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Adeno-associated viruses (AAVs) are widely used to deliver genetic material in vivo to distinct cell types such as neurons or glial cells, allowing for targeted manipulation. Transduction of microglia is mostly excluded from this strategy, likely due to the cells’ heterogeneous state upon environmental changes, which makes AAV design challenging. Here, we established the retina as a model system for microglial AAV validation and optimization. First, we show that AAV2/6 transduced microglia in both synaptic layers, where layer preference corresponds to the intravitreal or subretinal delivery method. Surprisingly, we observed significantly enhanced microglial transduction during photoreceptor degeneration. Thus, we modified the AAV6 capsid to reduce heparin binding by introducing four point mutations (K531E, R576Q, K493S, and K459S), resulting in increased microglial transduction in the outer plexiform layer. Finally, to improve microglial-specific transduction, we validated a Cre-dependent transgene delivery cassette for use in combination with the Cx3cr1CreERT2 mouse line. Together, our results provide a foundation for future studies optimizing AAV-mediated microglia transduction and highlight that environmental conditions influence microglial transduction efficiency., Graphical abstract, Targeting microglia in vivo using AAVs is neither robust nor reliable. Here, Maes et al. show that transduction of retinal microglia niches depends on the injection method and the degeneration environment. This informed development of a capsid-modified AAV2/6 that in combination with the Cre-loxP system improved microglia specificity.

Lyme disease is the most common vector-borne disease in the United States. While Lyme disease vectors are widespread, high incidence states are concentrated in the Northeast, North Central and Mid-Atlantic regions. Mapping the distribution of Lyme disease spirochetes in ticks may aid in providing data-driven explanations of epidemiological trends and recommendations for targeting prevention strategies to communities at risk. We compiled data from the literature, publicly available tickborne pathogen surveillance databases, and internal CDC pathogen testing databases to map the county-level distribution of Lyme disease spirochetes reported in host-seeking Ixodes pacificus and Ixodes scapularis across the contiguous United States. We report B. burgdorferi s.s.-infected I. scapularis from 384 counties spanning 26 eastern states located primarily in the North Central, Northeastern, and Mid-Atlantic regions, and in I. pacificus from 20 counties spanning 2 western states, with most records reported from northern and north-coastal California. Borrelia mayonii was reported in I. scapularis in 10 counties in Minnesota and Wisconsin in the North Central United States, where records of B. burgdorferi s.s. were also reported. In comparison to a broad distribution of vector ticks, the resulting map shows a more limited distribution of Lyme disease spirochetes.

Octopuses have evolved incredibly large and complex nervous systems that allow them to perform impressive behaviors, like plan ahead, navigate and solve puzzles. The nervous system of the common octopus (also known as Octopus vulgaris) contains over half a billion nerves cells called neurons, similar to the number found in small primates. Two thirds of these cells reside in the octopuses’ arms, while the rest make-up a central brain that sits between their eyes. Very little is known about how this central brain forms in the embryo, including where the cells originate and which molecular factors drive their maturation in to adult cells. To help answer these questions, Deryckere et al. studied the brain of Octopus vulgaris at different stages of early development using various cell staining and imaging techniques. The experiments identified an important pool of dividing cells which sit in an area outside the central brain called the ‘lateral lips’. In these cells, genes known to play a role in neural development in other animals are active, indicating that the cells had not reached their final, mature state. In contrast, the central brain did not seem to contain any of these immature cells at the point when it was growing the most. To investigate this further, Deryckere et al. used fluorescent markers to track the progeny of the dividing cells during development. This revealed that cells in the lateral lips take on a specific neuronal fate before migrating to their target region in the central brain. Newly matured neurons have also been shown to travel large distances in the embryos of vertebrates, suggesting that this mechanism may be a common strategy for building large, complex brains. Although the nervous system of the common octopus is comparable to mammals, they evolved from a very distant branch of the tree of life; indeed, their last common ancestor was a worm-like animal that lived about 600 million years ago. Studying the brain of the common octopus, as done here, could therefore provide new insights into how complex nervous systems, including our own, evolved over time.

We collected questing Haemaphysalis longicornis ticks from southeastern counties of Pennsylvania, USA. Of 263 ticks tested by PCR for pathogens, 1 adult female was positive for Borrelia burgdorferi sensu stricto, yielding a 0.4% infection rate. Continued monitoring of this invasive tick is essential to determine its public health role.

As the geographic distributions of medically important ticks and tick-borne pathogens continue to expand in the United States, the burden of tick-borne diseases continues to increase along with a growing risk of coinfections. Coinfection with multiple tick-borne pathogens may amplify severity of disease and complicate diagnosis and treatment. By testing 13,400 Ixodes ticks from 17 US states spanning five geographical regions for etiological agents of Lyme disease (Borrelia burgdorferi sensu stricto [s.s.] and Borrelia mayonii), Borrelia miyamotoi disease (Borrelia miyamotoi), anaplasmosis (Anaplasma phagocytophilum), and babesiosis (Babesia microti) we show that B. burgdorferi s.s. was the most prevalent and widespread pathogen. Borrelia miyamotoi, A. phagocytophilum, and B. microti were widespread but less prevalent than B. burgdorferi s.s. Coinfections with B. burgdorferi s.s. and A. phagocytophilum or B. microti were most common in the Northeast and occurred at rates higher than expected based on rates of single infections in that region.

