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The Trypanosoma theileri group includes several trypanosome species hardly distinguishable due to the lack of discriminating morphological characters. Trypanosomes belonging to this group have been isolated from different bovine, ovine, and cervids in Europe, Africa, Asia, and Americas. The principal vectors of the T. theileri group are considered tabanid flies; however, T. melophagium is transmitted exclusively by sheep keds. In 2016, 128 sand flies out of 2,728 trapped in Valsamoggia municipality, Italy, were individually dissected and an unknown trypanosome strain, named TrPhp1, was isolated from a female of the sand fly Phlebotomus perfiliewi . Sequence analysis placed this trypanosome in the T. theileri group with very high homology to other trypanosomes detected in European cervids. This is the first report of the T. theileri group isolation from a sand fly, and the possible role of this insect group in the trypanosome transmission cycle is discussed. Within the T. theileri group, the phylogenetic analysis distinguished several lineages, which, unfortunately, do not correspond with their host specificity and their taxonomic status remains ambiguous.

Over the last few years a considerable spread of ethnic foods was observed in Italy. Among them is the Döner kebab. During 2014-2015, in order to evaluate the effectiveness of traditional cooking process, raw product (defrosted), sliced cooked portions cut through electric knife and assembled sandwich were officially sampled in kebab houses and in a local industrial kebab producer in Reggio Emilia (a province in Italy). Microbiological researches for safety and hygienic microbiological indicators were carried out (research of Salmonella , Listeria monocytogenes , Campylobacter and Shiga toxin-producing Escherichia coli ; enumeration mesophilic aerobic bacteria, lactic acid bacteria, sulfite-reducing bacteria growing under anaerobic conditions, yeasts and molds). Between the raw and the cooked product an average of 3 log reduction in mesophilic aerobic bacteria counts was observed. In two out of three kebab houses sampled, which were supplied by the same local industrial producer, the presence of L. monocytogenes was detected. During the official inspection carried out at the production plant a contamination of L. monocytogenes was assessed in both ambient and instruments. Furthermore, 3 lots of products were analyzed and all were found to be contaminated by L. monocytogenes (always above 100 CFU/g). In order to verify the capability of the traditional cooking process to reduce the risk of contamination at an acceptable level, a batch of naturally contaminated kebab (4.5 log CFU/g) was cooked and sliced simulating a day work activity in a kebab shop. The product was then sampled during preparation and enumeration of L. monocytogenes was obtained. After an hour of cooking, the residual contamination was 1.8 log CFU/g, after two hours and a half L. monocytogenes was no longer detectable in the product, but half an hour later it was again detectable in 25g. At the end of the experiment, the contamination grown up to the same level enumerated after an hour of cooking (1.8 log CFU/g). Considering the microbiological results, traditional cooking obtained a rate of -2.40 log CFU/gh -1 , a D=26 min that corresponds to a temperature of maximum 60°C (z=6). In conclusion, our experiment demonstrates the traditional kebab cooking process could not always guarantee a complete product decontamination., Competing Interests: Conflict of interest: the authors declare no potential conflict of interest.

Genetic disruptions of spindle/centrosome-associated WD40-repeat protein 62 (WDR62) are causative for autosomal recessive primary microcephaly (MCPH) and a broader range of cortical malformations. Since the identification of WDR62 as encoded by the MCPH2 locus in 2010, recent studies that have deleted/depleted WDR62 in various animal models of cortical development have highlighted conserved functions in brain growth. Here, we provide a timely review of our current understanding of WDR62 contributions in the self-renewal, expansion and fate specification of neural stem and progenitor cells that are critical for neocortical development. Recent studies have revealed multiple functions for WDR62 in the regulation of spindle organization, mitotic progression and the duplication and biased inheritance of centrosomes during asymmetric divisions. We also discuss recently elaborated WDR62 interaction partners that include Aurora and c-Jun N-terminal kinases as part of complex signalling mechanisms that may define its neural functions. These studies provide new insights into the molecular and cellular processes that are required for brain formation and implicated in the genesis of primary microcephaly.

Sensory neurons of the dorsal root ganglia (DRG) are the primary responders to stimuli inducing feelings of touch, pain, temperature, vibration, pressure and muscle tension. They consist of multiple subpopulations based on their morphology, molecular and functional properties. Our understanding of DRG sensory neurons has been predominantly driven by rodent studies and using transformed cell lines, whereas less is known about human sensory DRG neurons simply because of limited availability of human tissue. Although these previous studies have been fundamental for our understanding of the sensory system, it is imperative to profile human DRG subpopulations as it is becoming evident that human sensory neurons do not share the identical molecular and functional properties found in other species. Furthermore, there are wide range of diseases and disorders that directly/indirectly cause sensory neuronal degeneration or dysfunctionality. Having an in vitro source of human DRG sensory neurons is paramount for studying their development, unique neuronal properties and for accelerating regenerative therapies to treat sensory neuropathies. Here we review the major studies describing generation of DRG sensory neurons from human pluripotent stem cells and fibroblasts and the gaps that need to be addressed for using in vitro-generated human DRG neurons to model human DRG tissue., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Progress in aeronautics and spaceflight technologies requires in parallel further research on how microgravity may affect human tissue. To date, little is known about the effects of microgravity on human development. In this study we used the rotary cell culture system to investigate whether microgravity supports the generation and maintenance of neural organoids derived from human embryonic stem cells (hESCs) as a model of human brain development. Our results show that although neural organoids could be generated and maintained in microgravity conditions, there were changes in expression of rostral-caudal neural patterning genes and cortical markers compared to organoids generated in standard conditions. This phenomenon was also observed in hESC-derived cortical organoids exposed to microgravity for relatively shorter periods. These results are one of the first for analyzing human neurogenesis in a microgravity environment.

Mutations in RAB39B are a known cause of X-linked early onset Parkinson's disease. Isogenic human embryonic stem cell lines carrying two independent deletions of RAB39B were generated using CRISPR/Cas9 genome editing tool. The deletions were confirmed by PCR and direct sequence analysis in two edited stem cell lines. Both cell lines showed pluripotency and displayed a normal karyotype. Further, they were able to form embryoid bodies in vitro, and express markers indicative of differentiation to the three germ layers., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

In this study, we explore the use of electrically active graphene foam as a scaffold for the culture of human-derived neurons. Human embryonic stem cell (hESC)-derived cortical neurons fated as either glutamatergic or GABAergic neuronal phenotypes were cultured on graphene foam. We show that graphene foam is biocompatible for the culture of human neurons, capable of supporting cell viability and differentiation of hESC-derived cortical neurons. Based on the findings, we propose that graphene foam represents a suitable scaffold for engineering neuronal tissue and warrants further investigation as a model for understanding neuronal maturation, function and circuit formation., Competing Interests: We declare we have no competing interests.

