohort was injected with vehicle or THC, and sperm DNAm was analyzed. Finally, THC-exposed and control adult male rats were mated with THC-naïve females. DNAm levels of target genes in brain tissues of the offspring were determined by pyrosequencing. RRBS identified 2,940 DM CpGs mapping to 627 genes. Significant hypermethylation was confirmed (p Por), involved in toxin processing and disorders of sexual development. Por hypermethylation was not observed after THC injection or in the subsequent generation. These results support that THC alters DNAm in sperm and that route of exposure can have differential effects. Although we did not observe evidence of intergenerational transmission of the DNAm change, larger studies are required to definitively exclude this possibility.

Background/Aims: Calcium-binding proteins in neurons buffer intracellular free Ca2+ ions, which interact with proteins controlling enzymatic and ion channel activity. The heterogeneous distribution of calretinin, calbindin, and parvalbumin influences calcium homeostasis, and calcium-related neuronal processes play an important role in neuronal aging and degeneration. This study evaluated age-related changes in calretinin, calbindin, and parvalbumin immune reactivity in spiral ganglion cells. Methods: A total of 16 C57BL/6J and 16 129/SvJ mice at different ages (2, 4, 7, and 12 months) were included in the study. Hearing thresholds were assessed using auditory brainstem response before inner ears were excised for further evaluation. Semiquantitative immunohistochemistry for the aforementioned calcium-binding proteins was performed at the cellular level. Results: The hearing thresholds of C57BL/6J and 129/SvJ mice increased significantly by 7 months of age. The average immune reactivity of calbin­din as well as the relative number of positive cells increased significantly with aging, but no significant alterations in calretinin or parvalbumin were observed. Conclusions: Upregulation of calbindin could serve as a protection to compensate for functional deficits that occur with aging. Expression of both calretinin and parvalbumin seem to be stabilizing factors in murine inner ears up to the age of 12 months in C57BL/6J and 129/SvJ mice.

Background: Alzheimer’s disease (AD) animal models have revealed neuroprotective actions of Bryostatin-1 mediated by activation of novel PKC isoforms, suppression of beta-amyloid and downregulation of inflammatory and angiogenic events, making Bryostatin-1 an attractive candidate for attenuating AD-associated neural, vascular, and cognitive disturbances. Objective: To further enhance Bryostatin-1 efficacy, nanoparticle-encapsulated Bryostatin-1 formulations were prepared. Methods: We compared nano-encapsulated and unmodified Bryostatin-1 in in vitro models of neuronal PKC-d, PKC-e isoforms, α-secretase and studied nano-encapsulated Bryostatin-1 in an AD mouse model of spatial memory (BC3-Tg (APPswe, PSEN1 dE9) 85Dbo/J mice). Results: We found that nanoencapsulated Bryostatin-1 formulations displayed activity greater or equal to that of unmodified Bryostatin-1 in PKC-δ and -ε and α-secretase activation assays. We next evaluated how treatment with a nanoencapsulated Bryostatin-1 formulation facilitated spatial learning in the Morris water maze. AD transgenic mice (6.5 to 8 months of age) were treated with nanoparticle encapsulated Bryostatin-1 formulation (1, 2.5, or 5 μg/mouse) three times the week before testing and then daily for each of the 5 days of testing. Across the acquisition phase, mice treated with nanoencapsulated Bryostatin-1 had shorter latencies, increased % time in the target zone and decreased % time in the opposite quadrant. The mice were given retention testing after a 2-week period without drug treatment. Mice treated with nanoencapsulated Bryostatin-1 had shorter latencies to find the escape platform, indicating retention of spatial memory. Conclusion: These data suggest that cognitive deficits associated with AD could be treated using highly potent nanoparticle-encapsulated formulations of Bryostatin-1.

The isolated anatomical position and blood-labyrinth barrier hampers systemic drug delivery to the mammalian inner ear. Intratympanic placement of drugs and permeation via the round- and oval window are established methods for local pharmaceutical treatment. Mechanisms of drug uptake and pathways for distribution within the inner ear are hard to predict. The complex microanatomy with fluid-filled spaces separated by tight- and leaky barriers compose various compartments that connect via active and passive transport mechanisms. Here we provide a review on the inner ear architecture at light- and electron microscopy level, relevant for drug delivery. Focus is laid on the human inner ear architecture. Some new data add information on the human inner ear fluid spaces generated with high resolution microcomputed tomography at 15 μm resolution. Perilymphatic spaces are connected with the central modiolus by active transport mechanisms of mesothelial cells that provide access to spiral ganglion neurons. Reports on leaky barriers between scala tympani and the so-called cortilymph compartment likely open the best path for hair cell targeting. The complex barrier system of tight junction proteins such as occludins, claudins and tricellulin isolates the endolymphatic space for most drugs. Comparison of relevant differences of barriers, target cells and cell types involved in drug spread between main animal models and humans shall provide some translational aspects for inner ear drug applications.

Mitochondrial function depends crucially on the maintenance of multiple mitochondrial DNA (mtDNA) copies. Surprisingly, the cellular mechanisms regulating mtDNA copy number remain poorly understood. Through a systematic high-throughput approach in Saccharomyces cerevisiae, we determined mtDNA-to-nuclear DNA ratios in 5148 strains lacking nonessential genes. The screen revealed MRX6, a largely uncharacterized gene, whose deletion resulted in a marked increase in mtDNA levels, while maintaining wild type-like mitochondrial structure and cell size. Quantitative superresolution imaging revealed that deletion of MRX6 alters both the size and the spatial distribution of mtDNA nucleoids. We demonstrate that Mrx6 partially colocalizes with mtDNA within mitochondria and interacts with the conserved Lon protease Pim1 in a complex that also includes Mam33 and the Mrx6-related protein Pet20. Acute depletion of Pim1 phenocopied the high mtDNA levels observed in Δmrx6 cells. No further increase in mtDNA copy number was observed upon depletion of Pim1 in Δmrx6 cells, revealing an epistatic relationship between Pim1 and Mrx6. Human and bacterial Lon proteases regulate DNA replication by degrading replication initiation factors, suggesting a model in which Pim1 acts similarly with the Mrx6 complex, providing a scaffold linking it to mtDNA.