Obesity and other lifestyle diseases in modern society can be related to historical dietary changes from diets balanced in omega-6 and omega-3 to the unbalanced "Western-type" diet. It is recognized that diet influences the murine and human gut microbiome, and most research indicates that microbial diversity and composition are altered by high-fat diets (HFDs). However, good knowledge about the effects of early exposure to HFD on the maturation and structure of the bacterial community is limited. Using mice as model, we hypothesized that an HFD alters the early dynamic of the gut bacterial community toward an unstable/unhealthy state. By sequencing the V3 and V4 regions of the 16S ribosomal ribonucleic acid gene, we investigated the bacterial community in fecal samples of mice fed a control diet and an HFD at weaning (sampling time 1) and after 8 weeks of dietary intervention (11weeks of age; sampling time 2). Natural temporal microbiome maturation was evidenced by a general increase in microbial diversity and shifts in microbial community between sampling times 1 and 2 toward a mature community. However, the HFD led to significant structural segregation of the microbiome compared with controls; the HFD diet repressed health-enhancing bacteria (eg, Bifidobacterium and Akkermansia) and promoted health-detracting bacteria (ie, those associated with gut disorders, eg, Dorea). We suggest that early-life consumption of HFD negatively impacts the natural gut bacterial community maturation leading toward a potentially persistent unhealthy stage.

SUMMARYEffective methods to increase awareness of preventable infectious diseases are key components of successful control programmes. Rabies is an example of a disease with significant impact, where public awareness is variable. A recent awareness campaign in a rabies endemic region of Azerbaijan provided a unique opportunity to assess the efficacy of such campaigns. A cluster cross-sectional survey concerning rabies was undertaken following the awareness campaign in 600 households in 38 randomly selected towns, in districts covered by the campaign and matched control regions. This survey demonstrated that the relatively simple awareness campaign was effective at improving knowledge of rabies symptoms and vaccination schedules. Crucially, those in the awareness campaign group were also 1·4 times more likely to report that they had vaccinated their pets, an essential component of human rabies prevention. In addition, low knowledge of appropriate post-exposure treatment and animal sources of rabies provide information useful for future public awareness campaigns in the region and other similar areas.

Background and purpose A Consensus document on palliative care and neurology has made recommendations on the care of people with chronic and progressive neurological disease. This study aimed to investigate whether these recommendations are understood by, acceptable to and used in practice by neurologists in Belgium. Methods An online survey was undertaken of 100 neurologists in Belgium, asking for their opinion on all of the recommendations in the Consensus document. Results Sixty-four of the neurologists replied. Overall, they expressed support for the recommendations, in particular open communication with patients, open assessment of patient and family needs, and discussion of dying. There was less understanding of the role of palliative care in the implementation of palliative care early in disease progression and the role of palliative care multidisciplinary teams. Conclusions The survey shows that many of the recommendations in the European Academy of Neurology/European Association for Palliative Care Taskforce on Neurology Consensus document are understood by neurologists, and several are now seen as part of normal clinical practice. However, there is still a need to develop a more collaborative approach between neurology and palliative care services, for the benefit of patients and families.

Borrelia mayonii is a newly described member of the Borrelia burgdorferi sensu lato complex that is vectored by the black-legged tick (Ixodes scapularis Say) and a cause of Lyme disease in Minnesota and Wisconsin. Vertebrate reservoir hosts involved in the enzootic maintenance of B. mayonii have not yet been identified. Here, we describe the first isolation of B. mayonii from naturally infected white-footed mice (Peromyscus leucopus Rafinesque) and an American red squirrel (Tamiasciurus hudsonicus Erxleben) from Minnesota, thus implicating these species as potential reservoir hosts for this newly described spirochete.

The natural flight response in shrimp is powered by rapid contractions of the abdominal muscle fibres to propel themselves backwards away from perceived danger. This muscle contraction is dependent on repetitive depolarization of muscle plasma membrane, triggering tightly spaced cytoplasmic [Ca2+] transients and rapidly rising tetanic force responses. To achieve such high amplitude and high frequency of Ca2+ transients requires a high abundance of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) to rapidly clear cytoplasmic Ca2+ between each transient and an efficient Ca2+ release system consisting of the Ryanodine Receptor (RyR), and voltage gated Ca2+ channels (CaVs). With the aim to expand our knowledge of muscle gene function and identify orthologous genes regulating muscle excitation-contraction (EC) coupling, this study assembled nine Penaeid shrimp muscle transcriptomes. On average, the nine transcriptomes contained 27,000 contigs, with an annotation rate of 36% and a BUSCO completeness of 70%. Despite maintaining their function, the crustacean RyR and CaV proteins showed evidence of significant diversification from mammalian orthologs, while SERCA remained more conserved. Several key components of protein interaction were conserved, while others showed distinct crustacean specific evolutionary adaptations. Lastly, this study revealed approximately 1,000 orthologous genes involved in muscle specific processes present across all nine species.