Background: Different phleboviruses are important pathogens for humans; most of these viruses are transmitted by sand flies. An increasing number of new phleboviruses have been reported over the past decade, especially in Mediterranean countries, mainly via their detection in sand flies., Results: At least five different phleboviruses co-circulated in sand flies that were collected in three sites in Emilia-Romagna (Italy) in the summer of 2013. The well-known Toscana virus (TOSV) was isolated; three new, closely related phleboviruses differing in their M segments and tentatively named Ponticelli I, Ponticelli II and Ponticelli III virus, respectively, were isolated; a fifth putative phlebovirus, related to the sand fly fever Naples phlebovirus species, was also detected. The co-circulation, in a restricted area, of three viruses characterized by different M segments, likely resulted from reassortment events. According to the phylogenetic analysis of complete genome sequences, the TOSV belongs to clade A, together with other Italian isolates, while the Ponticelli viruses fall within the Salehabad phlebovirus species., Conclusions: Results highlight an unexpected diversity of phleboviruses that co-circulate in the same area, suggesting that interactions likely occur amongst them, that can present challenges for their correct identification. The co-circulation of different phleboviruses appears to be common, and the bionomics of sand fly populations seem to play a relevant role. Such a complex situation emphasizes the need for detailed investigations of the biology of these viruses to better characterize their pathogenic potential for mammals, including humans.

Neurons release neurotransmitters such as glutamate to communicate with each other and to coordinate brain functioning. As increased glutamate release is indicative of neuronal maturation and activity, a system that can measure glutamate levels over time within the same tissue and/or culture system is highly advantageous for neurodevelopmental investigation. To address such challenges, we develop for the first time a convenient method to realize functionalized borosilicate glass capillaries with nanostructured texture as an electrochemical biosensor to detect glutamate release from cerebral organoids generated from human embryonic stem cells (hESC) that mimic various brain regions. The biosensor shows a clear catalytic activity toward the oxidation of glutamate with a sensitivity of 93 ± 9.5 nA·µM -1 ·cm -2 . It was found that the enzyme-modified microelectrodes can detect glutamate in a wide linear range from 5 µM to 0.5 mM with a limit of detection (LOD) down to 5.6 ± 0.2 µM. Measurements were performed within the organoids at different time points and consistent results were obtained. This data demonstrates the reliability of the biosensor as well as its usefulness in measuring glutamate levels across time within the same culture system., Competing Interests: The authors declare no conflict of interest.

The dorsal root ganglia (DRG) consist of a multitude of sensory neuronal subtypes that function to relay sensory stimuli, including temperature, pressure, pain and position to the central nervous system. Our knowledge of DRG sensory neurons have been predominantly driven by animal studies and considerably less is known about the human DRG. Human embryonic stem cells (hESC) are valuable resource to help close this gap. Our previous studies reported an efficient system for deriving neural crest and DRG sensory neurons from hESC. Here we show that this differentiation system gives rise to heterogeneous populations of sensory neuronal subtypes as demonstrated by phenotypic and functional analyses. Furthermore, using microelectrode arrays the maturation rate of the hESC-derived sensory neuronal cultures was monitored over 8 weeks in culture, showing their spontaneous firing activities starting at about 12 days post-differentiation and reaching maximum firing at about 6 weeks. These studies are highly valuable for developing an in vitro platform to study the diversity of sensory neuronal subtypes found within the human DRG.

Adrenomedullary chromaffin cells are catecholamine (CA)-producing cells originating from trunk neural crest (NC) via sympathoadrenal progenitors (SAPs). We generated NC and SAPs from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in vitro via BMP2/FGF2 exposure, ascertained by qPCR and immunoexpression of SOX10, ASCL1, TFAP2α, and PHOX2B, and by fluorescence-activated cell sorting selection for p75NTR and GD2, and confirmed their trunk-like HOX gene expression. We showed that continuing BMP4 and curtailing FGF2 in vitro, augmented with corticosteroid mimetic, induced these cells to upregulate the chromaffin cell-specific marker PNMT and other CA synthesis and storage markers, and we demonstrated noradrenaline and adrenaline by Faglu and high-performance liquid chromatography. We showed these human cells' SAP-like property of migration and differentiation into cells expressing chromaffin cell markers by implanting them into avian embryos in vivo and in chorio-allantoic membrane grafts. These cells have the potential for investigating differentiation of human chromaffin cells and for modeling diseases involving this cell type., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: While evidence for white matter and astrocytic abnormalities exist in autism, a detailed investigation of astrocytes has not been conducted. Such an investigation is further warranted by an increasing role for neuroinflammation in autism pathogenesis, with astrocytes being key players in this process. We present the first study of astrocyte density and morphology within the white matter of the dorsolateral prefrontal cortex (DLPFC) in individuals with autism., Methods: DLPFC formalin-fixed sections containing white matter from individuals with autism ( n  = 8, age = 4-51 years) and age-matched controls ( n  = 7, age = 4-46 years) were immunostained for glial fibrillary acidic protein (GFAP). Density of astrocytes and other glia were estimated via the optical fractionator, astrocyte somal size estimated via the nucleator, and astrocyte process length via the spaceballs probe., Results: We found no evidence for alteration in astrocyte density within DLPFC white matter of individuals with autism versus controls, together with no differences in astrocyte somal size and process length., Conclusion: Our results suggest that astrocyte abnormalities within the white matter in the DLPFC in autism may be less pronounced than previously thought. However, astrocytic dysregulation may still exist in autism, even in the absence of gross morphological changes. Our lack of evidence for astrocyte abnormalities could have been confounded to an extent by having a small sample size and wide age range, with pathological features potentially restricted to early stages of autism. Nonetheless, future investigations would benefit from assessing functional markers of astrocytes in light of the underlying pathophysiology of autism.

Usutu virus is an arbovirus closely related to West Nile virus (genus Flavivirus), which circulates between mosquitoes and wild birds. This virus has been increasingly reported in Europe, raising concerns for its possible pathogenic potential for wild birds and humans. This study reports the whole genome sequences of 15 strains of USUV, isolated between 2010 and 2014 from mosquitoes and wild birds in the course of West Nile virus surveillance in the Emilia-Romagna and Lombardy regions of Northern Italy. Both whole and 656 partial genome sequences, obtained from isolated viruses and field samples (mosquitoes and wild birds), were analyzed to describe the temporal and geographical spread of USUV in the surveyed area. The detected sequences belonged to two groups, with one circulating primarily in the northwestern part of the area and the other in the southeastern part. This pattern is likely the result of different routes of introduction from the North (over the Alps) and from the East, respectively. The phylogenetic analysis of obtained sequences and other European sequences demonstrated that the majority of European strains belonged to one main clade, while less common strains, mainly from Western Europe, fell in other two clades. This analysis strongly suggested an autochthonous evolution process of strains of the main clade from a common ancestor with an estimated time of arrival in Europe at the beginning of the 1990s. In addition to causing mass mortality in wild birds, Usutu virus can infect humans and can sporadically cause disease. These factors and the endemization of the Usutu virus in a large area of Europe, sustained by the obtained data, strongly support the need to adequately survey Usutu virus in areas in which its circulation is detected., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Usutu (USUV) and West Nile (WNV) are mosquito-borne Flavivirus emerged in Italy in 1996 and 1998, respectively, and reappeared 10 years later. The aim of this work is to review the Italian mosquito species found positive for WNV and USUV between 2008 and 2014. Moreover, the role of mosquitoes in promoting the overwintering of these viruses is discussed, as a result of the mosquito collections performed in Molise region between September 2010 and April 2011. Overall 99,000 mosquitoes were collected: 337 and 457 mosquito pools tested positive by real time reverse transcriptase polymerase chain reaction (real time RT-PCR) for WNV and USUV, respectively. West Nile virus was detected in pools of Culex pipiens s.l. (329), Ochlerotatus caspius (4), Culex modestus (2), and Culex spp. (2). Positive USUV pools were from Cx. pipiens s.l. (435), Aedes albopictus (12), Oc. caspius (5), Culex spp. (2), Anopheles maculipennis s.l. (1), Culiseta annulata (1), and Ochlerotatus detritus (1). In Molise region, 1,694 mosquitoes were collected, and USUV was identi ed in Cx. pipiens s.l., Cs. annulata, and Oc. detritus pools. This paper shows that Cx. pipiens s.l. is the mosquito species most involved in the WNV and USUV circulation in Italy, although other species would also support the spread of both the viruses during Winter.

