High Prevalence of Clonal Reproduction and Low Genetic Diversity in Scutellaria floridana, a Federally Threatened Florida-Endemic Mint.

The threatened mint Florida skullcap (Scutellaria floridana) is endemic to four counties in the Florida panhandle. Because development and habitat modification extirpated several historical occurrences, only 19 remain to date. To inform conservation management and delisting decisions, a comprehensive investigation of the genetic diversity and relatedness, population structure, and clonal diversity was conducted using SNP data generated by ddRAD. Compared with other Lamiaceae, we detected low genetic diversity (HE = 0.125-0.145), low to moderate evidence of inbreeding (FIS = -0.02-0.555), and moderate divergence (FST = 0.05-0.15). We identified eight populations with most of the genetic diversity, which should be protected in situ, and four populations with low genetic diversity and high clonality. Clonal reproduction in our circular plots and in 92% of the sites examined was substantial, with average clonal richness of 0.07 and 0.59, respectively. Scutellaria floridana appears to have experienced a continued decline in the number of extant populations since its listing under the Endangered Species Act; still, the combination of sexual and asexual reproduction may be advantageous for maintaining the viability of extant populations. However, the species will likely require ongoing monitoring, management, and increased public awareness to ensure its survival and effectively conserve its genetic diversity.

Focused ion beam-scanning electron microscopy links pathological myelin outfoldings to axonal changes in mice lacking Plp1 or Mag.

Healthy myelin sheaths consist of multiple compacted membrane layers closely encasing the underlying axon. The ultrastructure of CNS myelin requires specialized structural myelin proteins, including the transmembrane-tetraspan proteolipid protein (PLP) and the Ig-CAM myelin-associated glycoprotein (MAG). To better understand their functional relevance, we asked to what extent the axon/myelin-units display similar morphological changes if PLP or MAG are lacking. We thus used focused ion beam-scanning electron microscopy (FIB-SEM) to re-investigate axon/myelin-units side-by-side in Plp- and Mag-null mutant mice. By three-dimensional reconstruction and morphometric analyses, pathological myelin outfoldings extend up to 10 µm longitudinally along myelinated axons in both models. More than half of all assessed outfoldings emerge from internodal myelin. Unexpectedly, three-dimensional reconstructions demonstrated that both models displayed complex axonal pathology underneath the myelin outfoldings, including axonal sprouting. Axonal anastomosing was additionally observed in Plp-null mutant mice. Importantly, normal-appearing axon/myelin-units displayed significantly increased axonal diameters in both models according to quantitative assessment of electron micrographs. These results imply that healthy CNS myelin sheaths facilitate normal axonal diameters and shape, a function that is impaired when structural myelin proteins PLP or MAG are lacking.

Brain-derived neurotrophic factor stimulates the retrograde pathway for axonal autophagy.

Autophagy is a lysosomal degradation pathway important for neuronal development, function, and survival. How autophagy in axons is regulated by neurotrophins to impact neuronal viability and function is poorly understood. Here, we use live-cell imaging in primary neurons to investigate the regulation of axonal autophagy by the neurotrophin brain-derived neurotrophic factor (BDNF) and elucidate whether autophagosomes carry BDNF-mediated signaling information. We find that BDNF induces autophagic flux in primary neurons by stimulating the retrograde pathway for autophagy in axons. We observed an increase in autophagosome density and retrograde flux in axons, and a corresponding increase in autophagosome density in the soma. However, we find little evidence of autophagosomes comigrating with BDNF. In contrast, BDNF effectively engages its cognate receptor TrkB to undergo retrograde transport in the axon. These compartments, however, are distinct from LC3-positive autophagic organelles in the axon. Together, we find that BDNF stimulates autophagy in the axon, but retrograde autophagosomes do not appear to carry BDNF cargo. Thus, autophagosomes likely do not play a major role in relaying neurotrophic signaling information across the axon in the form of active BDNF/TrkB complexes. Rather, BDNF likely stimulates autophagy as a consequence of BDNF-induced processes that require canonical roles for autophagy in degradation.

Organization of the autophagy pathway in neurons.