Purpose Inhibition or targeted deletion of histone deacetylase 3 (HDAC3) is neuroprotective in a variety neurodegenerative conditions, including retinal ganglion cells (RGCs) after acute optic nerve damage. Consistent with this, induced HDAC3 expression in cultured cells shows selective toxicity to neurons. Despite an established role for HDAC3 in neuronal pathology, little is known regarding the mechanism of this pathology. Methods Induced expression of an HDAC3-mCherry fusion protein in mouse RGCs was accomplished by transduction with AAV2/2-Pgk-HDAC3-mCherry. Increased susceptibility to optic nerve damage in HDAC3-mCherry expressing RGCs was evaluated in transduced mice that received acute optic nerve crush surgery. Expression of HDAC3-FLAG or HDAC3-mCherry was induced by nucleofection or transfection of plasmids into differentiated or undifferentiated 661W tissue culture cells. Immunostaining for cleaved caspase 3, localization of a GFP-BAX fusion protein, and quantitative RT-PCR was used to evaluate HDAC3-induced damage. Results Induced expression of exogenous HDAC3 in RGCs by viral-mediated gene transfer resulted in modest levels of cell death but significantly increased the sensitivity of these neurons to axonal damage. Undifferentiated 661W retinal precursor cells were resilient to induced HDAC3 expression, but after differentiation, HDAC3 induced GFP-BAX recruitment to the mitochondria and BAX/BAK dependent activation of caspase 3. This was accompanied by an increase in accumulation of transcripts for the JNK2/3 kinases and the p53-regulated BH3-only gene Bbc3/Puma. Cell cycle arrest of undifferentiated 661W cells did not increase their sensitivity to HDAC3 expression. Conclusions Collectively, these results indicate that HDAC3-induced toxicity to neurons is mediated by the intrinsic apoptotic pathway.

Perineuronal nets (PNNs), components of the extracellular matrix, preferentially coat parvalbumin-positive interneurons and constrain critical-period plasticity in the adult cerebral cortex. Current strategies to remove PNN are long-lasting, invasive, and trigger neuropsychiatric symptoms. Here, we apply repeated anesthetic ketamine as a method with minimal behavioral effect. We find that this paradigm strongly reduces PNN coating in the healthy adult brain and promotes juvenile-like plasticity. Microglia are critically involved in PNN loss because they engage with parvalbumin-positive neurons in their defined cortical layer. We identify external 60-Hz light-flickering entrainment to recapitulate microglia-mediated PNN removal. Importantly, 40-Hz frequency, which is known to remove amyloid plaques, does not induce PNN loss, suggesting microglia might functionally tune to distinct brain frequencies. Thus, our 60-Hz light-entrainment strategy provides an alternative form of PNN intervention in the healthy adult brain.

The important roles of mitochondrial function and dysfunction in the process of neurodegeneration are widely acknowledged. Retinal ganglion cells (RGCs) appear to be a highly vulnerable neuronal cell type in the central nervous system with respect to mitochondrial dysfunction but the actual reasons for this are still incompletely understood. These cells have a unique circumstance where unmyelinated axons must bend nearly 90° to exit the eye and then cross a translaminar pressure gradient before becoming myelinated in the optic nerve. This region, the optic nerve head, contains some of the highest density of mitochondria present in these cells. Glaucoma represents a perfect storm of events occurring at this location, with a combination of changes in the translaminar pressure gradient and reassignment of the metabolic support functions of supporting glia, which appears to apply increased metabolic stress to the RGC axons leading to a failure of axonal transport mechanisms. However, RGCs themselves are also extremely sensitive to genetic mutations, particularly in genes affecting mitochondrial dynamics and mitochondrial clearance. These mutations, which systemically affect the mitochondria in every cell, often lead to an optic neuropathy as the sole pathologic defect in affected patients. This review summarizes knowledge of mitochondrial structure and function, the known energy demands of neurons in general, and places these in the context of normal and pathological characteristics of mitochondria attributed to RGCs.

Borrelia miyamotoi is an emerging, tick-borne human pathogen. In North America, it is primarily associated with Ixodes scapularis and Ixodes pacificus, two species known to bite humans. Here we describe the development and evaluation of a pair of real-time TaqMan PCR assays designed to detect B. miyamotoi in North American ticks. We sought to achieve sensitivity to B. miyamotoi strains associated with ticks throughout North America, the full genetic diversity of which is unknown, by targeting sequences that are largely conserved between B. miyamotoi strains from the eastern United States and genetically distinct B. miyamotoi strains from Japan. The two assays target different loci on the B. miyamotoi chromosome and can be run side by side under identical cycling conditions. One of the assays also includes a tick DNA target that can be used to verify the integrity of tick-derived samples. Using both recombinant plasmid controls and genomic DNA from North American and Japanese strains, we determined that both assays reliably detect as few as 5 copies of the B. miyamotoi genome. We verified that neither detects B. burgdorferi, B. lonestari or B. turicatae. This sensitive and specific pair of assays successfully detected B. miyamotoi in naturally-infected, colony-reared nymphs and in field-collected I. scapularis and I. pacificus from the Northeast and the Pacific Northwest respectively. These assays will be useful in screening field-collected Ixodes spp. from varied regions of North America to assess the risk of human exposure to this emerging pathogen.

