Alu elements, the most abundant human repeats. The methylation of Alu elements is an important mechanism to suppress Alu transcription and subsequent retrotransposition. Decades of studies revealed that Alu methylation is highly dynamic during early development and aging. Recently, many environmental factors were shown to have a great impact on Alu methylation. In addition, aberrant Alu methylation has been documented to be an early event in many tumors and Alu methylation levels have been associated with tumor aggressiveness. The assessment of the Alu methylation has become an important approach for early diagnosis and/or prognosis of cancer. This review focuses on the dynamic Alu methylation during development, aging, and tumor genesis. The cause and consequence of Alu methylation changes will be discussed.DNA methylation primarily occurs on CpG dinucleotides and plays an important role in transcriptional regulations during tissue development and cell differentiation. Over 25% of CpG dinucleotides in the human genome reside within  Alu repeats of short interspersed repetitive elements (SINEs) which comprise 13.7% of the human genome. A full-length Alu element is approximately 300 nucleotides in length and contains two almost identical monomers separated by an A-rich region. Most of the Alu repeats share extensive homology over the 300 nt sequence with a recognition site for the restriction enzyme AluI  Alu methadiation , 89, andadiation , 91. Intger Alus . A recenrticular . The racd groups . Alu methylation status and carcinogenesis has been established, many studies demonstrated the strong correlations between the global Alu methylation level and the clinical outcomes of patients with cancer [ Alu methylation from the adjacent normal salivary gland to the intermediate, mucous, and squamous cells [ Alu elements [ Alu elements gain or lose methylation in tumors. The Alu elements proximal to CpG islands tend to be hypermethylated in ependymomas, whereas those in intergenic regions are more likely to be hypomethylated. In addition, the methylation levels of some specific Alu elements, either hypermethylated or hypomethylated in tumors, were confirmed to be associated with tumor aggressiveness. Since some Alu elements become hypermethylated while some others become hypomethylated, the methylation analysis on a panel of specific Alu loci, rather than global Alu methylation, could greatly improve the specificity and sensitivity for cancer detection and prognosis. For instance, a study of the MLH1 gene in gastrointestinal cancer found that the methylation of an Alu element in the first intron spread to its promoter. In normal cells, the spread of Alu methylation is limited and a clear boundary is present between that Alu element and upstream promoter [ Alu elements remains to be addressed, in which methylation levels could be used as common diagnostic or prognostic indicators for a variety of tumors.Although no direct connection betweenh cancer , 96. A sus cells . Our recelements . Comparepromoter . The abepromoter . Whether Alu elements play important roles in the genome and transcriptome, the consequences of aberrant Alu methylation in tumors could be multifaceted  to initiate Pol III-dependent transcription. The binding activity of TFIIIC is the key of the transcription activity of Alu. Jang and Latchman found that ICP27 is responsible for the increase in TFIIIC binding and in turn produced the increase in Alu transcription [ Alu corepressor 1 (ACR1) [ Alu transcription in vitro. Sperm Alu binding protein (SABP) binding is sufficiently selective to protect Alu from in vitro methylation without significantly altering the methylation of adjacent CpGs and protect a region (positions 25–33) downstream from the A box promoter element for Pol III [ Alu-RNA containing nuclear RNP particles [ Alu and might participate in Alu expression regulation.cription . The intcription . Alu cor1 (ACR1)  and nucl1 (ACR1)  were dem Pol III . Lukyanoarticles . The p68 Alu RNAs may affect nearby gene expression, distal gene expression, and global translation. For instance, the expression of an Alu in the promoter of the epsilon-globin gene was found to negatively regulate globin gene expression by transcriptional interference [ Alu RNA was found to be a modular transacting repressor of mRNA transcription [ Alu RNAs also affect translational initiation and are found to form stable, discrete complexes with double-stranded RNA-activated kinase PKR and antagonized PKR activation [ Alu RNA processing causes the degradation of a subset of critical stem cell mRNAs  and modulates the exit from the proliferative stem cell state [ Alu RNA accumulation induces retinal pigmented epithelium cytotoxicity, thus leading to geographic atrophy and blindness [Being more than a dramatic source of insertional mutagens, the expression of freerference . Recentlcription . Importativation . DICER-dll state . Meanwhilindness , 117. Alu Pol III transcripts have also been associated with the miRNA transcription. Recognized as an important component of the transcriptome, miRNAs are approximately 22 nt noncoding RNAs mediating posttranscriptional regulation [ Alu transcripts are often comprised of 3′-flanking sequence prior to the Pol III transcription terminator “TTTT.” If located within the Alu or immediate 3′-flanking