Developing effective and affordable biomarkers for dementias is critical given the difficulty to achieve early diagnosis. In this sense, electroencephalographic (EEG) methods offer promising alternatives due to their low cost, portability, and growing robustness. Here, we relied on EEG signals and a novel information-sharing method to study resting-state connectivity in patients with behavioral variant frontotemporal dementia (bvFTD) and controls. To evaluate the specificity of our results, we also tested Alzheimer's disease (AD) patients. The classification power of the ensuing connectivity patterns was evaluated through a supervised classification algorithm (support vector machine). In addition, we compared the classification power yielded by (i) functional connectivity, (ii) relevant neuropsychological tests, and (iii) a combination of both. BvFTD patients exhibited a specific pattern of hypoconnectivity in mid-range frontotemporal links, which showed no alterations in AD patients. These functional connectivity alterations in bvFTD were replicated with a low-density EEG setting (20 electrodes). Moreover, while neuropsychological tests yielded acceptable discrimination between bvFTD and controls, the addition of connectivity results improved classification power. Finally, classification between bvFTD and AD patients was better when based on connectivity than on neuropsychological measures. Taken together, such findings underscore the relevance of EEG measures as potential biomarker signatures for clinical settings.

The capacity for induced pluripotent stem (iPS) cells to be differentiated into a wide range of neural cell types makes them an attractive donor source for autologous neural transplantation therapies aimed at brain repair. Translation to the in vivo setting has been difficult, however, with mixed results in a wide variety of preclinical models of brain injury and limited information on the basic in vivo properties of neural grafts generated from human iPS cells. Here we have generated a human iPS cell line constitutively expressing green fluorescent protein as a basis to identify and characterize grafts resulting from transplantation of neural progenitors into the adult rat brain. The results show that the grafts contain a mix of neural cell types, at various stages of differentiation, including neurons that establish extensive patterns of axonal growth and progressively develop functional properties over the course of 1 year after implantation. These findings form an important basis for the design and interpretation of preclinical studies using human stem cells for functional circuit re-construction in animal models of brain injury. Stem Cells Translational Medicine 2017;6:1547-1556., (© 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

We sought to identify the impacts of Friedreich's ataxia (FRDA) on cardiomyocytes. FRDA is an autosomal recessive degenerative condition with neuronal and non-neuronal manifestations, the latter including progressive cardiomyopathy of the left ventricle, the leading cause of death in FRDA. Little is known about the cellular pathogenesis of FRDA in cardiomyocytes. Induced pluripotent stem cells (iPSCs) were derived from three FRDA individuals with characterized GAA repeats. The cells were differentiated into cardiomyocytes to assess phenotypes. FRDA iPSC- cardiomyocytes retained low levels of FRATAXIN (FXN) mRNA and protein. Electrophysiology revealed an increased variation of FRDA- cardiomyocyte beating rates which was prevented by addition of nifedipine, suggestive of a calcium handling deficiency. Finally, calcium imaging was performed and we identified small amplitude, diastolic and systolic calcium transients confirming a deficiency in calcium handling. We defined a robust FRDA cardiac-specific electrophysiological profile in patient-derived iPSCs which could be used for high throughput compound screening. This cell-specific signature will contribute to the identification and screening of novel treatments for this life-threatening disease.

Primitive cardiac lymphoma (PCL) is a rare disease accounting for only 1-2% of primary cardiac tumors. Diffuse large B cell lymphoma is the most common type and shows a rapid progression with poor prognosis. The clinical presentation of PCL is nonspecific, and echocardiographic study is essential to the initial work-up. Magnetic resonance imaging and computed tomography scan are the methods of choice for the assessment of tumor extension. The definitive diagnosis is histopathology examination. Chemotherapy and radiotherapy represent the best treatment and should be started promptly after PCL diagnosis. We here report a case of PCL in a 59-year-old man complicated by pulmonary microembolism, atrial fibrillation and signs of right outflow tract obstruction.

Mutations in WD40-repeat protein 62 ( WDR62 ) are commonly associated with primary microcephaly and other developmental cortical malformations. We used human pluripotent stem cells (hPSC) to examine WDR62 function during human neural differentiation and model early stages of human corticogenesis. Neurospheres lacking WDR62 expression showed decreased expression of intermediate progenitor marker, TBR2, and also glial marker, S100 β . In contrast, inhibition of c-Jun N-terminal kinase (JNK) signalling during hPSC neural differentiation induced upregulation of WDR62 with a corresponding increase in neural and glial progenitor markers, PAX6 and EAAT1, respectively. These findings may signify a role of WDR62 in specifying intermediate neural and glial progenitors during human pluripotent stem cell differentiation.

The Culex pipiens complex includes species with reported differences in vector competence for arthropod-borne viruses, many of which are of significant importance to human health such as the West Nile virus and the Sindbis virus. This group of mosquitoes is difficult to distinguish morphologically; particularly as adult females. In Europe, the two species of the complex, Culex pipiens Linnaeus 1758 and Culex torrentium Martini 1925, are often found sympatrically. With the aim to characterize the presence and spread of both species in the Emilia-Romagna region, Northern Italy, mosquitoes of the complex - collected during the West Nile virus surveillance plans - were tested by multiplex real-time PCR for the detection of the two species Cx. pipiens and Cx. torrentium. A total of 24 165 mosquitoes, collected between 2012 and 2014 from 105 sites, and sorted in 204 pools, were tested. All tested pools were found to be composed of Cx. pipiens, whereas Cx. torrentium was not detected. These results indicate a likely absence of Cx. torrentium mosquitoes within the surveyed territory, whereas Cx. pipiens is widely distributed in the area mentioned. This is in line with previous reports, which describe a pre-alpine distribution of Cx. torrentium in Italy., (© 2016 The Royal Entomological Society.)