Purpose Gene therapy of retinal ganglion cells (RGCs) has promise as a powerful therapeutic for the rescue and regeneration of these cells after optic nerve damage. However, early after damage, RGCs undergo atrophic changes, including gene silencing. It is not known if these changes will deleteriously affect transduction and transgene expression, or if the therapeutic protein can influence reactivation of the endogenous genome. Methods Double-transgenic mice carrying a Rosa26-(LoxP)-tdTomato reporter, and a mutant allele for the proapoptotic Bax gene were reared. The Bax mutant blocks apoptosis, but RGCs still exhibit nuclear atrophy and gene silencing. At times ranging from 1 hour to 4 weeks after optic nerve crush (ONC), eyes received an intravitreal injection of AAV2 virus carrying the Cre recombinase. Successful transduction was monitored by expression of the tdTomato reporter. Immunostaining was used to localize tdTomato expression in select cell types. Results Successful transduction of RGCs was achieved at all time points after ONC using AAV2 expressing Cre from the phosphoglycerate kinase (Pgk) promoter, but not the CMV promoter. ONC promoted an increase in the transduction of cell types in the inner nuclear layer, including Muller cells and rod bipolar neurons. There was minimal evidence of transduction of amacrine cells and astrocytes in the inner retina or optic nerve. Conclusions Damaged RGCs can be transduced and at least some endogenous genes can be subsequently activated. Optic nerve damage may change retinal architecture to allow greater penetration of an AAV2 virus to transduce several additional cell types in the inner nuclear layer.

The distribution of I. scapularis, the tick vector of the bacteria that cause Lyme disease, has been expanding over the last two decades in the north-central United States in parallel with increasing incidence of human cases of Lyme disease in that region. However, assessments of residential risk for exposure to ticks are lacking from this region. Here, we measured the density of host-seeking I. scapularis nymphs in two suburban and two rural public recreational sites located in Washington County, Minnesota as well as in nearby residential properties. We sought to compare tick densities across land use types and to identify environmental factors that might impact nymphal density. We also assessed the prevalence of infection in the collected ticks with Lyme disease spirochetes (Borrelia burgdorferi sensu stricto, B. mayonii), and other I. scapularis-borne pathogens including B. miyamotoi, Babesia microti and Anaplasma phagocytophilum. Similar to studies from the eastern United States, on residential properties, I. scapularis nymphal densities were highest in the ecotonal areas between the forest edge and the lawn. Residences with the highest densities of nymphs were more likely to have a higher percentage of forest cover, log piles, and signs of deer on their property. In recreational areas, we found the highest nymphal densities both in the wooded areas next to trails as well as on mowed trails. Among the 303 host-seeking I. scapularis nymphs tested for pathogens, B. burgdorferi sensu stricto, A. phagocytophilum and B. miyamotoi were detected in 42 (13.8%), 14 (4.6%), and 2 (0.6%) nymphs, respectively.

Tick-borne pathogens transmitted by Ixodes scapularis Say (Acari: Ixodidae), also known as the deer tick or blacklegged tick, are increasing in incidence and geographic distribution in the United States. We examined the risk of tick-borne disease exposure in 9 national parks across six Northeastern and Mid-Atlantic States and the District of Columbia in 2014 and 2015. To assess the recreational risk to park visitors, we sampled for ticks along frequently used trails and calculated the density of I. scapularis nymphs (DON) and the density of infected nymphs (DIN). We determined the nymphal infection prevalence of I. scapularis with a suite of tick-borne pathogens including Borrelia burgdorferi, Borrelia miyamotoi, Anaplasma phagocytophilum, and Babesia microti. Ixodes scapularis nymphs were found in all national park units; DON ranged from 0.40 to 13.73 nymphs per 100 m2. Borrelia burgdorferi, the causative agent of Lyme disease, was found at all sites where I. scapularis was documented; DIN with B. burgdorferi ranged from 0.06 to 5.71 nymphs per 100 m2. Borrelia miyamotoi and A. phagocytophilum were documented at 60% and 70% of the parks, respectively, while Ba. microti occurred at just 20% of the parks. Ixodes scapularis is well established across much of the Northeastern and Mid-Atlantic States, and our results are generally consistent with previous studies conducted near the areas we sampled. Newly established I. scapularis populations were documented in two locations: Washington, D.C. (Rock Creek Park) and Greene County, Virginia (Shenandoah National Park). This research demonstrates the potential risk of tick-borne pathogen exposure in national parks and can be used to educate park visitors about the importance of preventative actions to minimize tick exposure.