regions, miRNAs may be expressed with Alu Pol III promoters. Chromosome 19 miRNA clusters (C19MC) were first shown to be transcribed by Pol III with upstream Alu promoters. Based on computational prediction and high-throughput small RNA sequencing, an additional twenty-three miRNAs at the downstream of the Alu elements were suggested to be transcribed by Pol III with Alu promoters [ AluSx is located in between the spliced MIR1975 but in the antisense orientation with respect to MIR1975 transcription. Thus, the following remains an open question: how frequently and under what condition would miRNA be transcribed with Alu promoters?Recently,gulation . Alu traromoters . Howeverromoters –122. Recromoters , 123 andAlu elements may serve as cis-regulatory elements in the control of neighboring gene expression. Based on the analysis of the Alu consensus sequence, Alu elements contain transcriptional factor binding sites for the zinc-finger Pol II transcription factors SP1 and YY1, activator and repressor, respectively, in a sequence-specific binding manner [Alu elements [Alu subfamilies, the methylation and deamination resulting from the CG to TG transition could generate the CATG motifs attractive to p53 for binding in vivo [Alu elements contain negative calcium response elements [Alu elements with conserved noncoding elements in mammalian genomes were found to be the enhancers, such as that of FGF8 and SATB2 genes, and are involved in brain development [Alu element in the last intron of T-cell-specific CD8α gene was found to serve as an enhancer with GATA3, bHLH protein, and LYF1 binding motifs [In addition to being part of the transcriptome, many g manner   ( Alu element containing the vitamin D receptor binding element in the promoter of CAMP gene mediates the vitamin D-cathelicidin pathway and becomes a key component of a novel innate immune response of human to infection [ Alu-DR (direct repeat) elements provide the binding sites for hepatocyte nuclear factor 4 alpha (HNF4α) which activates the transcription of a list of target genes [ Alu repeats, in particular AluS. Importantly, consensus Alu-DR2 elements arose predominantly via deamination of a methylated CpG dinucleotide [ Alus gain the palindromic inserted repeats separated by three base pairs and acquired the ability to function as estrogen receptor-dependent enhancers [Experimentally, somes (HREs) , includinfection . Alu-DR et genes . Laperricleotide . By acqunhancers , 145. Alu repeats, and, additionally, most RNA Pol IITF binding motifs cluster in the RNA Pol III promoter sequences, which provoke the thinking of the competition and cooperation among these binding proteins. On the other hand, how RNA Pol II and Pol III transcription machineries utilize Alu repeats in different tissues (such as SABP in sperm versus p68 in somatic cells) and under different microenvironments to recruit different transcription factors remains a mystery. Shankar found that a progressive loss of the Pol III transcriptional potential was observed with concomitant accumulation of Pol II regulatory sites [ Alu sequences is involved in the balanced activity between Pol II and Pol III. The methylation statuses of Alu elements would certainly determine the accessibility of Alu containing cis-elements and have a great impact on the binding of transcriptional factors. However, no genome-wide data has been generated so far to explore the Alu methylation patterns and the binding profiles of various factors on Alu elements.As shown in ry sites , while ary sites , and Polry sites . All of  Alu elements play important roles in the genome, from their contribution to the epigenome to their occurrence in a significant fraction of the human transcriptome. The time when they were considered to be nothing but junk is certainly long gone. DNA methylation is one of the key mechanisms controlling Alu expression. The dynamic Alu methylation during development and aging suggests Alu elements and their transcripts may be involved in the cell differentiation and tissue development. The methylation levels of a subset of Alu elements have been associated with tumor malignancy and aggressiveness. This indicates a potential biological and clinical relevance of the methylation of some Alu elements during tumorigenesis and progression. Since the interactions between genes and Alus are multidimensional, the impact of such epigenetic alterations on Alu elements may be multifold. Future integrative Alu epigenomic and transcriptomic studies will further elucidate the functional aspects of Alu elements and their transcripts.Decades of studies have demonstrated that