From 1 May 2015 to 31 October 2015 over 20 million visitors from all over the world visited the Universal Exhibition (EXPO) hosted by Milan (Lombardy region, Italy), raising concerns about the possible introduction of mosquito-borne diseases from endemic countries. The entomological surveillance protocol performed in Lombardy over the last three years was implemented in the EXPO area and in the two major regional airports using both Center for Disease Control CO₂ and Biogents Sentinel traps. This surveillance aimed to estimate the presence and densities of putative vectors, and also to support investigations, including the vector species involved and area of diffusion, on the local spread of Chikungunya, Dengue and West Nile viruses (WNV) by competent vectors. From 3544 mosquitoes belonging to five different species, 28 pools of Culex spp. and 45 pools of Aedes spp. were screened for the presence of WNV, and for both Chikungunya and flaviviruses, respectively. The entomological surveillance highlighted a low density of potential vectors in the surveyed areas and did not reveal the presence of Chikungunya or Dengue viruses in the local competent vectors inside the EXPO area or in the two airports. In addition, the surveillance reported a low density of Culex spp. mosquitoes, which all tested negative for WNV.

Several flaviviruses are important pathogens for humans and animals (Dengue viruses, Japanese encephalitis virus, Yellow-fever virus, Tick-borne encephalitis virus, West Nile virus). In recent years, numerous novel and related flaviviruses without known pathogenic capacity have been isolated worldwide in the natural mosquito population. However, phylogenetic studies have shown that genomic sequences of these viruses diverge from other flaviviruses. Moreover, these viruses seem to be exclusive of insects (they do not seem to grow on vertebrate cell lines), and were already defined as mosquito-only flaviviruses or insect-specific flaviviruses. At least eleven of these viruses were isolated worldwide, and sequences ascribable to other eleven putative viruses were detected in several mosquito species. A large part of the cycle of these viruses is not well known, and their persistence in the environment is poorly understood. These viruses are detected in a wide variety of distinct mosquito species and also in sandflies and chironomids worldwide; a single virus, or the genetic material ascribable to a virus, was detected in several mosquito species in different countries, often in different continents. Furthermore, some of these viruses are carried by invasive mosquitoes, and do not seem to have a depressive action on their fitness. The global distribution and the continuous detection of new viruses in this group point out the likely underestimation of their number, and raise interesting issues about their possible interactions with the pathogenic flaviviruses, and their influence on the bionomics of arthropod hosts. Some enigmatic features, as their integration in the mosquito genome, the recognition of their genetic material in DNA forms in field-collected mosquitoes, or the detection of the same virus in both mosquitoes and sandflies, indicate that the cycle of these viruses has unknown characteristics that could be of use to reach a deeper understanding of the cycle of related pathogenic flaviviruses., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Induced pluripotent stem cells (iPSCs) may serve as an autologous source of replacement neurons in the injured cochlea, if they can be successfully differentiated and reconnected with residual elements in the damaged auditory system. Here, we explored the potential of hiPSC-derived neurons to innervate early postnatal hair cells, using established in vitro assays. We compared two hiPSC lines against a well-characterized hESC line. After ten days' coculture in vitro, hiPSC-derived neural processes contacted inner and outer hair cells in whole cochlear explant cultures. Neural processes from hiPSC-derived neurons also made contact with hair cells in denervated sensory epithelia explants and expressed synapsin at these points of contact. Interestingly, hiPSC-derived neurons cocultured with hair cells at an early stage of differentiation formed synapses with a higher number of hair cells, compared to hiPSC-derived neurons cocultured at a later stage of differentiation. Notable differences in the innervation potentials of the hiPSC-derived neurons were also observed and variations existed between the hiPSC lines in their innervation efficiencies. Collectively, these data illustrate the promise of hiPSCs for auditory neuron replacement and highlight the need to develop methods to mitigate variabilities observed amongst hiPSC lines, in order to achieve reliable clinical improvements for patients.

Background & Aims: Hirschsprung disease (HSCR) is caused by failure of cells derived from the neural crest (NC) to colonize the distal bowel in early embryogenesis, resulting in absence of the enteric nervous system (ENS) and failure of intestinal transit postnatally. Treatment is by distal bowel resection, but neural cell replacement may be an alternative. We tested whether aneuronal (aganglionic) colon tissue from patients may be colonized by autologous ENS-derived cells., Methods: Cells were obtained and cryopreserved from 31 HSCR patients from the proximal resection margin of colon, and ENS cells were isolated using flow cytometry for the NC marker p75 (nine patients). Aneuronal colon tissue was obtained from the distal resection margin (23 patients). ENS cells were assessed for NC markers immunohistologically and by quantitative reverse-transcription polymerase chain reaction, and mitosis was detected by ethynyl-2'-deoxyuridine labeling. The ability of human HSCR postnatal ENS-derived cells to colonize the embryonic intestine was demonstrated by organ coculture with avian embryo gut, and the ability of human postnatal HSCR aneuronal colon muscle to support ENS formation was tested by organ coculture with embryonic mouse ENS cells. Finally, the ability of HSCR patient ENS cells to colonize autologous aneuronal colon muscle tissue was assessed., Results: ENS-derived p75-sorted cells from patients expressed multiple NC progenitor and differentiation markers and proliferated in culture under conditions simulating Wnt signaling. In organ culture, patient ENS cells migrated appropriately in aneural quail embryo gut, and mouse embryo ENS cells rapidly spread, differentiated, and extended axons in patient aneuronal colon muscle tissue. Postnatal ENS cells derived from HSCR patients colonized autologous aneuronal colon tissue in cocultures, proliferating and differentiating as neurons and glia., Conclusions: NC-lineage cells can be obtained from HSCR patient colon and can form ENS-like structures in aneuronal colonic muscle from the same patient.

West Nile virus (WNV) is a recently re-emerged health problem in Europe. In Italy, an increasing number of outbreaks of West Nile disease, with occurrences of human cases, have been reported since 2008. This is particularly true in northern Italy, where entomological surveillance systems have been implemented at a regional level. The aim of this study was to use, for the first time, all the entomological data collected in the five regions undergoing surveillance for WNV in northern Italy to characterize the viral circulation (at a spatial and temporal scale), identify potential mosquito vectors, and specify relationships between virus circulation and meteorological conditions. In 2013, 286 sites covering the entire Pianura Padana area were monitored. A total of 757,461 mosquitoes were sampled. Of these, 562,079 were tested by real-time PCR in 9,268 pools, of which 180 (1.9%) were positive for WNV. The largest part of the detected WNV sequences belonged to lineage II, demonstrating that, unlike those in the past, the 2013 outbreak was mainly sustained by this WNV lineage. This surveillance also detected the Usutu virus, a WNV-related flavivirus, in 241 (2.6%) pools. The WNV surveillance systems precisely identified the area affected by the virus and detected the viral circulation approximately two weeks before the occurrence of onset of human cases. Ninety percent of the sampled mosquitoes were Culex pipiens, and 178/180 WNV-positive pools were composed of only this species, suggesting this mosquito is the main WNV vector in northern Italy. A significantly higher abundance of the vector was recorded in the WNV circulation area, which was characterized by warmer and less rainy conditions and greater evapotranspiration compared to the rest of the Pianura Padana, suggesting that areas exposed to these conditions are more suitable for WNV circulation. This observation highlights warmer and less rainy conditions as factors able to enhance WNV circulation and cause virus spillover outside the sylvatic cycle.