Plague, a primarily flea-borne disease caused by Yersinia pestis, is characterized by rapidly spreading epizootics separated by periods of quiescence. Little is known about how and where Y. pestis persists between epizootics. It is commonly proposed, however, that Y. pestis is maintained during interepizootic periods in enzootic cycles involving flea vectors and relatively resistant host populations. According to this model, while susceptible individuals serve as infectious sources for feeding fleas and subsequently die of infection, resistant hosts survive infection, develop antibodies to the plague bacterium, and continue to provide bloodmeals to infected fleas. For Y. pestis to persist under this scenario, fleas must remain infected after feeding on hosts carrying antibodies to Y. pestis. Studies of other vector-borne pathogens suggest that host immunity may negatively impact pathogen survival in the vector. Here, we report infection rates and bacterial loads for fleas (both Xenopsylla cheopis (Rothschild) and Oropsylla montana (Baker)) that consumed an infectious bloodmeal and subsequently fed on an immunized or age-matched naive mouse. We demonstrate that neither the proportion of infected fleas nor the bacterial loads in infected fleas were significantly lower within 3 d of feeding on immunized versus naive mice. Our findings thus provide support for one assumption underlying the enzootic host model of interepizootic maintenance of Y. pestis.

The genus Flavivirus includes both vector-borne and no known vector (NKV) species, but the molecular determinants of transmission mode are not known. Conserved sequence differences between the two groups occur in 5′ and 3′ UTRs. To investigate the impact of these differences on transmission, chimeric genomes were generated, in which UTRs, UTRs+capsid, or the upper 3′ UTR stem–loop of mosquito-borne dengue virus (DENV) were replaced with homologous regions from NKV Modoc virus (MODV); the conserved pentanucleotide sequence (CPS) was also deleted from the DENV genome. Virus was not recovered following transfection of these genomes in three different cell types. However, DENV genomes in which the CPS or variable region (VR) of the 3′ UTR were replaced with MODV sequences were recovered and infected Aedes aegypti mosquitoes with similar efficiencies to DENV. These results demonstrate that neither vector-borne CPS nor VR is required for vector-borne transmission.

The potential of a novel class of prebiotics, arabinoxylan oligosaccharides (AXOS), was investigated on growth performance and gut microbiota of juvenile Acipenser baerii. Two independent feeding trials of 10 or 12 weeks were performed with basal diets supplemented with 2% or 4% AXOS-32-0.30 (trial 1) and 2% AXOS-32-0.30 or AXOS-3-0.25 (trial 2), respectively. Growth performance was improved by feeding 2% AXOS-32-0.30 in both trials, although not significantly. Microbial community profiles were determined using 454-pyrosequencing with barcoded primers targeting the V3 region of the 16S rRNA gene. AXOS significantly affected the relative abundance of bacteria at the phylum, family, genus and species level. The consumption of 2% AXOS-32-0.30 increased the relative abundance of Eubacteriaceae, Clostridiaceae, Streptococcaceae and Lactobacillaceae, while the abundance of Bacillaceae was greater in response to 4% AXOS-32-0.30 and 2% AXOS-3-0.25. The abundance of Lactobacillus spp. and Lactococcus lactis was greater after 2% AXOS-32-0.30 intake. Redundancy analysis showed a distinct and significant clustering of the gut microbiota of individuals consuming an AXOS diet. In both trials, concentration of acetate, butyrate and total short-chain fatty acids (SCFAs) increased in fish fed 2% AXOS-32-0.30. Our data demonstrate a shift in the hindgut microbiome of fish consuming different preparation of AXOS, with potential application as prebiotics.

Estimating connectivity between juvenile and adult fish habitats can provide an important contribution to effective fisheries management, through a better understanding of population resilience to harvesting pressure. Indirect methods for quantifying connectivity, such as geochemical or genetic techniques, allow us to assign adults from various sampling regions to their natal location, provided that natal origin data can be defined. The elemental composition of otoliths from juvenile sole Solea solea collected at 4 sampling locations in the Southern Bight of the North Sea was measured using laser-ablation inductively-coupled plasma mass spectrometry (LA-ICPMS), to determine elemental fingerprints indicative of distinct nursery grounds. Significant differences in elemental composition were detected among the 4 locations, with Na, Sr, Ba, Mn and RI) concentrations varying the most between groups. A discriminant model resulted in high assignment proportions of the juvenile fish to their respective nursery grounds with a total jackknife reclassification success of 88%. Even though some interannual variability in otolith chemistry was observed in juveniles from the Scheldt estuary, spatial patterns seemed to dominate. Our results constitute a firm basis for future investigations on nursery area contributions and quality, adult dispersal history and applications of population traceability.

SUMMARYHuman anthrax cases reported in the country of Georgia increased 75% from 2011 (n= 81) to 2012 (n= 142). This increase prompted a case-control investigation using 67 culture- or PCR-confirmed cases and 134 controls matched by residence and gender to investigate risk factor(s) for infection during the month before case onset. Independent predictors most strongly associated with disease in the multivariable modelling were slaughtering animals [odds ratio (OR) 7·3, 95% confidence interval (CI) 2·9–18·1,P< 0·001] and disposing of dead animals (OR 13·6, 95% CI 1·5–119·8,P= 0·02). Participants owning or working with livestock (n= 131) were additionally interviewed about livestock management practices during the previous 6 months: 53 (44%) of 121 respondents vaccinated livestock against anthrax; 19 (16%) of 116 moved livestock >1 km; 15 (12%) of 125 had sick livestock; and 11 (9%) of 128 respondents reported finding dead livestock. We recommend joint public health and veterinary anthrax case investigations to identify areas of increased risk for livestock anthrax outbreaks, annual anthrax vaccination of livestock in those areas, and public awareness education.