The United States is swiftly moving toward increased legalization of medical and recreational cannabis. Currently considered the most commonly used illicit psychoactive drug, recreational cannabis is legal in 11 states and Washington, DC, and male use is an important and understudied concern. Questions remain, however, about the potential long-term consequences of this exposure and how cannabis might impact the epigenetic integrity of sperm in such a way that could influence the health and development of offspring. This review summarizes cannabis use and potency in the USA, provides a brief overview of DNA methylation as an epigenetic mechanism that is vulnerable in sperm to environmental exposures including cannabis, and summarizes studies that have examined the effects of parental exposure to cannabis or delta-9 tetrahydrocannabinol (THC, the main psychoactive component of cannabis) on the epigenetic profile of the gametes and behavior of offspring. These studies have demonstrated significant changes to the sperm DNA methylome following cannabis use in humans, and THC exposure in rats. Furthermore, the use of rodent models has shown methylation and behavioral changes in rats born to fathers exposed to THC or synthetic cannabinoids, or to parents who were both exposed to THC. These data substantiate an urgent need for additional studies assessing the effects of cannabis exposure on childhood health and development. This is especially true given the current growing state of cannabis use in the USA.

The outstanding capacity of Geobacter sulfurreducens cells to directly connect their inner electron transport chain to a polarized electrode have prompted the application of a wide arrange of electrochemical techniques to explore their metabolism and current production possibilities. In this work we use very basic electrochemical assays as open circuit potential, voltammetric and chronopotentiometric measurements to obtain fundamental information on bacterial electrochemical characteristics of importance to interpret bacterial functioning. Specifically, we obtained information on G. sulfurreducens biofilm capacity to store charge in three major molecular reservoirs bridging the gap between NADH and the electrode, demonstrating, at the same time, the value of OCP measurements as a reporter of internal redox state of cells. Fil: Schrott, Germán David. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; Argentina Fil: Bonanni, Pablo Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; Argentina Fil: Busalmen, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; Argentina

Globally 360 million people have disabling hearing loss and, of these, 32 million are children. Human hearing relies on 15,000 hair cells that transduce mechanical vibrations to electrical signals in the auditory nerve. The process is powered by the endo-cochlear potential, which is produced by a vascularized epithelium that actively transports ions in conjunction with a gap junction (GJ) system. This "battery" is located "off-site" in the lateral wall of the cochlea. The GJ syncytium contains the GJ protein genes beta 2 (GJB2/connexin26 (Cx26)) and 6 (GJB6/connexin30 (Cx30)), which are commonly involved in hereditary deafness. Because the molecular arrangement of these proteins is obscure, we analyze GJ protein expression (Cx26/30) in human cochleae by using super-resolution structured illumination microscopy. At this resolution, the Cx26 and Cx30 proteins were visible as separate plaques, rather than being co-localized in heterotypic channels, as previously suggested. The Cx26 and Cx30 proteins thus seem not to be co-expressed but to form closely associated assemblies of GJ plaques. These results could assist in the development of strategies to treat genetic hearing loss in the future.

Little is known about the reproductive effects of paternal cannabis exposure. We evaluated associations between cannabis or tetrahydrocannabinol (THC) exposure and altered DNA methylation in sperm from humans and rats, respectively. DNA methylation, measured by reduced representation bisulfite sequencing, differed in the sperm of human users from non-users by at least 10% at 3,979 CpG sites. Pathway analyses indicated Hippo Signaling and Pathways in Cancer as enriched with altered genes (Bonferroni p

Background Hyperglycemia leading to increased oxidative stress is implicated in the increased risk for the development of macrovascular and microvascular complications in patients with type 1 diabetes mellitus. Methods and Results A random subcohort of 349 participants was selected from the DCCT / EDIC (Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications) cohort. This included 320 controls and 29 cardiovascular disease cases that were augmented with 98 additional known cases to yield a case cohort of 447 participants (320 controls, 127 cases). Biosamples from DCCT baseline, year 1, and closeout of DCCT , and 1 to 2 years post‐ DCCT ( EDIC years 1 and 2) were measured for markers of oxidative stress, including plasma myeloperoxidase, paraoxonase activity, urinary F 2α isoprostanes, and its metabolite, 2,3 dinor‐8 iso prostaglandin F 2α . Following adjustment for glycated hemoblobin and weighting the observations inversely proportional to the sampling selection probabilities, higher paraoxonase activity, reflective of antioxidant activity, and 2,3 dinor‐8 iso prostaglandin F 2α , an oxidative marker, were significantly associated with lower risk of cardiovascular disease (−4.5% risk for 10% higher paraoxonase, P iso prostaglandin F 2α , P =0.0092). In contrast, the oxidative markers myeloperoxidase and F 2α isoprostanes were not significantly associated with cardiovascular disease after adjustment for glycated hemoblobin. There were no significant differences between DCCT intensive and conventional treatment groups in the change in all biomarkers across time segments. Conclusions Heightened antioxidant activity (rather than diminished oxidative stress markers) is associated with lower cardiovascular disease risk in type 1 diabetes mellitus, but these biomarkers did not change over time with intensification of glycemic control. Clinical Trial Registration URL : https://www.clinicaltrials.gov . Unique identifiers: NCT 00360815 and NCT 00360893.

An electrical model able to decouple the electron pathway from microbial cell machinery impedance terms is introduced. In this context, capacitance characteristics of the biofilm are clearly resolved. In other words, the model allows separating, according to the advantage of frequency and spectroscopic response approach, the different terms controlling the performance of the microbial biofilm respiratory process and thus the directly related electricity production process. The model can be accurately fitted to voltammetry measurements obtained under steady-state conditions and also to biofilm discharge amperometric measurements. The implications of biological aspects of the electrochemical or redox capacitance are discussed theoretically in the context of current knowledge with regard to structure and physiological activity of microbial Geobacter biofilms. Fil: Bueno, Paulo R.. Universidade Estadual Paulista. Institute of Chemistry; Brasil Fil: Schrott, Germán David. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación En Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentina Fil: Bonanni, P. Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación En Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentina Fil: Simison, Silvia Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación En Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentina Fil: Busalmen, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación En Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentina

Highlights • Pre- and post-somatic segments of type I spiral ganglion neurons (SGNs) are unmyelinated in man. • Following hair cell loss and retrograde nerve degeneration SGNs survive as “mono-polar” cells in human deafness. • Non-myelinated Schwann cells may consolidate the neural cell bodies and protect SGNs from further degeneration. • Human SGNs can persist as electrically excitable mono-polar cells even after long-time deafness. • Robust survival of human SGNs is a prerequisite for cochlear implant function., Human auditory nerve afferents consist of two separate systems; one is represented by the large type I cells innervating the inner hair cells and the other one by the small type II cells innervating the outer hair cells. Type I spiral ganglion neurons (SGNs) constitute 96% of the afferent nerve population and, in contrast to other mammals, their soma and pre- and post-somatic segments are unmyelinated. Type II nerve soma and fibers are unmyelinated. Histopathology and clinical experience imply that human SGNs can persist electrically excitable without dendrites, thus lacking connection to the organ of Corti. The biological background to this phenomenon remains elusive. We analyzed the pre- and post-somatic segments of the type I human SGNs using immunohistochemistry and transmission electron microscopy (TEM) in normal and pathological conditions. These segments were found surrounded by non-myelinated Schwann cells (NMSCs) showing strong intracellular expression of laminin-β2/collagen IV. These cells also bordered the perikaryal entry zone and disclosed surface rugosities outlined by a folded basement membrane (BM) expressing laminin-β2 and collagen IV. It is presumed that human large SGNs are demarcated by three cell categories: (a) myelinated Schwann cells, (b) NMSCs and (c) satellite glial cells (SGCs). Their BMs express laminin-β2/collagen IV and reaches the BM of the sensory epithelium at the habenula perforata. We speculate that the NMSCs protect SGNs from further degeneration following dendrite loss. It may give further explanation why SGNs can persist as electrically excitable monopolar cells even after long-time deafness, a blessing for the deaf treated with cochlear implantation.

Anemia is a very common blood disorder that affects the lives of billions of people worldwide. Anemia is caused by the loss of blood, increased destruction of red blood cells (RBCs), or reduced production of RBCs. Erythropoiesis is the complex process of RBC differentiation and maturation, in which protein degradation plays a crucial role. Protein ubiquitination regulates programmed protein degradation, which can be reversed by deubiquitinating enzymes (DUBs); however, the role of DUBs in erythropoiesis has not been well studied. We examined the expression of DUBs during erythropoiesis using an ex vivo human CD34+ hematopoietic progenitor cell culture system. Here we show that ubiquitin-specific protease 50 (USP50) levels are increased during erythropoiesis. USP50 mRNA levels are significantly increased on day 3 and protein levels are elevated on day 9 of erythroid differentiation. Coimmunoprecipitation and proteomics analyses reveal that Ku70, a DNA-binding protein, is associated with USP50. Overexpression of USP50 has no effect on Ku70 mRNA levels, while it reduces Ku70 protein levels by promoting Ku70 degradation, suggesting that USP50 may indirectly regulate Ku70 protein stability. USP50 protein is also not stable. USP50 protein degradation is independent of the proteasomal and the lysosomal degradation systems. This study suggests that DUBs like USP50 may regulate protein stability during erythropoiesis; however, more investigation is warranted.

Severe sepsis is known to result in various neurological long-term deficits in human. Recently, a link between severe, lethal sepsis and significant hearing loss with correlating histomorphological inner ear changes in mice (C57BL/6) was observed. However, if similar observations can be made in severe, non-lethal sepsis in mice is unclear. This study evaluates mice after severe, non-lethal sepsis for analogue functional and histomorphological alterations of the inner ear. A total of 63 C57BL/6 mice were included in the study. All underwent an initial hearing test with auditory brainstem response on day 1. In 35 mice sepsis was induced by cecal ligation and puncture (CLP), in 15 sham surgery was performed and 13 served as healthy control. A second hearing test was performed on day 7. All mice were sacrificed afterwards for further histomorphological evaluation of the inner ears. Immunohistochemical analysis with apoptotic markers Cleaved-caspase 3, BAX and BCL-2 were performed to identify structural inner ear damage. Of all CLP mice, 21/35 (60.0%) died due to the induced sepsis. Of the surviving CLP mice, 14/35 (40.0%), post-treatment hearing thresholds differed significantly from the sham and control mice (P0.05). The present data suggests that severe, non-lethal sepsis in mice results in significantly elevated hearing thresholds. A positive labelling for the pro-apoptotic markers BAX and Cleaved-caspase 3 suggested the induction of apoptosis in inner ear., Summary: This study shows that a mild sepsis leads to a significant hearing impairment due to apoptosis of different inner ear structures.

Background: Due to their biochemical versatility, nanoparticles (NPs) have become one of the most important future carriers for drugs and genes. NP-mediated delivery could enable an effective pharmacotherapy to the inner ear and combat hearing loss. Aims: This study investigates the endocytic trafficking of silica NPs within HEI-OC1 cells, a cell line derived from the inner ear. Materials & methods: To investigate the interaction between 50-, 70- and 100-nm silica NPs and the cells, the authors employed a set of commonly available methods involving light and electron microscopy, and sample processing methods, which preserve the native cell shape and the fragile endocytic structures. Results: The authors observed that 50-nm NPs were the most efficiently internalized. They also identified macropinocytosis as the dominant mechanism of uptake, showed localization of NPs in the early endosome and observed that silica NPs were delayed during trafficking to the lysosomes, where these NPs stayed confined, showing no endosomal escape. Conclusion: These silica NPs mostly rely on macropinocytosis for internalization. A successful use of silica NPs as vectors would involve smaller NPs and an endosomal escape strategy. Original submitted 21 December 2011; Revised submitted 23 May 2012; Published online 14 August 2012

Background: Mutations in the GJB2 gene, which encodes the Connexin26 (Cx26) protein, are the most common cause of childhood hearing loss in American and European populations. The cochlea contains a gap junction (GJ) network in the sensory epithelium and two connective tissue networks in the lateral wall and spiral limbus. The syncytia contain the GJ proteins beta 2 (GJB2/Cx26) and beta 6 (GJB6/Cx30). Our knowledge of their expression in humans is insufficient due to the limited availability of tissue. Here, we sought to establish the molecular arrangement of GJs in the epithelial network of the human cochlea using surgically obtained samples. Methods: We analyzed Cx26 and Cx30 expression in GJ networks in well-preserved adult human auditory sensory epithelium using confocal, electron, and super -resolution structured illumination microscopy (SR-SIM). Results: Cx30 plaques (