Macroautophagy (hereafter referred to as autophagy) is an essential quality-control pathway in neurons, which face unique functional and morphological challenges in maintaining the integrity of organelles and the proteome. To overcome these challenges, neurons have developed compartment-specific pathways for autophagy. In this review, we discuss the organization of the autophagy pathway, from autophagosome biogenesis, trafficking, to clearance, in the neuron. We dissect the compartment-specific mechanisms and functions of autophagy in axons, dendrites, and the soma. Furthermore, we highlight examples of how steps along the autophagy pathway are impaired in the context of aging and neurodegenerative disease, which underscore the critical importance of autophagy in maintaining neuronal function and survival.

Autophagy: Identification of MTMR5 as a neuron-enriched suppressor.

A puzzle of autophagy in neurons is that, unlike in other cells, it is not robustly induced by inhibition of mammalian target of rapamycin (mTOR). A new study now solves this conundrum and establishes that myotubularin-related phosphatase 5 limits the induction of neuronal autophagy by mTOR inhibitors.

Progressive axonopathy when oligodendrocytes lack the myelin protein CMTM5.

Oligodendrocytes facilitate rapid impulse propagation along the axons they myelinate and support their long-term integrity. However, the functional relevance of many myelin proteins has remained unknown. Here, we find that expression of the tetraspan-transmembrane protein CMTM5 (chemokine-like factor-like MARVEL-transmembrane domain containing protein 5) is highly enriched in oligodendrocytes and central nervous system (CNS) myelin. Genetic disruption of the Cmtm5 gene in oligodendrocytes of mice does not impair the development or ultrastructure of CNS myelin. However, oligodendroglial Cmtm5 deficiency causes an early-onset progressive axonopathy, which we also observe in global and tamoxifen-induced oligodendroglial Cmtm5 mutants. Presence of the WldS mutation ameliorates the axonopathy, implying a Wallerian degeneration-like pathomechanism. These results indicate that CMTM5 is involved in the function of oligodendrocytes to maintain axonal integrity rather than myelin biogenesis.

Synaptic activity controls autophagic vacuole motility and function in dendrites.

Macroautophagy (hereafter "autophagy") is a lysosomal degradation pathway that is important for learning and memory, suggesting critical roles for autophagy at the neuronal synapse. Little is known, however, about the molecular details of how autophagy is regulated with synaptic activity. Here, we used live-cell confocal microscopy to define the autophagy pathway in primary hippocampal neurons under various paradigms of synaptic activity. We found that synaptic activity regulates the motility of autophagic vacuoles (AVs) in dendrites. Stimulation of synaptic activity dampens AV motility, whereas silencing synaptic activity induces AV motility. Activity-dependent effects on dendritic AV motility are local and reversible. Importantly, these effects are compartment specific, occurring in dendrites and not in axons. Most strikingly, synaptic activity increases the presence of degradative autolysosomes in dendrites and not in axons. On the basis of our findings, we propose a model whereby synaptic activity locally controls AV dynamics and function within dendrites that may regulate the synaptic proteome.

Microtubules orchestrate local translation to enable cardiac growth.

Hypertension, exercise, and pregnancy are common triggers of cardiac remodeling, which occurs primarily through the hypertrophy of individual cardiomyocytes. During hypertrophy, stress-induced signal transduction increases cardiomyocyte transcription and translation, which promotes the addition of new contractile units through poorly understood mechanisms. The cardiomyocyte microtubule network is also implicated in hypertrophy, but via an unknown role. Here, we show that microtubules are indispensable for cardiac growth via spatiotemporal control of the translational machinery. We find that the microtubule motor Kinesin-1 distributes mRNAs and ribosomes along microtubule tracks to discrete domains within the cardiomyocyte. Upon hypertrophic stimulation, microtubules redistribute mRNAs and new protein synthesis to sites of growth at the cell periphery. If the microtubule network is disrupted, mRNAs and ribosomes collapse around the nucleus, which results in mislocalized protein synthesis, the rapid degradation of new proteins, and a failure of growth, despite normally increased translation rates. Together, these data indicate that mRNAs and ribosomes are actively transported to specific sites to facilitate local translation and assembly of contractile units, and suggest that properly localized translation - and not simply translation rate - is a critical determinant of cardiac hypertrophy. In this work, we find that microtubule based-transport is essential to couple augmented transcription and translation to productive cardiomyocyte growth during cardiac stress.