The pro-apoptotic BCL2 gene family member, BAX, plays a pivotal role in the intrinsic apoptotic pathway. Under cellular stress, BAX recruitment to the mitochondria occurs when activated BAX forms dimers, then oligomers, to initiate mitochondria outer membrane permeabilization (MOMP), a process critical for apoptotic progression. The activation and recruitment of BAX to form oligomers has been studied for two decades using fusion proteins with a fluorescent reporter attached in-frame to the BAX N-terminus. We applied high-speed live cell imaging to monitor the recruitment of BAX fusion proteins in dying cells. Data from time-lapse imaging was validated against the activity of endogenous BAX in cells, and analyzed using sigmoid mathematical functions to obtain detail of the kinetic parameters of the recruitment process at individual mitochondrial foci. BAX fusion proteins behave like endogenous BAX during apoptosis. Kinetic studies show that fusion protein recruitment is also minimally affected in cells lacking endogenous BAK or BAX genes, but that the kinetics are moderately, but significantly, different with different fluorescent tags in the fusion constructs. In experiments testing BAX recruitment in 3 different cell lines, our results show that regardless of cell type, once activated, BAX recruitment initiates simultaneously within a cell, but exhibits varying rates of recruitment at individual mitochondrial foci. Very early during BAX recruitment, pro-apoptotic molecules are released in the process of MOMP, but different molecules are released at different times and rates relative to the time of BAX recruitment initiation. These results provide a method for BAX kinetic analysis in living cells and yield greater detail of multiple characteristics of BAX-induced MOMP in living cells that were initially observed in cell free studies.

OBJECTIVES: This study assessed measles vaccination rates and risk factors for lack of vaccination among preschool children enrolled in the Special Supplemental Food Program for Women, Infants, and Children (WIC) during the 1991 measles epidemic in New York City. METHODS: Children aged 12 to 59 months presenting for WIC certification between April 1 and September 30, 1991, at six volunteer WIC sites in New York City were surveyed. RESULTS: Of the 6181 children enrolled in the study, measles immunization status was ascertained for 6074 (98%). Overall measles coverage was 86% (95% confidence interval [CI] = +/- 1%) and at least 90% by 21 months of age (95% CI = +/- 1%). Young age of the child, use of a private provider, and Medicaid as a source of health care payment were risk factors for lack of vaccination (P < .001). CONCLUSIONS: During the peak of a measles epidemic, measles immunization rates were more than 80% by 24 months of age in a sample of WIC children. The ease of ascertaining immunization status and the size of the total WIC population underscore the importance of WIC immunization initiatives.

The four major flavivirus clades are transmitted by mosquitoes, ticks, directly between vertebrates or directly between arthropods, respectively, but the molecular determinants of mode of transmission in flaviviruses are unknown. To assess the role of the UTRs in transmission, we generated chimeric genomes in which the 5′ UTR, capsid and/or 3′ UTR of mosquito-borne dengue virus serotype 4 (rDENV-4) were replaced, separately or in combination, with those of tick-borne Langat virus (rLGTV). None of the chimeric genomes yielded detectable virus following transfection. Replacement of the variable region (VR) in the rDENV-4 3′ UTR with that of rLGTV generated virus rDENV-4-rLGTswapVR, which showed lower replication than its wild-type parents in mammalian but not mosquito cells in culture and was able to infect mosquitoes in vivo. Neither rDENV-4 nor rDENV-4-rLGTswapVR could infect larval Ixodes scapularis ticks immersed in virus, while rLGTV was highly infectious via this route.

Archived otolith collections represent an invaluable source of information to study demographic and genetic changes in commercially important fish populations. Studies combining both approaches are however rare and reliable extraction of molecular and population demographic data from the same collection of otoliths has never been assessed in the endangered European eel ( Anguilla anguilla L.). Here we evaluate various DNA extraction protocols to compare DNA yield, microsatellite amplification success, genotype integrity and precision of age determination for eel otoliths that have been archived for 4 weeks, and for 28 and 48 years. Our results show a high amplification success and an equal genotype integrity for DNA fragments extracted from both recently sampled otoliths and high quality reference DNA tissue. Although historical samples yielded low amounts of DNA, PCR amplification was successful and genotyping reliable for short fragments, but decreased significantly with PCR fragment size. None of the extraction protocols caused physical damage to the otoliths and precision of age determination was high for both treated and untreated otoliths. Hence, the methodology can be applied as a standard for the further joint analysis of past demographic and genetic changes during the last century in the highly exploited European eel and in other fish requiring urgent conservation measures.