GTF2IRD1 is one of the three members of the GTF2I gene family, clustered on chromosome 7 within a 1.8 Mb region that is prone to duplications and deletions in humans. Hemizygous deletions cause Williams-Beuren syndrome (WBS) and duplications cause WBS duplication syndrome. These copy number variations disturb a variety of developmental systems and neurological functions. Human mapping data and analyses of knockout mice show that GTF2IRD1 and GTF2I underpin the craniofacial abnormalities, mental retardation, visuospatial deficits and hypersociability of WBS. However, the cellular role of the GTF2IRD1 protein is poorly understood due to its very low abundance and a paucity of reagents. Here, for the first time, we show that endogenous GTF2IRD1 has a punctate pattern in the nuclei of cultured human cell lines and neurons. To probe the functional relationships of GTF2IRD1 in an unbiased manner, yeast two-hybrid libraries were screened, isolating 38 novel interaction partners, which were validated in mammalian cell lines. These relationships illustrate GTF2IRD1 function, as the isolated partners are mostly involved in chromatin modification and transcriptional regulation, whilst others indicate an unexpected role in connection with the primary cilium. Mapping of the sites of protein interaction also indicates key features regarding the evolution of the GTF2IRD1 protein. These data provide a visual and molecular basis for GTF2IRD1 nuclear function that will lead to an understanding of its role in brain, behaviour and human disease.

A new method for the fabrication of a label-free electrochemical immunosensor based on vertical nanowires (VNWs) is proposed. The VNWs are functionalized to detect antibodies against a major astrocytic structural protein component, glial fibrillary acidic protein (GFAP). It is revealed that the interaction of GFAP-antibody with functionalized VNWs leads to a clear change in device conductance and the corresponding capacitance., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3β and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named "caudal neural progenitors" (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube., (© 2015 AlphaMed Press.)

Toscana virus (TOSV), transmitted by phlebotomine sandflies, is recognised as one of the most important causes of viral meningitis in summer in Mediterranean countries. A surveillance plan based on both human and entomological surveys was started in 2010 in the Emilia-Romagna region, Italy. Clinical samples from patients with neurological manifestations were collected during 2010 to 2012. The surveillance protocol was improved during these years, allowing the detection of 65 human infections. Most of these infections were recorded in hilly areas, where sandflies reach the highest density. Entomological sampling around the homes of the patients resulted in a low number of captured sandflies, while later sampling in a hilly area with high number of human cases (n=21) resulted in a larger number of captured sandflies. Using this approach, 25,653 sandflies were sampled, of which there were 21,157 females, which were sorted into 287 pools. TOSV RNA was detected by real-time PCR in 33 of the pools. The results highlighted the role of Phlebotomus perfiliewi as the main vector of TOSV and a potential link between vector density and virus circulation. This integrated system shows that an interdisciplinary approach improves the sensitiveness and effectiveness of health surveillance.

Predicting West Nile virus (WNV) circulation and the risk of WNV epidemics is difficult due to complex interactions of multiple factors involved. Surveillance systems that timely detect virus activity in targeted areas, and allow evidence-based risk assessments may therefore be necessary. Since 2009, a system integrating environmental (mosquitoes and birds) and human surveillance has been implemented and progressively improved in the Emilia-Romagna region, Italy. The objective is to increase knowledge of WNV circulation and to reduce the probability of virus transmission via blood, tissue and organ donation. As of 2013, the system has shown highly satisfactory results in terms of early detection capacity (the environmental surveillance component allowed detection of WNV circulation 3–4 weeks before human cases of West Nile neuroinvasive disease (WNND) occurred), sensitivity (capacity to detect virus circulation even at the enzootic level) and area specificity (capacity to indicate the spatial distribution of the risk for WNND). Strong correlations were observed between the vector index values and the number of human WNND cases registered at the province level. Taking into consideration two scenarios of surveillance, the first with environmental surveillance and the second without, the total costs for the period from 2009 to 2013 were reduced when environmental surveillance was considered (EUR 2.093 million for the first scenario vs EUR 2.560 million for the second). Environmental surveillance helped to reduce costs by enabling a more targeted blood unit testing strategy. The inclusion of environmental surveillance also increased the efficiency of detecting infected blood units and further allowed evidence-based adoption of preventative public health measures.

Emerging therapies for sensorineural hearing loss include replacing damaged auditory neurons (ANs) using stem cells. Ultimately, it is important that these replacement cells can be patient-matched to avoid immunorejection. As human induced pluripotent stem cells (hiPSCs) can be obtained directly from the patient, they offer an opportunity to generate patient-matched neurons for transplantation. Here, we used an established neural induction protocol to differentiate two hiPSC lines (iPS1 and iPS2) and one human embryonic stem cell line (hESC; H9) toward a neurosensory lineage in vitro. Immunocytochemistry and qRT-PCR were used to analyze the expression of key markers involved in AN development at defined time points of differentiation. The hiPSC- and hESC-derived neurosensory progenitors expressed the dorsal hindbrain marker (PAX7), otic placodal marker (PAX2), proneurosensory marker (SOX2), ganglion neuronal markers (NEUROD1, BRN3A, ISLET1, ßIII-tubulin, Neurofilament kDa 160), and sensory AN markers (GATA3 and VGLUT1) over the time course examined. The hiPSC- and hESC-derived neurosensory progenitors had the highest expression levels of the sensory neural markers at 35 days in vitro. Furthermore, the neurons generated from this assay were found to be electrically active. While all cell lines analyzed produced functional neurosensory-like progenitors, variabilities in the levels of marker expression were observed between hiPSC lines and within samples of the same cell line, when compared with the hESC controls. Overall, these findings indicate that this neural assay was capable of differentiating hiPSCs toward a neurosensory lineage but emphasize the need for improving the consistency in the differentiation of hiPSCs into the required lineages.

Neurodegenerative disorders such as Friedreich ataxia (FRDA) present significant challenges in developing effective therapeutic intervention. Current treatments aim to manage symptoms and thus improve quality of life, but none can cure, nor are proven to slow, the neurodegeneration inherent to this disease. The primary clinical features of FRDA include progressive ataxia and shortened life span, with complications of cardiomyopathy being the major cause of death. FRDA is most commonly caused by an expanded GAA trinucleotide repeat in the first intron of FXN that leads to reduced levels of frataxin, a mitochondrial protein important for iron metabolism. The GAA expansion in FRDA does not alter the coding sequence of FXN. It results in reduced production of structurally normal frataxin, and hence any increase in protein level is expected to be therapeutically beneficial. Recently, there has been increased interest in developing novel therapeutic applications like cell and/or gene therapies, and these cutting-edge applications could provide effective treatment options for FRDA. Importantly, since individuals with FRDA produce frataxin at low levels, increased expression should not elicit an immune response. Here we review the advances to date and highlight the future potential for cell and gene therapy to treat this debilitating disease.