The expression patterns of the neurotrophin, brain-derived neurotrophic factor, BDNF, and the neurotrophic receptors-p75NTR and Trk receptors-in the developing human fetal inner ear between the gestational weeks (GW) 9 to 12 are examined via in situ hybridization and immunohistochemistry. BDNF mRNA expression was highest in the cochlea at GW 9 but declined in the course of development. In contrast to embryonic murine specimens, a decline in BDNF expression from the apical to the basal turn of the cochlea could not be observed. p75NTR immunostaining was most prominent in the nerve fibers that penetrate into the sensory epithelia of the cochlea, the urticule and the saccule as gestational age progresses. TrkB and TrkC expression intensified towards GW 12, at which point the BDNF mRNA localization was at its lowest. TrkA expression was limited to fiber subpopulations of the facial nerve at GW 10. In the adult human inner ear, we observed BDNF mRNA expression in the apical poles of the cochlear hair cells and supporting cells, while in the adult human utricle, the expression was localized in the vestibular hair cells. We demonstrate the highly specific staining patterns of BDNF mRNA and its putative receptors over a developmental period in which multiple hearing disorders are manifested. Our findings suggest that BDNF and neurotrophin receptors are important players during early human inner ear development. In particular, they seem to be important for the survival of the afferent sensory neurons.

Background Proteomic analysis of cerebrospinal fluid (CSF) has shown great promise in identifying potential markers of injury in neurodegenerative diseases [1–13]. Here we compared CSF proteomes in healthy individuals, with patients diagnosed with traumatic brain injury (TBI) and subarachnoid hemorrhage (SAH) in order to characterize molecular biomarkers which might identify these different clinical states and describe different molecular mechanisms active in each disease state. Methods Patients presenting to the Neurosurgery service at the Louisiana State University Hospital-Shreveport with an admitting diagnosis of TBI or SAH were prospectively enrolled. Patients undergoing CSF sampling for diagnostic procedures were also enrolled as controls. CSF aliquots were subjected to 2-dimensional gel electrophoresis (2D GE) and spot percentage densities analyzed. Increased or decreased spot expression (compared to controls) was defined in terms of in spot percentages, with spots showing consistent expression change across TBI or SAH specimens being followed up by Matrix-Assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS). Polypeptide masses generated were matched to known standards using a search of the NCBI and/or GenPept databases for protein matches. Eight hundred fifteen separately identifiable polypeptide migration spots were identified on 2D GE gels. MALDI-MS successfully identified 13 of 22 selected 2D GE spots as recognizable polypeptides. Results Statistically significant changes were noted in the expression of fibrinogen, carbonic anhydrase-I (CA-I), peroxiredoxin-2 (Prx-2), both α and β chains of hemoglobin, serotransferrin (Tf) and N-terminal haptoglobin (Hp) in TBI and SAH specimens, as compared to controls. The greatest mean fold change among all specimens was seen in CA-I and Hp at 30.7 and −25.7, respectively. TBI specimens trended toward greater mean increases in CA-I and Prx-2 and greater mean decreases in Hp and Tf. Conclusions Consistent CSF elevation of CA-I and Prx-2 with concurrent depletion of Hp and Tf may represent a useful combination of biomarkers for the prediction of severity and prognosis following brain injury.

While actual models explaining electron conduction in electricity producing biofilms have evolved separately to apparent irreconcilable conceptual positions, finding cytochrome complexes in the external matrix of Geobacter biofilms supports the proposal of a new functional model, that takes fundamental elements from confronting theories. In this model electrons expelled by cells are conducted to the collecting electrode along a network of supramolecular cytochrome arrangements interconnected by semiconducting pilus fibres that provide equipotential conditions within physically distant points. This arrangement resembles, from our point of view, a relay network for Geobacter biofilm, which allows a concerted physiological response of the entire population to any local redox change. Fil: Ordoñez, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; Argentina Fil: Schrott, Germán David. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; Argentina Fil: Massazza, Diego Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; Argentina Fil: Busalmen, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; Argentina

Grasslands are often grazed by cattle and many grassland birds nest on the ground, potentially exposing nests to trampling. We tested for trampling risk introduced by cattle to nests of endangered Florida Grasshopper Sparrows (Ammodramus savannarum floridanus) using experimentally paired grids of artificial nests (i.e., clay targets) similar in size to nests of Florida Grasshopper Sparrows and counted the number of clay targets that were broken in paired grazed and ungrazed enclosures. Clay targets in grazed grids were trampled 3.9% more often than their respective ungrazed grids, and measurements of cattle presence or density were correlated with the number of broken clay targets, suggesting that excluding cattle during breeding is an important management recommendation for the Florida Grasshopper Sparrow. Trampling rates within grazed enclosures were spatially homogeneous with respect to cattle infrastructure such as supplemental feeding troughs and fences, and forests and stocking density were poor predictors of trampling rates when excluding ungrazed grids. We used population viability analysis to compare quasi-extinction rates, intrinsic growth rates, and median abundance in grazed and ungrazed Florida Grasshopper Sparrow aggregations to further understand the biological significance of management aimed at reducing trampling rates during the breeding season. Simulations indicated that trampling from grazing increased quasi-extinction rates by 41% while reducing intrinsic growth rates by 0.048, and reducing median abundance by an average of 214 singing males after 50 years. Management should avoid grazing enclosures occupied by Florida Grasshopper Sparrows during the nesting season to minimize trampling rates. Our methods that combine trampling experiments with population viability analysis provide a framework for testing effects from trampling on other grassland ground-nesting birds, and can directly inform conservation and management of the Florida Grasshopper Sparrow.

The mechanism of electron transport in Geobacter sulfurreducensbiofilms is a topic under intense study and debate. Although some proteins were found to be essential for current production, the specific role that each one plays in electron transport to the electrode remains to be elucidated and a consensus on the mechanism of electron transport has not been reached. In the present paper, to understand the state of the art in the topic, electron transport from inside of the cell to the electrode in Geobacter sulfurreducens biofilms is analysed, reviewing genetic studies, biofilm conductivity assays and electrochemical and spectro- electrochemical experiments. Furthermore, crucial data still required to achieve a deeper understanding are highlighted. Fil: Bonanni, Pablo Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; Argentina Fil: Schrott, Germán David. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; Argentina Fil: Busalmen, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; Argentina

In this paper, we report on the development of a continuous microfluidic reaction system for hydrolysis of soybean oil catalyzed with Thermomyces lanuginosus lipase (Lipolase 100 L). The microfluidic reaction system generates water–oil emulsions in the form of a hydrodynamically well controlled slug flow and automatically separates the oil phase after the hydrolysis by employing two microfluidic separators. All elements of this reaction system were tested at different hydrodynamic conditions and showed the ability to operate in a wide range of reactant flow rates. 25–30% conversion of triglyceride was reached in setting the residence time of the emulsion mixture to 10 min. This conversion increased to almost 50% for the residence time of 1 h. These results are comparable with those published for the same enzyme reaction system. This feature along with the benefits stemming from the use of microfluidics make our developed system a useful, easy to control and easy to scale-up technology for fast production of fine chemicals. Moreover, our calculations indicate that our slug flow system allows for significant savings of the mechanical energy.