As cell-based therapies begin to enter clinical trials, human pluripotent stem cells (PSCs) present a reliable and robust source of cells for differentiation into all cell types in the human body. PSC mayMost PSC suspension expansion to date has been performed in benchtop stirred-tank bioreactors operating at volumes of 50-200mL. Little bioprocess optimization has been conducted at these scales likely because of the high cost of PSC growth media and labour requirements for bioreactor growth. This study describes the use of a small-scale bioreactor system for Design-of-Experiments based PSC expansion process development. These process developments inform strategies to bring PSC-based processes into clinical production.Human embryonic stem cells (HES2 cell line) were cultured in adherent conditions on Geltrex-coated plates. Cells were fed daily with Nutristem PSC XF Media, and were passaged with TrypLE every 5-6 days at a passage ratio of 1:24. Cells were dissociated to single cells for bioreactor seeding with TrypLE, strained through a 40μm strainer, and seeded into Nutristem media with Y-27632 ROCK inhibitor prior to bioreactor seeding. Culture media was exchanged at regular intervals by allowing aggregates to settle, removing half of the culture media, and replacing fresh media (without ROCK inhibitor).To study the effects of feeding frequency , dissolved oxygen level , and seeding density  on PSC expansion, a three level, three factor Box-Behnken experimental design was developed including three centrepoint replicates . Cell expansion was strongly dependent on feeding frequency Figure c: daily The final composition of PSCs in the bioreactor-expanded cells was quantified by flow cytometry. All conditions tested retained >90% Oct4+ Nanog+ pluripotent PSCs (representative plot in Figure PSC were observed to spontaneously form uniform aggregates when seeded into the bioreactor as single cells, which expanded to uniform PSCs aggregates at 4 and 6 days of expansion Figure g. AggregThe process optimum found in this study raises several interesting questions for future experimentation. The most significant factor was feeding frequency, with more frequent feeding increasing yields; however, it is unknown what drives the dramatic effects of media supplementation on the resultant increased yields. If growth factors are being depleted, repeating these experiments with culture media that is more enriched in the necessary growth factors could generate different trends than those observed here. Similarly, if secreted factors or metabolites impede cell growth, modified media exchange strategies may be appropriate.While the initial experimental design investigated the effects of oxygen tension on cell production, this parameter was found to be insignificant in these studies. Previous literature indicates that hypoxic conditions are beneficial for PSC growth , as wellFor future experimentation, the effects of increasing culture volume and the resulting effects on the shear stress that cells are exposed to should be explored. Not only will this increase understanding of bioprocess parameters in this platform, but increasing cell volume above the 2mL point, which is the low end of the operating range of the device, could decrease experimental variability.The Micro-24 system represents a tool useful for relating media components, cellular parameters, and bioprocess parameters to cell manufacturing in the cell therapy industry. The results of this experiment can guide the development of process design spaces for PSC expansion processes.


Autophagy was originally described as a highly conserved system for the degradation of cytosol through a lysosome-dependent pathway. In response to starvation, autophagy degrades organelles and proteins to provide metabolites and energy for its pro-survival effects. Autophagy is recognized as playing a role in the pathogenesis of disease either directly or indirectly, through the regulation of vital processes such as programmed cell death, inflammation, and adaptive immune mechanisms. Recent studies have demonstrated that autophagy is not only a simple metabolite recycling system, but also has the ability to degrade specific cellular targets, such as mitochondria, cilia, and invading bacteria. In addition, selective autophagy has also been implicated in vesicle trafficking pathways, with potential roles in secretion and other intracellular transport processes. Selective autophagy has drawn the attention of researchers because of its potential importance in clinical diseases. Therapeutic strategies to target selective autophagy rather than general autophagy may maximize clinical benefit by enhancing selectivity. In this review, we outline the principle components of selective autophagy processes and their emerging importance in human disease, with an emphasis on pulmonary diseases. Autophagy is a lysosomal degradation system by which the cell can recycle its cytoplasmic components . At presAutophagy proceeds through sequential steps, beginning with the generation of autophagosomes from an isolation membrane and followed by elongation to form a mature autophagosome which captures cytosolic cargo . GeneticN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) has been identified as the regulator of autophagosome–lysosome fusion (The membrane origin of autophagosomes remains unclear. Although the endoplasmic reticulum (ER), mitochondria and plasma membrane have been reported as the membrane source, recent studies have also suggested that the ER-mitochondria contact site is important in autophagosome formation . Subseque fusion .Once, autophagy was simply regarded as a non-specific degradation system, however, recent research shows that autophagy can selectively degrade specific targets in processes referred to as “selective autophagy” . Each sePrevious studies suggest that autophagy is relevant to human diseases, including pulmonary diseases . FurtherSelective autophagy can deliver a wide range of cargo to the lysosome, including protein aggregates, whole