Peptidomimetic-Based Vesicles Inhibit Amyloid-ß Fibrillation and Attenuate Cytotoxicity.

An interruption in Aß homeostasis leads to the deposit of neurotoxic amyloid plaques and is associated with Alzheimer's disease. A supramolecular strategy based on the assembly of peptidomimetic agents into functional vesicles has been conceived for the simultaneous inhibition of Aß42 fibrillation and expedited clearance of Aß42 aggregates. Tris-pyrrolamide peptidomimetic, ADH-353, contains one hydrophobic N-butyl and two hydrophilic N-propylamine side chains and readily forms vesicles under physiological conditions. These vesicles completely rescue both mouse neuroblastoma N2a and human neuroblastoma SH-SY5Y cells from the cytotoxicity that follows from Aß42 misfolding likely in mitochondria. Biophysical studies, including confocal imaging, demonstrate the biocompatibility and selectivity of the approach toward this aberrant protein assembly in cellular milieu.

Sulfidation of ZVI/AC composite leads to highly corrosion-resistant nanoremediation particles with extended life-time.

Nanoscale zero-valent iron (nZVI) is a powerful reductant for many water pollutants. The lifetime of nZVI in aqueous environments is one of its limitations. Sulfidation of the nZVI surface by reduced sulfur species is known to significantly modify the particle properties. In the present study we examined various post-synthesis sulfidation methods applied on Carbo-Iron, a composite material where iron nanostructures are embedded in colloidal activated carbon (AC) particles. In such cases, where ZVI is surrounded by carbon, sulfidation largely inhibits the anaerobic corrosion of ZVI in water whereas its dechlorination activity was slightly increased. Even at a very low molar S/Fe ratio of 0.004 a strong decrease of the corrosion rate by a factor of 65 was achieved, while concurrently dechlorination of tetrachloroethene (PCE) was accelerated by a factor of three compared to the untreated particles. As a consequence, over 98% of the reduction equivalents of the sulfidated ZVI were utilized for the reduction of the target contaminant (33 mg L-1 PCE) under simulated groundwater conditions. In a long-term experiment over 160 days the extended life-time and the preservation of the reduction capacity of the embedded ZVI were confirmed. Reasons for the modified reaction behavior of Carbo-Iron after sulfidation compared to previously studied nZVI are discussed. We hypothesize that the structure of the carbon-embedded iron is decisive for the observed reaction behavior. In addition to reaction rates, the product pattern is vastly different compared to that of sulfidated nZVI. The triple combination of ZVI, AC and sulfur makes the composite particle very suitable for practical in-situ applications.

Acceleration of microiron-based dechlorination in water by contact with fibrous activated carbon.

Zero-valent iron (ZVI) is widely applied for reduction of chlorohydrocarbons in water. Since the dechlorination occurs at the iron surface, marked differences in rate constants are commonly found for nanoscale and microscale ZVI. It has already been shown for trichloroethene (TCE) adsorbed to activated carbon (AC) that the dechlorination reaction is shifted to the carbon surface simply by contacting the AC with highly reactive nanoscale ZVI particles. Transfer of reactive species to the adsorbed pollutant was discussed. The present study shows that even low price and very low reactive microscale ZVI can also be utilized for an effective dechlorination process. Compared to the reaction rate at the iron surface itself, an enormous acceleration of the dechlorination rate for chlorinated ethenes was observed, reaching activity levels such as known for nanoscale ZVI. When fibrous AC is brought into direct contact with microscale ZVI the iron-surface-normalised dechlorination rate constants increased by up to four orders of magnitude. This implies that the dechlorination reaction is fully transferred to the AC surface. At the same time, the anaerobic corrosion of the same material was not substantially affected. Thus, the utilization of iron's reduction equivalents towards dechlorination (dechlorination efficiency) can be considerably enhanced. A screening with various AC types showed that the extent of rate acceleration depends strongly on the surface chemistry of the AC. By means of temperature-programmed desorption, it could be shown that concentration and type of oxygen surface groups determine the redox-mediation properties. Quinone/hydroquinone groups were identified as being the main drivers for electron-transfer processes, but to some extent other redox-active groups such as chromene and pyrone can also act as redox mediators. AC overall plays the role of a catalyst rather than a reactant. The present study derives recommendations for practical application of the findings in water-treatment approaches.