Highlights ► We classified four different populations among human spiral ganglion cells. ► Spatial organization of human spiral ganglion cells and nuclei along the cochlea was determined. ► Size of spiral ganglion perikarya influences action potential initiation site. ► Simulation of single nerve fiber response showed different stimulation threshold. ► Temporal parameters of the AP are affected by perikaryal size., The unique, unmyelinated perikarya of spiral ganglion cells (SGCs) in the human cochlea are often arranged in functional units covered by common satellite glial cells. This micro anatomical peculiarity presents a crucial barrier for an action potential (AP) travelling from the sensory receptors to the brain. Confocal microscopy was used to acquire systematically volumetric data on perikarya and corresponding nuclei in their full dimension along the cochlea of two individuals. Four populations of SGCs within the human inner ear of two different specimens were identified using agglomerative hierarchical clustering, contrary to the present distinction of two groups of SGCs. Furthermore, we found evidence of a spatial arrangement of perikarya and their accordant nuclei along the cochlea spiral. In this arrangement, the most uniform sizes of cell bodies are located in the middle turn, which represents the majority of phonational frequencies. Since single-cell recordings from other mammalians may not be representative to humans and human SGCs are not accessible for physiological measurements, computer simulation has been used to quantify the effect of varying soma size on single neuron response to electrical micro stimulation. Results show that temporal parameters of the spiking pattern are affected by the size of the cell body. Cathodic stimulation was found to induce stronger variations of spikes while also leading to the lowest thresholds and longest latencies. Therefore, anodic stimulation leads to a more uniform excitation profile among SGCs with different cell body size.

Activation of tyrosine kinase receptor B (TrkB), a neurotrophin receptor, has been shown to increase neuronal cell survival and promote regeneration. Stimulation of the TrkB receptor by neurotrophic growth factors has been identified as a possible therapeutic target for the treatment of neurodegenerative disorders. However, growth factor delivery is problematic because of a short half-life in vivo. We have conjugated hNgf-EE, a short peptide mimetic of NGFβ to the surface of polymersome nanoparticles and shown that they are capable of activating the TrkB receptor in vitro in the SHSY-G7 cell line. We propose that polymersomes could act as a scaffold for the delivery of TrkB activating moieties and that the polymersome size and polyethylene glycol surface have been shown to increase in vivo retention time. These multifunctional nanoparticles have potential for the treatment of neurodegenerative disorders by TrkB activation. From the Clinical Editor Tyrosine kinase receptor B activation has been shown to promote regeneration and survival of neurons. However, growth factor delivery to stimulate these receptors remains problematic. The authors demonstrate that a peptide mimetic of NGFβ conjugated to the surface of polymersome nanoparticles is capable of activating the TrkB receptors. These nanoparticles may offer a novel treatment strategy for a variety of neurodegenerative disorders.

Mechanisms underlying the unique survival property of human spiral neurons are yet to be explored. P75 (p75(NTR)) is a low affinity receptor for neurotrophins and is known to interact with Trk receptors to modulate ligand binding and signaling. Up-regulation of this receptor was found to be associated with apoptosis as well as with cell proliferation. Its distribution and injury-induced change in expression pattern in the cochlea have been mainly studied in rodents. There is still no report concerning p75(NTR) in post-natal human inner ear. We analyzed, for the first time, p75(NTR) expression in five freshly fixed human cochleae by using immunohistochemistry techniques, including myelin basic protein (MBP) as a myelin sheath marker and TrkB as the human spiral neuron marker, and by using thin optical sectioning of laser confocal microscopy. The inner ear specimens were obtained from adult patients who had normal pure tone thresholds before the surgical procedures, via a trans-cochlear approach for removal of giant posterior cranial fossa meningioma. The expression of p75(NTR) was investigated and localized in the glial cells, including Schwann cells and satellite glial cells in the Rosenthal canal, in the central nerve bundles within the modiolus, and in the osseous spiral lamina of the human cochleae. The biological significance of p75(NTR) in human cochlea is discussed.

Improved spread of transduction in the central nervous system (CNS) was achieved from intravenous administration of adeno-associated virus serotype-9 (AAV9) to neonatal rats. Spinal lower motor neuron transduction efficiency was estimated to be 78% using the highest vector dose tested at a 12-week interval. The widespread expression could aid studying diseases that affect both the spinal cord and brain, such as amyotrophic lateral sclerosis (ALS). The protein most relevant to neuropathology in ALS is transactive response DNA-binding protein 43 (TDP-43). When expressed in rats, human wild-type TDP-43 rapidly produced symptoms germane to ALS including paralysis of the hindlimbs and muscle wasting, and mortality over 4 weeks that did not occur in controls. The hindlimb atrophy and weakness was evidenced by assessments of rotarod, rearing, overall locomotion, muscle mass, and histology. The muscle wasting suggested denervation, but there was only 14% loss of motor neurons in the TDP-43 rats. Tissues were negative for ubiquitinated, cytoplasmic TDP-43 pathology, suggesting that altering TDP-43's nuclear function was sufficient to cause the disease state. Other relevant pathology in the rats included microgliosis and degenerating neuronal perikarya positive for phospho-neurofilament. The expression pattern encompassed the distribution of neuropathology of ALS, and could provide a rapid, relevant screening assay for TDP-43 variants and other disease-related proteins.