organelles , and intracellular pathogens . AlthougStaphylococcus aureus-induced acute lung injury (ALI). PINK1 was found to interact with an alternative ubiquitin E3 ligase component, F-box only protein 15 (Fbxo15), which promoted mitochondrial instability in this model  and optineurin recognize ubiquitinated pathogens and target them to autophagosomes . Besideslication . These blication .Recently, we have demonstrated that an autophagy-dependent pathway regulates cilia length , a procein vivo emphysema model, genetic deletion of specific autophagy proteins reduced airspace enlargement . In addition, as dose-response effects of autophagy/mitophagy have been proposed, we cannot completely exclude the possibility that mitophagy may also contribute to mitochondrial quality control for its pro-survival role during mild CS exposure (Chronic obstructive pulmonary disease (COPD) contributes significantly to the global burden of disease as the fourth leading cause of mortality worldwide, however, the pathogenesis of this disease remains incompletely understood . We prevargement . More reargement . Cigaretexposure . FurtherFigure 1B; Becn1+/- or Map1lc3B-/-) mice, as well as tracheal epithelial cells isolated from these mice, resisted CS-induced cilia shortening. We identified the cytosolic deacetylase HDAC6 as a critical regulator of autophagy-mediated cilia shortening during CS exposure  infection is well-characterized. The lungs are the major site for Mtb infection. Mtb employs a unique strategy for survival that interferes with the fusion between phagosomal compartments containing Mtb and lysosomes . Hypoxic PH is a progressive and often fatal complication of chronic lung disease . ChronicThe mitochondrial outer-membrane protein FUN14 domain-containing protein 1 (FUNDC1) mediates hypoxia-induced mitophagy in mammalian cells . FUNDC1 Although autophagy originally was considered as a simple bulk degradation system for cellular components, accumulating evidence demonstrates that autophagy can selectively degrade specific targets. Selective autophagy plays a complex role in human diseases where it can have both protective and injurious effects. However, when viewed in the light of evidence that many cellular functions can have both protective and injurious effects, it is likely that selective autophagy may also act as a double-edged-sword in the pathogenesis of human diseases. For future clinical applications, rather than intervention strategies to target general autophagy, the specific targeting of selective autophagy pathways may enhance the efficacy of therapeutic strategies. Further research into selective autophagy in the lung and other organs will allow for the development new therapeutic interventions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The derivation of canine embryonic stem cells and generation of canine-induced pluripotent stem cells are significant achievements that have unlocked the potential for developing novel cell-based disease models, drug discovery platforms, and transplantation therapies in the dog. A progression from concept to cure in this clinically relevant companion animal will not only help our canine patients but also help advance human regenerative medicine. Nevertheless, many issues remain to be resolved before pluripotent cells can be used clinically in a safe and reproducible manner. Limited proliferative potential was observed for several cESC lines tested by a few research groups (in vivo (We were among a number of research groups between 2007 and 2009 who derived numerous canine embryonic stem cell (cESC) lines from blastocyst stage embryos –6 Table. These ch groups , 2, whil, whereas others were three-dimensional, round, tightly packed colonies that resemble mouse ESCs (mESCs). Interestingly, our mESC-like cESCs could not be successfully propagated long-term in KnockOut serum (KSR)-containing medium supplemented with leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) , while tin vivo differentiation capabilities  plus LIN28 and NANOG, however germ cell-like tumors were formed upon engraftment into NOD/SCID mice  with disease-specific alleles and the derivation of cell types afflicted by the disease promises the development of novel cellular disease models, drug screening platforms, and potential regenerative therapies. It seems that cPSC technologies may be on the verge of clinical translation in the dog , 26, 32,−4% . This po−4% , 27, prein vitro  need to be further developed  can be adapted for the derivation and maintenance of cPSCs . Moreoveeveloped .Combining patient-specific ciPSC generation with targeted genome editing technologies  will enaRealizing the therapeutic potential of PSCs for clinical applications remain a central goal for the veterinary and scientific communities. Since at least half of all heritable canine diseases are known to have human equivalents , the dogThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Classical bovine spongiform encephalopathy (c-BSE) is the only animal prion disease reputed to be zoonotic, causing variant Creutzfeldt-Jakob disease (vCJD) in humans and having guided protective measures for animal and human health against animal prion diseases. Recently, partial transmissions to humanized mice showed that the zoonotic potential of scrapie might be similar to c-BSE. We