Combined chemical and microbiological degradation of tetrachloroethene during the application of Carbo-Iron at a contaminated field site.

After the injection of Carbo-Iron® into an aquifer contaminated with tetrachloroethene (PCE), combined chemical and microbiological contaminant degradation processes were found in a long-term study of the field site in Lower Saxony (Germany). The applied composite material Carbo-Iron, which consists of colloidal activated carbon and embedded nanoscale zero-valent iron (ZVI) structures, functioned as intended: accumulating the pollutants and promoting their reductive dechlorination. Furthermore, the particles decreased the redox potential of the groundwater due to their reaction with oxygen and to the ZVI-corrosion-induced formation of molecular hydrogen up to 190 days after the injection, the latter promoting sulphate-reducing conditions. The emergence of cis-dichloroethene (cis-DCE), which was only found in trace quantities before the injection of Carbo-Iron, together with the presence of organisms related to Sulfospirillum multivorans, Desulfitobacterium spp. and Dehalococcoides mccartyi, indicate that Carbo-Iron is also able to support microbial degradation of PCE. However, cis-DCE did not accumulate in the present case study, although it is often observed at sites with active microbial dechlorination. The results of compound-specific isotope analysis in combination with pyrosequencing data suggested the oxidative degradation of cis-DCE by an organism related to Polaromonas sp. strain JS666. Consequently, the formation of the carcinogenic degradation intermediate vinyl chloride was circumvented. Overall, the moderate and slow change of environmental conditions mediated by Carbo-Iron not only supported organohalide-respiring bacteria, but also created the basis for a subsequent microbial oxidation step.

Teaching an old scaffold new recognition tricks: oligopyrrolamide antagonists of IAPP aggregation.

A library of N-substituted oligopyrrolamides was designed to modulate the aggregation kinetics of islet amyloid polypeptide (IAPP). IAPP is a hormonal peptide, co-secreted with insulin in the pancreatic ß-cells. IAPP samples a variety of conformations, starting from a native random coil to membrane-associated α-helical intermediates and eventually terminates in the amyloid plaques rich in ß-sheet structures. A growing body of evidence suggests that membrane bound α-helical intermediates are the key cytotoxic species that impair the functionality and viability of ß-cells and contribute to the onset of type 2 diabetes mellitus (DM2). The N-substituted oligopyrrolamides were screened against the aggregation of IAPP using amyloid kinetic assays. A tripyrrole, ADH-101, was the most effective antagonist of IAPP fibrillation in a physiologically relevant lipid membrane system as well as under de novo conditions. ADH-101 induces/stabilizes a secondary structure in IAPP which potentially affects its downstream functions. ADH-101 efficiently affects IAPP-mediated liposome leakage and cell toxicity in insulin secreting cells. ADH-101 inhibits the elongation process potentially binding to the monomeric IAPP and attenuating its access to the preformed fibers. More importantly, oligopyrrolamides are better inhibitors of IAPP aggregation than analogous oligopyridylamides and have more desirable biological properties reflected by their partition coefficients. In essence, an oligopyrrolamide scaffold has been designed which modulates the membrane bound helical intermediates of IAPP and affects their downstream functions such as oligomerization, membrane poration, and cytotoxicity.

Hexokinase II-derived cell-penetrating peptide targets mitochondria and triggers apoptosis in cancer cells.