The federally Endangered Florida Grasshopper Sparrow (FGSP, Ammodramus savannarum floridanus) is endemic to dry prairie habitat in central Florida and is currently only found at three public management areas (Avon Park Air Force Range, APAFR; Kissimmee Prairie Preserve State Park, KPPSP; and Three Lakes Wildlife Management Area, TLWMA). We analyzed long-term (1991–2008) point-count data to compare population trends of FGSPs at these management areas to determine if they function as independent populations by using Spearman's rank correlation to test for independence between annual trends. We also examined banding and resighting data to infer metapopulation structure. Populations fluctuated across years at all three sites, declining significantly at APAFR (rs=−0.89, P < 0.001, N= 13) and KPPSP (rs=−0.78, P= 0.004, N= 11) and remaining stable at TLWMA (rs= 0.19, P= 0.46, N= 17). Population trends among the three management areas appeared independent (absolute value of rs≤ 0.50, P≥ 0.12). Previous studies indicated that sparrows in the three areas were not genetically differentiated, and two cases of dispersal between APAFR and KPPSP have been documented. However, dispersal rates among areas appear to be too low to influence demographic dynamics within individual areas. Within APAFR, FGSPs are aggregated into three spatially distinct habitat patches previously considered separate populations, but dispersal among these patches is more frequent than previously reported and population trends among these patches are correlated (rs≥ 0.91, P < 0.001). These patterns suggest that a single metapopulation of FGSPs exists consisting of three distinct populations (APAFR, KPPSP, and TLWMA), and the spatially distinct aggregations at APAFR constitute a single population using several habitat patches. The population at APAFR is at risk of extirpation, and immediate action is needed if that population is to recover. Taking broader metapopulation dynamics into account will be useful for guiding management efforts aimed at conserving the FGSP in the broader central Florida landscape. RESUMEN Ammodramus savannarum floridanus es clasificado como En Peligro y es endemico a los pastizales secos del centro de la Florida. Es actualmente encontrado solamente en tres areas bajo manejo publico (Avon Park Air Force Range, APAFR; Kissimmee Prairie Preserve State Park, KPPSP; y Three Lakes Wildlife Management Area, TLWMA). Analizamos datos de puntos de conteo a largo plazo (1991–2008) para comparar las tendencias poblacionales de A. s. floridanus en estos sitios para determinar si funcionan como poblaciones independientes. Usamos el coeficiente de correlacion de Spearman para determinar si habia independencia entre patrones anuales. Tambien examinamos datos de anillamiento y de observaciones de individuos anillados para hacer inferencias sobre la estructura metapoblacional. Las poblaciones fluctuaron entre anos en los tres sitios de estudio, disminuyendo significativamente en APAFR (rs=−0.89, P < 0.001, N= 13) y KPPSP (rs=−0.78, P= 0.004, N= 11) y manteniendose estable en TLWMA (rs= 0.19, P= 0.46, N= 17). Las tendencias poblacionales entre los tres sitios parecio independiente (valor absoluto de rs≤ 0.50, P≥ 0.12). Estudios previos indicaron que A. s. floridanus no son geneticamente diferenciados entre las tres areas, y dos casos de dispersion entre APAFR y KPPSP han sido documentados. Sin embargo, las tasas de dispersion entre los sitios parecen ser demasiadas bajas para influenciar las dinamicas demograficas en cada uno de los sitios. En APAFR, A. s. floridanus es concentrado en tres parches de habitat espacialmente distintos. Estos fueron previamente considerados como poblaciones separados, pero la dispersion entre estos parches es mas frecuente de lo previamente reportado y las tendencias poblaciones entre estos parches estan correlacionadas (rs≥ 0.91, P < 0.001). Estos patrones sugieren que existe una metapoblacion de A. s. floridanus cual consiste de tres poblaciones distintas (APAFR, KPPSP, y TLWMA), y que las agregaciones espacialmente distintas en APAFR constituyen una poblacion cual utiliza diferentes parches de habitat. La poblacion en APAFR esta bajo riesgo de extirpacion, y se necesita de accion inmediata para recuperarla. Tomando en cuenta las dinamicas metapoblaciones mas amplias serautil para guiar los esfuerzos de manejo enfocados en la conservacion de A. s. floridanus en el paisaje del centro de la Florida.

We measured densities of Solenopsis invicta (Red Imported Fire Ant) mounds at sites occupied by Ammodramus savannarum floridanus (Florida Grasshopper Sparrow), a federally endangered subspecies, at Avon Park Air Force Range (APAFR), Kissimmee Prairie Preserve State Park (KPPSP), and Three Lakes Wildlife Management Area (TLWMA). Our objective was to compare densities of fire ant mounds among areas with active cattle grazing programs (two areas of native dry prairie habitat at APAFR and a tamed pasture at KPPSP) and areas without active grazing programs (native dry prairie habitat at KPPSP and TLWMA). Densities of fire ant mounds differed among the five areas examined and were greater in areas with active grazing programs than in areas without active grazing programs. We measured densities of fire ant mounds inside and outside of a cattle exclosure, but the total numbers detected were insufficient for analysis. We also placed bait stations inside and outside the exclosure. Fire ants were detected a...

Cell specific targeting is an emerging field in nanomedicine. Homing of the multifunctional nanoparticles (MFNPs) is achieved by the conjugation of targeting moieties on the nanoparticle surface. The inner ear is an attractive target for new drug delivery strategies as it is hard to access and hearing loss is a significant worldwide problem. In this work we investigated the utility of a Nerve Growth Factor-derived peptide (hNgf_EE) functionalized nanoparticles (NPs) to target cells of the inner ear. These functionalized NPs were introduced to organotypic explant cultures of the mouse inner ear and to PC-12 rat pheochromocytoma cells. The NPs did not show any signs of toxicity. Specific targeting and higher binding affinity to spiral ganglion neurons, Schwann cells and nerve fibers of the explant cultures were achieved through ligand mediated multivalent binding to tyrosine kinase receptors and to p75 neurotrophin receptors. Unspecific uptake of NPs was investigated using NPs conjugated with scrambled hNgf_EE peptide. Our results indicate a selective cochlear cell targeting by MFNPs, which may be a potential tool for cell specific drug and gene delivery to the inner ear.