Friedreich ataxia (FRDA) is an autosomal recessive disease characterised by neurodegeneration and cardiomyopathy that is caused by an insufficiency of the mitochondrial protein, frataxin. Our previous studies described the generation of FRDA induced pluripotent stem cell lines (FA3 and FA4 iPS) that retained genetic characteristics of this disease. Here we extend these studies, showing that neural derivatives of FA iPS cells are able to differentiate into functional neurons, which don't show altered susceptibility to cell death, and have normal mitochondrial function. Furthermore, FA iPS-derived neural progenitors are able to differentiate into functional neurons and integrate in the nervous system when transplanted into the cerebellar regions of host adult rodent brain. These are the first studies to describe both in vitro and in vivo characterization of FA iPS-derived neurons and demonstrate their capacity to survive long term. These findings are highly significant for developing FRDA therapies using patient-derived stem cells.

Background and Aims: Hypothermia provides neuroprotection after cardiac arrest, hypoxic-ischemic encephalopathy, and in animal models of ischemic stroke. However, as drug development for stroke has been beset by translational failure, we sought additional evidence that hypothermia protects human neurons against ischemic injury., Methods: Human embryonic stem cells were cultured and differentiated to provide a source of neurons expressing β III tubulin, microtubule-associated protein 2, and the Neuronal Nuclei antigen. Oxygen deprivation, oxygen-glucose deprivation, and H2 O2 -induced oxidative stress were used to induce relevant injury., Results: Hypothermia to 33°C protected these human neurons against H2 O2 -induced oxidative stress reducing lactate dehydrogenase release and Terminal deoxynucleotidyl transferase dUTP nick end labeling-staining by 53% (P ≤ 0·0001; 95% confidence interval 34·8-71·04) and 42% (P ≤ 0·0001; 95% confidence interval 27·5-56·6), respectively, after 24 h in culture. Hypothermia provided similar protection against oxygen-glucose deprivation (42%, P ≤ 0·001, 95% confidence interval 18·3-71·3 and 26%, P ≤ 0·001; 95% confidence interval 12·4-52·2, respectively) but provided no protection against oxygen deprivation alone. Protection (21%) persisted against H2 O2 -induced oxidative stress even when hypothermia was initiated six-hours after onset of injury (P ≤ 0·05; 95% confidence interval 0·57-43·1)., Conclusion: We conclude that hypothermia protects stem cell-derived human neurons against insults relevant to stroke over a clinically relevant time frame. Protection against H2 O2 -induced injury and combined oxygen and glucose deprivation but not against oxygen deprivation alone suggests an interaction in which protection benefits from reduction in available glucose under some but not all circumstances., (© 2014 The Authors. International Journal of Stroke published by John Wiley & Sons Ltd on behalf of World Stroke Organization.)

Traditionally, Huntington's disease (HD) has been known as a movement disorder, characterized by motor, psychiatric, and cognitive impairments. Recent studies have shown that motor and action-language processes are neurally associated. The cognitive mechanisms underlying this interaction have been investigated through the action compatibility effect (ACE) paradigm, which induces a contextual coupling of ongoing motor actions and verbal processing. The present study is the first to use the ACE paradigm to evaluate action-word processing in HD patients (HDP) and their families. Specifically, we tested three groups: HDP, healthy first-degree relatives (HDR), and non-relative healthy controls. The results showed that ACE was abolished in HDP as well as HDR, but not in controls. Furthermore, we found that the processing deficits were primarily linguistic, given that they did not correlate executive function measurements. Our overall results underscore the role of cortico-basal ganglia circuits in action-word processing and indicate that the ACE task is a sensitive and robust early biomarker of HD and familial vulnerability.

Introduction: Npas4 is a calcium-dependent transcription factor expressed within neurons of the brain where it regulates the expression of several genes that are important for neuronal survival and synaptic plasticity. It is known that in the adult brain Npas4 plays an important role in several key aspects of neurobiology including inhibitory synapse formation, neuroprotection and memory, yet very little is known about the role of Npas4 during neurodevelopment. The aim of this study was to examine the expression and function of Npas4 during nervous system development by using a combination of in vivo experiments in the developing mouse embryo and neural differentiation of embryonic stem cells (ESCs) as an in vitro model of the early stages of embryogenesis., Methods: Two different neural differentiation paradigms were used to investigate Npas4 expression during neurodevelopment in vitro; adherent monolayer differentiation of mouse ESCs in N2B27 medium and Noggin-induced differentiation of human ESCs. This work was complemented by direct analysis of Npas4 expression in the mouse embryo. The function of Npas4 in the context of neurodevelopment was investigated using loss-of-function experiments in vitro. We created several mouse ESC lines in which Npas4 expression was reduced during neural differentiation through RNA interference and we then analyzed the ability of these Npas4 knockdown mouse ESCs lines to undergo neural differentiation., Results: We found that while Npas4 is not expressed in undifferentiated ESCs, it becomes transiently up-regulated during neural differentiation of both mouse and human ESCs at a stage of differentiation that is characterized by proliferation of neural progenitor cells. This was corroborated by analysis of Npas4 expression in the mouse embryo where the Npas4 transcript was detected specifically in the developing forebrain beginning at embryonic day 9.5. Finally, knockdown of Npas4 expression in mouse ESCs undergoing neural differentiation affected their ability to differentiate appropriately, resulting in delayed neural differentiation., Conclusions: Here we provide the first evidence that Npas4 is expressed during embryonic development and that it may have a developmental role that is unrelated to its function in the adult brain.

In severe cases of sensorineural hearing loss where the numbers of auditory neurons are significantly depleted, stem cell-derived neurons may provide a potential source of replacement cells. The success of such a therapy relies upon producing a population of functional neurons from stem cells, to enable precise encoding of sound information to the brainstem. Using our established differentiation assay to produce sensory neurons from human stem cells, patch-clamp recordings indicated that all neurons examined generated action potentials and displayed both transient sodium and sustained potassium currents. Stem cell-derived neurons reliably entrained to stimuli up to 20 pulses per second (pps), with 50% entrainment at 50 pps. A comparison with cultured primary auditory neurons indicated similar firing precision during low-frequency stimuli, but significant differences after 50 pps due to differences in action potential latency and width. The firing properties of stem cell-derived neurons were also considered relative to time in culture (31-56 days) and revealed no change in resting membrane potential, threshold or firing latency over time. Thus, while stem cell-derived neurons did not entrain to high frequency stimulation as effectively as mammalian auditory neurons, their electrical phenotype was stable in culture and consistent with that reported for embryonic auditory neurons., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This study investigates the pupal development times of the blow fly Calliphora vicina, which were studied in the laboratory at six different constant temperatures (15, 20, 23, 25, 28, and 30°C each ± 1°C). Lower thresholds (tL ) for development were estimated from the linear regression of the developmental rates on each temperature. These data have made it possible to calculate the accumulated degree days (ADD) necessary for C. vicina to complete the larval stage and to achieve adult emergence. The minimal duration of development from oviposition to adult emergence was found to be inversely related to temperature. Additionally, six landmarks in pupal development are showed, and for each of the landmarks, the ADD value was calculated for every rearing temperature involved. These data assist in calculating the duration of the pupal stage based on morphological characteristics and would be of great value for future forensic entomological casework., (© 2013 American Academy of Forensic Sciences.)