The growing accessibility to genomic resources using next-generation sequencing (NGS) technologies has revolutionized the application of molecular genetic tools to ecology and evolutionary studies in non-model organisms. Here we present the case study of the European hake (Merluccius merluccius), one of the most important demersal resources of European fisheries. Two sequencing platforms, the Roche 454 FLX (454) and the Illumina Genome Analyzer (GAII), were used for Single Nucleotide Polymorphisms (SNPs) discovery in the hake muscle transcriptome. De novo transcriptome assembly into unique contigs, annotation, and in silico SNP detection were carried out in parallel for 454 and GAII sequence data. High-throughput genotyping using the Illumina GoldenGate assay was performed for validating 1,536 putative SNPs. Validation results were analysed to compare the performances of 454 and GAII methods and to evaluate the role of several variables (e.g. sequencing depth, intron-exon structure, sequence quality and annotation). Despite well-known differences in sequence length and throughput, the two approaches showed similar assay conversion rates (approximately 43%) and percentages of polymorphic loci (67.5% and 63.3% for GAII and 454, respectively). Both NGS platforms therefore demonstrated to be suitable for large scale identification of SNPs in transcribed regions of non-model species, although the lack of a reference genome profoundly affects the genotyping success rate. The overall efficiency, however, can be improved using strict quality and filtering criteria for SNP selection (sequence quality, intron-exon structure, target region score).

Historical otolith collections are crucial in assessing the evolutionary consequences of natural and anthropogenic changes on the demography and connectivity of commercially important fish species. Hence, it is important to define optimal protocols for purifying DNA from such valuable information sources while avoiding any damage to the physical structure of the otolith. Before being able to conclude on the harmlessness of a method, it is important to validate protocols on different kinds of otoliths by testing purification methodologies under standardized conditions. Here we compare the effect of two DNA extraction methods on the success in identifying the age in an overexploited marine fish, the common sole (Solea solea L.). To ensure optimal future population genetic and demographic analyses, we assessed DNA quantity and tested the DNA quality by investigating the amplification success of a mitochondrial and nuclear marker. Our results show that the choice of the DNA extraction method had a significant effect on the success of using these otoliths in age and growth analyses. Standard commercial and published protocols resulted in a severe damaging of the otolith structure, hampering accurate preparation and analyses of the morphological structures of the otoliths. Shortening the lysis time and lowering the EDTA (ethylene diamine tetraacetic acid) and SDS (sodium dodecylsulphate) concentration turned out to be beneficial for the stability of otolith structure, while maintaining an overall high DNA quality measured through polymerase chain reaction amplification success. We therefore recommend that care should be taken when choosing the extraction method for a molecular study on archived samples, in order to enable the maximal use of information embedded in historical material.

The Rgcs1 quantitative trait locus, on mouse chromosome 5, influences susceptibility of retinal ganglion cells to acute damage of the optic nerve. Normally resistant mice (DBA/2J) congenic for the susceptible allele from BALB/cByJ mice exhibit susceptibility to ganglion cells, not only in acute optic nerve crush, but also to chronic inherited glaucoma that is characteristic of the DBA/2J strain as they age. SNP mapping of this QTL has narrowed the region of interest to 1 Mb. In this region, a single gene (Spink2) is the most likely candidate for this effect. Spink2 is expressed in retinal ganglion cells and is increased after optic nerve damage. This gene is also polymorphic between resistant and susceptible strains, containing a single conserved amino acid change (threonine to serine) and a 220 bp deletion in intron 1 that may quantitatively alter endogenous expression levels between strains. Overexpression of the different variants of Spink2 in D407 tissue culture cells also increases their susceptibility to the apoptosis-inducing agent staurosporine in a manner consistent with the differential susceptibility between the DBA/2J and BALB/cByJ strains.

Two orthopedic surgeons performed 27 knee arthroscopies on 26 patients using the 1.44 micrometers Nd:YAG. Mean patient age was 52.6 years. Six patients had their entire procedure done using the Nd:YAG alone and 20 had their procedures done using both the laser and a conventional arthroscopic side-shaver. Three patients had an additional knee procedure done immediately following their arthroscopy. The mean operative procedure time was 47.25 minutes (SD equals 14.75). The mean energy setting used was 2.2 Joules/Pulse (SD equals 0.89), mean frequency 16.6 Hz (SD equals 6.4), and mean total energy delivered was 9418.7 Joules (SD equals 6032.5). There were 15 patients with a minimum follow-up of at least 4 weeks (mean 8.26 weeks). The final results showed 35% returned to normal baseline, 43% improved from pre-op condition, and 21% were the same as pre-op. None of the patients were worse. The 1.44 micrometers Nd:YAG has a water absorption coefficient nearly identical to the 2.1 micrometers Ho:YAG, which is currently a popular arthroscopic tool. We conclude that the 1.44 micrometers Nd:YAG is an effective alternative for arthroscopic procedures of the knee.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

A new flexible handheld delivery system for the Erbium:YAG laser has recently been developed. We studied the ability of this system to deliver energy levels sufficient to cut human cadaveric femoral condylar bone and meniscal tissue, and evaluated the histologic effects and quality of those cuts. Furrowing cuts were made with the 2.9-micrometers Erbium:YAG laser in human cadaveric femoral condylar bone and meniscal tissue. Multiple cuts were delivered through a flexible handpiece with a focusing tip using five different energy settings ranging from 200 mj to 1000 mj at 10 Hz. The tissue samples were fixed and stained with HE and Trichrome. Microscopic analysis was completed and data is reported as direct measurements of histologic damage based on differential staining characteristics. This study shows that sufficient energy to cut cartilage and bone can be delivered through a flexible handheld device. The cut surfaces showed outstanding quality and minimal tissue damage, especially when compared to the Ho:YAG, Nd:YAG, and the CO2 lasers, none of which adequately cut bone at the present time.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