Objective: Prestin is an anion-transporter-related protein highly expressed in mammalian outer hair cells (OHCs). It is associated with the OHC basolateral plasma membrane and responsible for cell body contraction, increasing cochlear sensitivity and frequency resolution. Here, we analysed the expression and distribution of prestin in the human cochlea. Study Design: Immunohistochemistry including confocal microscopy and SEM were performed on EDTA-decalcified human cochlea removed during petro-clival meningioma surgery. Results: Prestin was found to be expressed solely in OHCs. No staining was seen in IHCs. Prestin immunolabelling framed the OHCs along the sensory region of the human cochlea. Staining was most prominent in the lateral cell membrane with less expression in the sub- and peri-nuclear part of the cell. Prestin immunostaining was also detected in the OHC cytosol, presumably reflecting intracellular molecular trafficking. Conclusion: This study describes for the first time the localizat...

Hypothesis Growth hormones have beneficial effects on increasing height in adults with Turner syndrome (TS) and may also affect auditory function. Background Turner syndrome is the most common sex-linked chromosomal abnormality in female conceptions. Epidermal growth factor and its receptor (EGFR) affect differentiation, proliferation, and migration of epithelial cells and function as survival factors. The expression of EGFR is found in the developing and juvenile inner ear of experimental animals but is absent in adults. Methods To determine whether EGFR plays a role in TS, its expression was analyzed in the cochlea of healthy fetus and fetus with TS and in healthy adults. Results In healthy fetuses, EGFR protein expression was localized to the inner and outer hair cells and the Reissner membrane. The fetuses with TS on the 13th gestational week (GW) showed a similar pattern of immunoreactivity as the normal 16th and 20th GW cochlea. By the 23rd GW, EGFR immunoreactivity was not detectable in the TS hair cells or the Reissner membrane, and less intensive staining was found in the surrounding fibrocytes of the spiral ganglion. Conclusion This is the first demonstration of EGFR immunoreactivity in the human cochlea and illustrates how EGFR expression is altered during development in TS. These findings indicate the importance of growth hormone receptors for inner ear development in humans.

Primary ciliary dyskinesia (PCD) is an autosomal recessive inherited disease characterized by abnormal ciliary motion and impaired mucociliary clearance. The prevalence of PCD is approximately 1 : 15 000 - 1 : 20 000 in live births. Cilia dysfunction is also implicated in a wider spectrum of diseases due to impaired organ genesis and body symmetry. Cilia are highly conserved in animals and show complex structures containing more than 250 proteins for their formation. Recent studies have begun to locate the PCD genes in the genome and characterize functional mutations. Specific diagnosis of the ciliary dysfunction requires physiological measurements as well as light- and electron microscopy. Abnormalities in ciliary motion and ultrastructural studies can be performed with nasal mucosal epithelium.

Young naive domestic chicks readily attack green insects and avoid insects painted red but show no discrimination of the same colours when applied to fruit-like objects, a discrimination that has been depicted as context-dependent preference. To study the neural representation of such preference we performed bilateral telencephalectomy on 1-day-old domestic chicks and tested them on an unlearned prey discrimination paradigm. Here we show that following complete decerebration, young domestic chicks preferentially peck at red fruit versus red insects and tend to choose green insects over green fruit indistinguishably from unoperated chicks. The present study provides the first direct evidence that sophisticated context-dependent, unlearned colour preference is processed by subtelencephalic areas of an amniote species.

Deafferentation of the auditory nerve from loss of sensory cells is associated with degeneration of nerve fibers and spiral ganglion neurons (SGN). SGN survival following deafferentation can be enhanced by application of neurotrophic factors (NTF), and NTF can induce the regrowth of SGN peripheral processes. Cochlear prostheses could provide targets for regrowth of afferent peripheral processes, enhancing neural integration of the implant, decreasing stimulation thresholds, and increasing specificity of stimulation. The present study analyzed distribution of afferent and efferent nerve fibers following deafness in guinea pigs using specific markers (parvalbumin for afferents, synaptophysin for efferent fibers) and the effect of brain derived neurotrophic factor (BDNF) in combination with acidic fibroblast growth factor (aFGF). Immediate treatment following deafness was compared with 3-week-delayed NTF treatment. Histology of the cochlea with immunohistochemical techniques allowed quantitative analysis of neuron and axonal changes. Effects of NTF were assessed at the light and electron microscopic levels. Chronic BDNF/aFGF resulted in a significantly increased number of afferent peripheral processes in both immediate- and delayed-treatment groups. Outgrowth of afferent nerve fibers into the scala tympani were observed, and SGN densities were found to be higher than in normal hearing animals. These new SGN might have developed from endogenous progenitor/stem cells, recently reported in human and mouse cochlea, under these experimental conditions of deafferentation-induced stress and NTF treatment. NTF treatment provided no enhanced maintenance of efferent fibers, although some synaptophysin-positive fibers were detected at atypical sites, suggesting some sprouting of efferent fibers.

Background The roles of the neurotrophins NGF (Neurotrophic growth factor) and BDNF (brain-derived neurotrophic factor) in neuronal growth and development are already known. Meanwhile, the neurotrophin receptors TrkA (tropomyosin related kinase A), TrkB, and p75 are important for determining the fate of cells. In endometriosis, this complex system has not been fully elucidated yet. The aim of this study was to evaluate the expression and location of these neurotrophins and their receptors in peritoneal (PE) and deep infiltrating endometriotic (DIE) tissues and to measure and compare the density of nerve fibers in the disease subtypes. Methods PE lesions (n = 20) and DIE lesions (n = 22) were immunostained and analyzed on serial slides with anti-BDNF, −NGF, −TrkA, −TrkB, −p75,-protein gene product 9.5 (PGP9.5, intact nerve fibers) and -tyrosine hydroxylase (TH, sympathetic nerve fibers) antibodies. Result There was an equally high percentage (greater than 75 %) of BDNF-positive immunostaining cells in both PE and DIE. TrkB (major BDNF receptor) and p75 showed a higher percentage of immunostaining cells in DIE compared to in PE in stroma only (p

Hypothesis: To analyze if adult-dissociated spiral ganglion cells may be propagated in vitro for later use in transplantation models to form integrated neural networks. Background: Hearing loss is often associated with primary or secondary spiral ganglion cell degeneration. New strategies for cell repair and tissue engineering warrants further elucidation of the regenerative capacity of the auditory nerve. Methods: We used in vitro/in video microscopy in combination with immunocytochemistry and field emission scanning electron microscopy to analyze neural development and network formation from dissociated adult guinea pig spiral ganglion cells. Cells were cultured in serum-free medium and in the presence of brain-derived neurotrophic factor, neurotrophin 3, and glia cell line-derived neurotrophic factor for up to 8 weeks. Results: Time-lapse video microscopy and scanning electron microscopy exposed the propagation of auditory neurons and the role of neural growth cones in axon locomotion, fasciculation, and nuclear migration, often ensuing in cell congregation (ganglion-like formations) during network formation. Axons were sometimes ensheathed by adjoining S-100/glia fibrillary acidic protein-expressing cells. A few expanding neurons were nestin positive and sometimes incorporated the markers of proliferating cells Ki67 and 5'-bromo-2-deoxyuridine. Neurons expressed the markers and transcription factors for neural development neurogenin 1, neurogenic differentiation factor 1, Brn3a, and GATA binding protein 3, as well as the neural markers beta-III tubulin, NeuN, and neurofilament 160 during this process. Conclusion: This method of culturing and expanding spiral ganglion neurons in vitro may be useful in further studies of cell transplantation models aiming to restore the injured inner ear.