A wide range of arthropod-borne viruses threaten both human and animal health either through their presence in Europe or through risk of introduction. Prominent among these is West Nile virus (WNV), primarily an avian virus, which has caused multiple outbreaks associated with human and equine mortality. Endemic outbreaks of West Nile fever have been reported in Italy, Greece, France, Romania, Hungary, Russia and Spain, with further spread expected. Most outbreaks in Western Europe have been due to infection with WNV Lineage 1. In Eastern Europe WNV Lineage 2 has been responsible for human and bird mortality, particularly in Greece, which has experienced extensive outbreaks over three consecutive years. Italy has experienced co-circulation with both virus lineages. The ability to manage this threat in a cost-effective way is dependent on early detection. Targeted surveillance for pathogens within mosquito populations offers the ability to detect viruses prior to their emergence in livestock, equine species or human populations. In addition, it can establish a baseline of mosquito-borne virus activity and allow monitoring of change to this over time. Early detection offers the opportunity to raise disease awareness, initiate vector control and preventative vaccination, now available for horses, and encourage personal protection against mosquito bites. This would have major benefits through financial savings and reduction in equid morbidity/mortality. However, effective surveillance that predicts virus outbreaks is challenged by a range of factors including limited resources, variation in mosquito capture rates (too few or too many), difficulties in mosquito identification, often reliant on specialist entomologists, and the sensitive, rapid detection of viruses in mosquito pools. Surveillance for WNV and other arboviruses within mosquito populations varies between European countries in the extent and focus of the surveillance. This study reviews the current status of WNV in mosquito populations across Europe and how this is informing our understanding of virus epidemiology. Key findings such as detection of virus, presence of vector species and invasive mosquito species are summarized, and some of the difficulties encountered when applying a cost-effective surveillance programme are highlighted.

Over a 7-day period beginning 8 August 2011, a large number of wild birds of several species were found dead or with neurologic clinical signs along the shore of Crostolo stream, in the Emilia Romagna region, Italy. Twenty-eight Mallards (Anas platyrhynchos), two Hooded Crows (Corvus corone cornix), and three coypus (Myocastor coypus) were found moribund on the Crostolo stream bank, collected, and sent to Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Reggio Emilia Section. The cause of mortality was determined to be Clostridium botulinum type C toxin. The toxin was identified by a mouse bioassay for botulinum toxins and confirmed in bird sera and blowfly larvae (Lucilia caesar) collected from the stomachs of birds.

West Nile virus (WNV) is one of the most serious public health threats that Europe and the Mediterranean countries are currently facing. In Italy, WNV emerged in 1998 and has been circulating since 2008. To tackle its continuous incursions, Italian national and regional institutions set up a surveillance program, which includes the serological screening of sentinel horses, sentinel-chickens and backyard poultry flocks and the surveillance on all equine neurological cases, resident captured and wild dead birds, and vectors. This communication aims to assess the importance of the entomological surveillance program as an early warning system for WNV circulation. In the province of Modena, the circulation of WNV lineage 2 strains was first detected in pools of Culex pipiens on July the 3rd, 42 days prior to the onset of the first 2013 human WNV neuroinvasive case reported in the same province. Similarly in Veneto, WNV was first detected on July 3rd in a pool of Cx. pipiens collected in the province of Venezia. The first human neuroinvasive case in this region occurred in the Rovigo province on July the 24th, seven days after the detection of WNV lineage 2 in a mosquito pool collected in the same province. Up to the end of July 2013, WNV circulation was further detected in several other pools of Cx. pipiens mosquitoes collected in Emilia-Romagna, Veneto and Lombardia. According to the NS3 partial sequence alignments including all recent European and Italian Lineage 2 strains, the new circulating WNV lineage 2 strains share high nt homology with the Hungarian and with the previous lineage 2 strains isolated in Veneto and Sardegna in 2011 and 2012. These data provide a clear and practical demonstration of the relevance of a reliable entomological surveillance program to early detect WNV in Italy.

Background: The circulation of West Nile virus and Usutu virus was detected in the Emilia-Romagna region in 2008 and 2009. To evaluate the extent of circulation of both viruses, environmental surveillance, based on bird and mosquito testing, was conducted in 2008 and gradually improved over the years., Methods: In February-March 2009-2011, 5,993 hibernating mosquitoes were manually sampled, out of which 80.1% were Culex pipiens; none tested positive for the viruses. From 2008 to 2011, 946,213 mosquitoes, sampled between May and October, were tested; 86.5% were Cx. pipiens. West Nile virus was detected in 32 Cx. pipiens pools, and Usutu virus was detected in 229 mosquito pools (217 Cx. pipiens, 10 Aedes albopictus, one Anopheles maculipennis s.l., and one Aedes caspius). From 2009 to 2011, of 4,546 birds collected, 42 tested positive for West Nile virus and 48 for Usutu virus. West Nile virus and Usutu virus showed different patterns of activity during the 2008-2011 surveillance period. West Nile virus was detected in 2008, 2009, and 2010, but not in 2011. Usutu virus, however, was continuously active throughout 2009, 2010, and 2011., Conclusions: The data strongly suggest that both viruses overwinter in the surveyed area rather than being continually reintroduced every season. The lack of hibernating mosquitoes testing positive for the viruses and the presence of positive birds sampled early in the season support the hypothesis that the viruses overwinter in birds rather than in mosquitoes. Herd immunity in key bird species could explain the decline of West Nile virus observed in 2011, while the persistence of Usutu virus may be explained by not yet identified reservoirs. Reported results are comparable with a peri-Mediterranean circulation of the West Nile virus lineage 1 related strain, which became undetectable in the environment after two to three years of obvious circulation.

We previously reported that lysophosphatidic acid (LPA) inhibits the neuronal differentiation of human embryonic stem cells (hESC). We extended these studies by analyzing LPA's effects on the expansion of neural stem/progenitor cells (NS/PC) derived from hESCs and human induced pluripotent stem cells (iPSC), and we assessed whether data obtained on the neural differentiation of hESCs were relevant to iPSCs. We showed that hESCs and iPSCs exhibited comparable mRNA expression profiles of LPA receptors and producing enzymes upon neural differentiation. We demonstrated that LPA inhibited the expansion of NS/PCs of both origins, mainly by increased apoptosis in a Rho/Rho-associated kinase (ROCK)-dependent mechanism. Furthermore, LPA inhibited the neuronal differentiation of iPSCs. Lastly, LPA induced neurite retraction of NS/PC-derived early neurons through Rho/ROCK, which was accompanied by myosin light chain (MLC) phosphorylation. Our data demonstrate the consistency of LPA effects across various sources of human NS/PCs, rendering hESCs and iPSCs valuable models for studying lysophospholipid signaling in human neural cells. Our data also highlight the importance of the Rho/ROCK pathway in human NS/PCs. As LPA levels are increased in the central nervous system (CNS) following injury, LPA-mediated effects on NS/PCs and early neurons could contribute to the poor neurogenesis observed in the CNS following injury.