Overexpression of mitochondria-bound hexokinase II (HKII) in cancer cells plays an important role in their metabolic reprogramming and protects them against apoptosis, thereby facilitating their growth and proliferation. Here, we show that covalently coupling a peptide corresponding to the mitochondrial membrane-binding N-terminal 15 aa of HKII (pHK) to a short, penetration-accelerating sequence (PAS) enhances the cellular uptake, mitochondrial localization, and cytotoxicity of the peptide in HeLa cells. Further analysis revealed that pHK-PAS depolarized mitochondrial membrane potential, inhibited mitochondrial respiration and glycolysis, and depleted intracellular ATP levels. The effects of pHK-PAS were correlated with dissociation of endogenous full-length HKII from mitochondria and release of cytochrome c Of significance, pHK-PAS treatment of noncancerous HEK293 cells resulted in substantially lower cytotoxicity. Thus, pHK-PAS effectively disrupted the mitochondria-HKII association in cancer cells, which led to mitochondrial dysfunction and, finally, apoptosis. Our results demonstrate the potential of the pHK-PAS cell-penetrating peptide as a novel therapeutic strategy in cancer.-Woldetsadik, A. D., Vogel, M. C., Rabeh, W. M., Magzoub, M. Hexokinase II-derived cell-penetrating peptide targets mitochondria and triggers apoptosis in cancer cells.

Cytotoxicity of prion protein-derived cell-penetrating peptides is modulated by pH but independent of amyloid formation.

Prion diseases are associated with conversion of cellular prion protein (PrPC) into an abnormally folded and infectious scrapie isoform (PrPSc). We previously showed that peptides derived from the unprocessed N-termini of mouse and bovine prion proteins, mPrP1-28 and bPrP1-30, function as cell-penetrating peptides (CPPs), and destabilize model membrane systems, which could explain the infectivity and toxicity of prion diseases. However, subsequent studies revealed that treatment with mPrP1-28 or bPrP1-30 significantly reduce PrPSc levels in prion-infected cells. To explain these seemingly contradictory results, we correlated the aggregation, membrane perturbation and cytotoxicity of the peptides with their cellular uptake and intracellular localization. Although the peptides have a similar primary sequence, mPrP1-28 is amyloidogenic, whereas bPrP1-30 forms smaller oligomeric or non-fibrillar aggregates. Surprisingly, bPrP1-30 induces much higher cytotoxicity than mPrP1-28, indicating that amyloid formation and toxicity are independent. The toxicity is correlated with prolonged residence at the plasma membrane and membrane perturbation. Both ordered aggregation and toxicity of the peptides are inhibited by low pH. Under non-toxic conditions, the peptides are internalized by lipid-raft dependent macropinocytosis and localize to acidic lysosomal compartments. Our results shed light on the antiprion mechanism of the prion protein-derived CPPs and identify a potential site for PrPSc formation.

Metabolic transit of dietary methylglyoxal.

Methylglyoxal (MGO) is responsible for the pronounced antibacterial activity of manuka honey, in which it may reach concentrations up to 800 mg/kg. As MGO formed in vivo is discussed to play a role in diabetic complications, the metabolic transit of dietary MGO was studied within a 3 day dietary recall with four healthy volunteers. Determination of MGO in 24 h urine was performed with GC-MS after derivatization with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine, and D-lactate was quantified enzymatically. Following a diet virtually free from MGO and other glycation compounds, a defined amount of MGO (500 µmol in manuka honey) was administered in the morning of day 2. Renal excretion was between 0.1 and 0.4 µmol/day for MGO and between 50 and 220 µmol/day for D-lactate. No influence on excretion of both compounds was observed following administration of MGO. To investigate the stability of MGO under physiological conditions, a simulated in vitro gastrointestinal digestion was performed with MGO-containing honey. After 8 h of in vitro digestion, only 5-20% of the initial methylglyoxal was recovered. This indicates that dietary MGO is rapidly degraded during the digestion process in the intestine and, therefore, exerts no influence on the MGO level in vivo.