SummaryAmidolytic assay of antithrombin III on capillary blood can validly substitute for similar assays performed on venous blood, as an excellent correlation exists (r = 0.95).For the amidolytic assays of heparin cofactor activity, a much less satisfactory correlation is found (r = 0.81). Results are far more dispersed and a decrease is observed in late capillary samples.Using a low heparin concentration to assay heparin cofactor activity leads to surprisingly high activities for capillary blood. The same type of discrepancy is observed during the earliest stages of clotting of venous blood.

Single pancreatic B cells are purified by autofluorescence-activated cell sorting, and their secretory activity is measured after overnight culture. Compared to intact islets, the isolated cells release 2-fold more insulin under basal conditions and 5-fold less during nutrient stimulation. Their secretory activity can be induced by glucose, leucine, or arginine, but only 0.3-1.7% of their hormone content is liberated at 20 mM nutrient concentrations. This poor nutrient-induced insulin release from purified B cells is attributed to their low cAMP levels and is markedly increased after addition of (Bu)2cAMP, of glucagon, or of pancreatic A cells. These results strongly support the concept that the potent in vivo insulin-releasing action of glucose and leucine is not only dependent on their fuel capacity in pancreatic B cells but also on the concurrent cAMP levels in these cells. In isolated islets, endogenously released glucagon apparently determines the cAMP production in B cells and thus participates in the nutrient-induced secretory process. Somatostatin and epinephrine were shown to exert their suppressive effects via the glucagon-dependent messenger system. It is concluded that nutrients and hormones interact with two different messenger systems which amplify each others' stimulatory effect upon insulin release. cAMP might represent the hormone-induced messenger which sets the B cell's sensitivity and secretory capacity for nutrient stimuli such as glucose. The higher insulin secretory response observed after reaggregation of single B cells could not be attributed to an altered activity in the nutrient or hormonal regulatory units, raising the possibility that the aggregated state of the cells is rather responsible for a better organization or cooperation of the secretory effector unit.

A 50 year old man is described in whom primary hypothyroidism and isolated ACTH deficiency leading to adrenocortical insufficiency occurred simultaneously. Low thyroid hormone levels, elevated serum TSH values and high titers of antithyroid antibodies (ATA) were consistent with primary hypothyroidism. The diagnosis of secondary adrenocortical insufficiency was based on low cortisol and ACTH morning levels, low urinary steroids, a significant increase of cortisol levels after 0.25 mg tetracosactide iv, an absence of ACTH and cortisol release after insulin-induced hypoglycemia and a similar absence of ACTH and 11-deoxycortisol increase after metyrapone administration. After one year treatment and five days withdrawal of cortisol administration, the insulin-induced hypoglycemia still failed to elicit secretory responses of ACTH and cortisol, despite the normalization of thyroid hormone levels. The etiopathogenesis of these two coexisting endocrine deficiencies is discussed.

A case of MEN type I a is described showing among other symptoms, three attacks of transient neurological symptoms. These were attributed to a localized, transient cerebral ischemia. The latter probably developed as a consequence of the concomitant occurrence of different phenomena: the existence of hypercalcemia and on the other hand the secretion of vasoactive substances by one of the three tumours forming the MEN type II a syndrome. The pheochromocytoma played an essential role because the symptoms did not recur after the bilateral adrenalectomy.

The function of the heart is characterized by nonuniform wall motions working coordinately to generate a smooth and effective pump action; however, the functional importance of these heterogeneous motions are largely unknown. To bridge the understanding of the basis for regional variations in contraction, we have analyzed left ventricular free-wall motion using echocardiography and sonomicrometry and quantified the expression of protein levels and post-translational modifications using gel electrophoresis. Porcine myocardium was used for investigation and a stress test, i.e. dobutamine infusion, was performed to amplify transmural contractile gradients during beta-adrenergic stimulation. Here we report greater segmental shortening, strain and strain rate in the endocardium compared to the epicardium in both the longitudinal and circumferential directions (p

Highlights • Background of lentiviral and adeno-associated virus vector design. • Reviewing of literature that reported in vivo microglial transduction. • Challenges to overcome low microglial transduction efficiency and specificity. • Guidelines for reporting viral transduction in microglia., Microglia have emerged as a critical component of neurodegenerative diseases. Genetic manipulation of microglia can elucidate their functional impact in disease. In neuroscience, recombinant viruses such as lentiviruses and adeno-associated viruses (AAVs) have been successfully used to target various cell types in the brain, although effective transduction of microglia is rare. In this review, we provide a short background of lentiviruses and AAVs, and strategies for designing recombinant viral vectors. Then, we will summarize recent literature on successful microglial transductions in vitro and in vivo, and discuss the current challenges. Finally, we provide guidelines for reporting the efficiency and specificity of viral targeting in microglia, which will enable the microglial research community to assess and improve methodologies for future studies.