Huntington's disease (HD) is an incurable neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the Huntingtin (HTT) gene. Recently, induced pluripotent stem cell (iPSC) lines carrying atypical and aggressive (CAG60+) HD variants have been generated and exhibit disparate molecular pathologies. Here we investigate two human embryonic stem cell (hESC) lines carrying CAG37 and CAG51 typical late-onset repeat expansions in comparison to wildtype control lines during undifferentiated states and throughout forebrain neuronal differentiation. Pluripotent HD lines demonstrate growth, viability, pluripotent gene expression, mitochondrial activity and forebrain specification that is indistinguishable from control lines. Expression profiles of crucial genes known to be dysregulated in HD remain unperturbed in the presence of mutant protein and throughout differentiation; however, elevated glutamate-evoked responses were observed in HD CAG51 neurons. These findings suggest typical late-onset HD mutations do not alter pluripotent parameters or the capacity to generate forebrain neurons, but that such progeny may recapitulate hallmarks observed in established HD model systems. Such HD models will help further our understanding of the cascade of pathological events leading to disease onset and progression, while simultaneously facilitating the identification of candidate HD therapeutics.

In mammals, the sensory hair cells and auditory neurons do not spontaneously regenerate and their loss results in permanent hearing impairment. Stem cell therapy is one emerging strategy that is being investigated to overcome the loss of sensory cells after hearing loss. To successfully replace auditory neurons, stem cell-derived neurons must be electrically active, capable of organized outgrowth of processes, and of making functional connections with appropriate tissues. We have developed an in vitro assay to test these parameters using cocultures of developing cochlear explants together with neural progenitors derived from human embryonic stem cells (hESCs). We found that these neural progenitors are electrically active and extend their neurites toward the sensory hair cells in cochlear explants. Importantly, this neurite extension was found to be significantly greater when neural progenitors were predifferentiated toward a neural crest-like lineage. When grown in coculture with hair cells only (denervated cochlear explants), stem cell-derived processes were capable of locating and growing along the hair cell rows in an en passant-like manner. Many presynaptic terminals (synapsin 1-positive) were observed between hair cells and stem cell-derived processes in vitro. These results suggest that differentiated hESC-derived neural progenitors may be useful for developing therapies directed at auditory nerve replacement, including complementing emerging hair cell regeneration therapies.

Recent studies have reported direct reprogramming of human fibroblasts to mature neurons by the introduction of defined neural genes. This technology has potential use in the areas of neurological disease modeling and drug development. However, use of induced neurons for large-scale drug screening and cell-based replacement strategies is limited due to their inability to expand once reprogrammed. We propose it would be more desirable to induce expandable neural precursor cells directly from human fibroblasts. To date several pluripotent and neural transcription factors have been shown to be capable of converting mouse fibroblasts to neural stem/precursor-like cells when delivered by viral vectors. Here we extend these findings and demonstrate that transient ectopic insertion of the transcription factors SOX2 and PAX6 to adult human fibroblasts through use of non-viral plasmid transfection or protein transduction allows the generation of induced neural precursor (iNP) colonies expressing a range of neural stem and pro-neural genes. Upon differentiation, iNP cells give rise to neurons exhibiting typical neuronal morphologies and expressing multiple neuronal markers including tyrosine hydroxylase and GAD65/67. Importantly, iNP-derived neurons demonstrate electrophysiological properties of functionally mature neurons with the capacity to generate action potentials. In addition, iNP cells are capable of differentiating into glial fibrillary acidic protein (GFAP)-expressing astrocytes. This study represents a novel virusfree approach for direct reprogramming of human fibroblasts to a neural precursor fate.

The floor plate is one of the major organizers of the developing nervous system through its secretion of sonic hedgehog (Shh). Although the floor plate is located within the neural tube, the derivation of the floor plate during development is still debatable and some studies suggest that floor plate cells are specified by Shh in a temporarily restricted window different to neuroepithelial cells. Using human embryonic stem cells (hESC) as a model of neurogenesis, we sought to determine how floor plate cells may be temporarily specified by SHH signaling during human embryogenesis. We found that inhibition of both GSK3β and activin/nodal pathways in hESC induces a cellular state of SOX2+/PAX6- expression, we describe as "pre-neuroepithelial." Exposure of SHH during this pre-neuroepithelial period causes the expression of GLI transcription factors to function as activators and consequently upregulate expression of the floor plate marker, FOXA2, while also supressing PAX6 expression to inhibit neuroepithelial fate. FOXA2+ cells were able to efficiently generate mesencephalic dopaminergic neurons, a floor plate derivative. Overall, this study demonstrates a highly efficient system for generating floor plate cells from hESC and, most importantly, reveals that specification of floor plate cells is temporally dependent, whereby it occurs prior to the onset of PAX6 expression, within a pre-neuroepithelial stage., (Copyright © 2012 AlphaMed Press.)

According to 2010 estimates from The National Institute on Deafness and other Communication Disorders, approximately 17% (36 million) American adults have reported some degree of hearing loss. Currently, the only clinical treatment available for those with severe-to-profound hearing loss is a cochlear implant, which is designed to electrically stimulate the auditory nerve in the absence of hair cells. Whilst the cochlear implant has been revolutionary in terms of providing hearing to the severe-to-profoundly deaf, there are variations in cochlear implant performance which may be related to the degree of degeneration of auditory neurons following hearing loss. Hence, numerous experimental studies have focused on enhancing the efficacy of cochlear implants by using neurotrophins to preserve the auditory neurons, and more recently, attempting to replace these dying cells with new neurons derived from stem cells. As a result, several groups are now investigating the potential for both embryonic and adult stem cells to replace the degenerating sensory elements in the deaf cochlea. Recent advances in our knowledge of stem cells and the development of induced pluripotency by Takahashi and Yamanaka in 2006, have opened a new realm of science focused on the use of induced pluripotent stem (iPS) cells for therapeutic purposes. This review will provide a broad overview of the potential benefits and challenges of using iPS cells in combination with a cochlear implant for the treatment of hearing loss, including differentiation of iPS cells into an auditory neural lineage and clinically relevant transplantation approaches.

Background: Since 1999, field evidence of transplacental infection by porcine circovirus type 2 (PCV2) and reproductive failure has been reported in pigs. The objective of this study was to evaluate the clinical and pathological consequences of PCV2 infection in conventional PCV2-seropositive gilts by insemination with PCV2b-spiked semen., Results: Six PCV2 seropositive gilts were inseminated with PCV2b-supplemented semen (infected) and three animals with semen and cell culture medium (controls). Only three out of the six infected animals were pregnant by ultrasonography on day 29 after insemination, while two out of the three controls were pregnant. One control gilt aborted on day 23 after insemination but not due to PVC2. Viraemia was demonstrated in four out of six infected and in one control gilt that became infected with PCV2a. Anti-PCV2 antibody titres showed dynamic variations in the infected group throughout the study. Among infected gilts, the animal with the lowest anti-PCV2 titre (1/100) at the beginning of the experiment and another that reached a similar low value during the experiment showed evident seroconversion over time and had also PCV2 positive foetuses. One placenta displayed mild focal necrosis of the chorionic epithelium positively stained by immunohistochemistry for PCV2 antigen., Conclusions: PCV2-seropositive gilts can be infected with PCV2 after intrauterine exposure and low maternal antibody titre may increase the probability of a foetal infection.