Parasitosis intestinales y características epidemiológicas en niños de 1 a 12 años de edad: ambulatorio urbano II Laura Labellarte, Barquisimeto, Venezuela / Intestinal parasites and features epidemilógicas in children 1-12 years of age: urban outpatient II Laura Labellarte, Barquisimeto, Venezuela

Las parasitosis intestinales son aún un problema de salud pública que requiere atención de los organismos la salud. Determinar la frecuencia de parasitosis intestinales y características epidemiológicas en niños de 1 a 12 años que acudieron al Ambulatorio Urbano Tipo II “Laura Labellarte”. Se realizó un estudio descriptivo de corte transversal, con muestra no probabilística por conveniencia, constituida por 139 niños. Previo consentimiento informado se realizó una entrevista, un examen de heces directo y concentrado y método de Graham. Los datos se procesaron en el Programa EPI-INFO 2007. Se calcularon porcentajes y proporciones, Chi cuadrado y test de Fisher con intervalo de confianza de 95%. Se encontró parasitado 49,6%, sin predilección por edad ni sexo. Se identificó Blastocystis hominis (43,5%), Enterobius vermicularis (39,1%), Giardia lamblia (33,3%), Entamoeba histolytica (10,1%) y Ascaris lumbricoides (1,4%). Se encontró comensales en 15%. El mayor número de parasitados se observó en quienes tenían inadecuadas disposición de excretas (71%), conservación de los alimentos (57,1%), calidad del agua de consumo (53,8%) y frecuencia de recolección de basura (50%), así como en los sintomáticos (51,3%), siendo más frecuente el dolor abdominal (66,7%). Se demostró asociación de vectores con Blastocystis hominis, moscas con Giardia lamblia y roedores con todos los agentes hallados. La elevada frecuencia de parasitos intestinales, especialmente Blastocystis hominis, con predominio entre quienes viven con fallas en la disposición de excretas, conservación de alimentos y la calidad del agua para consumo, demuestran la persistencia de las parasitosis intestinales como problema de salud pública

The intestinal parasitisms are still a problem of public health that requires attention of health organisms. To determine the frequency of intestinal parasitism and epidemiological characteristics in children 1 to 12 years old that attended the “Laura Labellarte” Urban Type II Ambulatory. A cross-sectional descriptive study was performed, with a nonprobabilistic by convenience sample, constituted by 139 children. With a previous informed consent, parents were interviewed and a direct and concentrate fecal analysis and a Graham´s test were performed. The data was processed in EPI-INFO 2007 Program. Percentage, proportions, Chi square and Fisher`s test with confidence interval of 95% were calculated. 49,6% of patients were parasited, with no predilection by age or sex. Blastocystis hominis (43,5%), Enterobius vermicularis (39,1%), Giardia lamblia (33,3%), Entamoeba histolytica (10,1%) and Ascaris lumbricoides (1,4%) were identified. The greater number of parasited patients was observed in those who had inadequate disposition of feces (71%), food storage (57,1%), quality of drinking water (53,8%) and garbage collection (50%) as well as in symptomatic children (51,3%). The most frequent symptom was abdominal pain (66,7%). Association of vectors with Blastocystis hominis, flies with Giardia lamblia and rodents with all causal agents was demonstrated. The high intestinal parasitisms frequency, especially Blastocystis hominis, with predominance among those who live with faults in feces disposition, foods storage and quality of drinking water, demonstrates the persistence of intestinal parasitisms as a public health problem

Molecular characterization of Brazilian infectious bursal disease virus isolated from 1997 to 2005.

This retrospective study concerned 41 infectious bursal disease virus (IBDV) isolates obtained from Brazilian broiler and layers flocks by reverse transcription-polymerase chain reaction. Twenty-five of them were identified as very virulent (vv) by restriction enzyme analysis and by further nucleotide and phylogenetic analysis. All of them had the typical amino acid residues, and all clustered in a phylogenetic tree with the vvIBDV strains. Four amino acid substitutions, at positions D213N, G254D, S317R, and D323E, were common to 3 vv isolates, Br/03/DB, Br/03/CK, and Br/04/CR, and differed from other vv isolates and strains. These isolates came from the same locale, but were collected in different years, indicating that the vvIBDVs circulating on Brazilian farms are undergoing slight but continuous exchanges.