Cochlear histopathological findings of a patient affected by Alport syndrome (AS) are presented and the main histopathological, genetic and clinical aspects of this condition are reviewed. AS is an inherited disorder of the basement membranes, arising from mutations in type IV collagen (COL4 genes that code for the α3, α4, and α5 chains of type IV collagen), the major collagenous constituent of these structures. Our histopathological findings partly coincide with those of recent studies and reveal a separation of the sensory organ and basilar membrane and some loss of fibrocytes in the spiral ligament in the apical turn accompanied by signs of endolymphatic hydrops. A ‘narrow’ tunnel of Corti, with cells filling this space as described recently, and hypothesized to represent a premature stage of development, could not be verified. We suggest that this filling of open spaces within the sensory organ may represent preparation artefacts. Thicker and more tangential sectioning often mimics cells to be present...

Interleukin-33 (IL-33) is a member of the IL-1 cytokine superfamily that potently drives production of a variety of cytokines and contributes to the pathogenesis of inflammatory diseases. The IL-33 is a nuclear protein and is released from apoptotic or necrotic cells. Serum IL-33 levels are increased in various diseases, such as atopic dermatitis, chronic hepatitis C infection, and asthma. Here, we show that red blood cells (RBCs) are one of the major sources of plasma IL-33. The IL-33 levels are significantly increased in supernatants from lysed RBCs. Plasma IL-33 levels are increased in patients during hemolysis, and plasma IL-33 levels show a positive correlation with degree of hemolysis. The IL-33 protein and messenger RNA levels were detected in the late stages of differentiation in ex vivo primary human erythroid progenitor cell cultures, suggesting that IL-33 is expressed during maturation of RBCs. Furthermore, hemoglobin depleted red cell lysates induced IL-8 expression in human epithelial cells. This effect was attenuated in IL-33 decoy receptor expressing cells and was enhanced in IL-33 receptor expressing cells. These results suggest that erythroid progenitor cells produce IL-33 and circulating RBCs represent a major source of IL-33 that is released upon hemolysis.

Background The measurement of HBV DNA levels has become the most direct and reliable method used for accurate diagnosis and prognosis of acute and chronic HBV infection [ Mommeja-Marin R, Mondou E, Blum MR, Rousseau F. Serum HBV DNA as a marker of efficacy during therapy for chronic HBV infection: analysis and review of the literature. Hepatology 2003;37:1309–19 ]. Nucleic acid amplification testing (NAT detection) also reduces the pre-seroconversion window period. The method can be used to aid in the management of HBV infection, by the identification of individuals with high levels of viral replication who might benefit from antiviral therapy, monitoring patients on therapy, and identification of the development of resistance. Objectives Evaluation of the novel COBAS ® AmpliPrep/COBAS ® TaqMan ® HBV Test, which combines automated extraction of DNA on the COBAS ® AmpliPrep Instrument (CAP), coupled with real-time PCR on the COBAS ® TaqMan ® Analyzer (CTM), thus greatly reducing hands-on time during sample preparation and amplification/detection. The assay fulfils the current requirements: a highly sensitive HBV DNA detection reagent which is calibrated to the International WHO Standard to reliably quantify HBV genotypes A–G in plasma with a very broad measuring range, thereby minimizing laborious repeat testing. Study design The test was evaluated for sensitivity, dynamic range, precision, clinical and analytical specificity, genotype inclusivity, interfering substances, and correlation with other tests for the detection of HBV DNA (COBAS ® Amplicor HBV Monitor Test, COBAS ® TaqMan ® HBV Test for Use with the High Pure System and VERSANT HBV DNA 3.0 Assay). Results and conclusion A fully automated system for sample preparation, amplification and quantitation of HBV DNA was developed that demonstrates a nearly 7-log dynamic range up to 1.1E+08 IU/mL, an assay sensitivity (95% hit-rate) of 4–12 IU/mL and an equivalent detection of genotypes A–G plus a prevalent pre-core mutant. The results obtained with the new test show a good correlation to titers obtained with other platforms.

Habitat restoration is often recommended in conservation without first evaluating whether pop- ulations are in fact habitat limited and thus whether declining populations can be stabilized or recovered through habitat restoration. We used a spatially structured demographic model coupled with a dynamic neu- tral landscape model to evaluate whether habitat restoration could rescue populations of several generic migratory songbirds that differed in their sensitivity to habitat fragmentation (i.e., severity of edge effects on nesting success). Simulating a best-case scenario, landscapes were instantly restored to 100% habitat before, at, or after habitat loss exceeded the species' vulnerability threshold. The vulnerability threshold is a measure of extinction risk, in which the change in population growth rate (∆λ) scaled to the rate of habitat loss (∆h )f alls below −1% (∆λ/∆h ≤− 0.01). Habitat restoration was most effective for species with low-to-moderate edge sensitivities and in landscapes that had not previously experienced extensive fragmentation. To stabilize pop- ulations of species that were highly edge sensitive or any species in heavily fragmented landscapes, restoration needed to be initiated long before the vulnerability threshold was reached. In practice, habitat restoration is generally not initiated until a population is at risk of extinction, but our model results demonstrate that some populations cannot be recovered at this point through habitat restoration alone. At this stage, habitat loss and fragmentation have seriously eroded the species' demographic potential such that halting population declines is limited more by demographic factors than the amount of available habitat. Evidence that populations de- cline in response to habitat loss is thus not sufficient to conclude that habitat restoration will be sufficient to rescue declining populations.