here report the direct transmission of a natural classical scrapie isolate to cynomolgus macaque, a highly relevant model for human prion diseases, after a 10-year silent incubation period, with features similar to those reported for human cases of sporadic CJD. Scrapie is thus actually transmissible to primates with incubation periods compatible with their life expectancy, although fourfold longer than BSE. Long-term experimental transmission studies are necessary to better assess the zoonotic potential of other prion diseases with high prevalence, notably Chronic Wasting Disease of deer and elk and atypical/Nor98 scrapie. For several protein-misfolding neurodegenerative diseases , a minority of cases are linked to mutations of specific proteins  but the great majority are considered to be sporadic and idiopathic, i.e. without identified origin. Among these proteinopathies, prion diseases constitute a peculiar group as they naturally occur in both humans and animals and are transmissible through natural routes of exposure. In humans, Kuru was transmitted through cannibalistic funeral ritesIn animals, classical scrapie (c-scrapie) of sheep and goats and chronic wasting disease (CWD) in farmed and wild cervids are transmitted through direct contact between animals and through grazing in prion-contaminated environments, which leads to persistence of these diseases at an enzootic level. In contrast, the bovine spongiform encephalopathy  epizootic was maintained through feeding cattle with prion-contaminated meat and bone meals.Animal prion diseases were considered to be innocuous for humans until the diagnosis of the first cases of variant CJD in young British patientsPrior to the recognition of c-BSE, c-scrapie and sCJD were the main reported prion diseases. The long-held belief that scrapie does not transmit to humans has been based on the lack of significant spatial correlation between c-scrapie and sCJD clusters6Historical transmission studies of scrapie to primates have shown variable results189Transgenic mouse models of human prion diseases complement studies performed in different species of non-human primates, the latter model providing the first evidence of human prion disease transmission144141517We, and others, have used the macaque model to assess the zoonotic potential of other ruminant prion strains in comparison to c-BSE. The more recently identified atypical L-type BSE strain was also transmissible to macaques1921The successful transmission of two bovine prion strains into macaques after relatively short incubation periods was suggestive of a low cattle-to-primate species barrier and we have thus continued to explore the zoonotic potential of cattle-derived prion isolates (including CWD after passage through cattle). As controls, we used two sheep prion isolates  both reputed to be non-transmissible and devoid of risk to humans. We report here that, surprisingly, the isolate of sheep c-scrapie led to clinical prion disease in a cynomolgus macaque a decade after exposure (two to four times longer than the incubation period observed with bovine prion strains) indicating the need for continued research in this area to clarify any potential risks of scrapie to human health and implying that very long term experiments are necessary to properly assess this risk.We inoculated cynomolgus macaques through the intracerebral route with different inocula of animal prion diseases. For some of them, inoculation in tonsils also was performed to model oral exposure. The survival periods are shown in table I and compared to the results of c-BSE and L-type BSE experiments previously described by our and other groups, including oral exposures1416192023At postmortem examination, the brain was atrophic, most evident in the temporal lobes . MicroscA peculiar lesion was present in the medial nucleus of the thalamus including a massive loss of parenchymal elements associated with spongiform change and gliosis  vs 1H bupars lateralis of the globus pallidus, the brain stem and the lower spinal cord. Reactive astrocytosis was especially intense in the cerebellum. Iron deposits were noted in the lenticular nucleus, predominant in globus pallidus . According to the cerebral atrophy, Alzheimer’s markers were also investigated but were negative (data not shown).PrPgregates , whereasechnique . Depositechnique . PrPSc ires in brainstem in comparison to the cerebral cortices or cerebellum  as expected. Moreover, the N-terminal part of PrPres was resistant to stringent conditions of proteolysis in brain material derived from the scrapie-infected macaque, as revealed with SAF-32 anti-PrP antibody: this is in contrast to PrPres derived from c-BSE or L-type BSE-infected animals. We did not detect accumulation of PrPres in peripheral organs  of this animal exposed through the intracerebral route.Western blot analyses confirmed that there were lower amounts of PrPrebellum . The molAt the time of this writing, the macaque exposed to the atypical (Nor98) scrapie isolate remains asymptomatic (>7 years post-inoculation). Similarly, we have not observed clinical signs in three macaques exposed to CWD isolates derived from naturally infected white-tailed deer or experimentally infected cattle (7 years post-inoculation). Amongst bovine prion strains, classical BSE  induced prion disease was observed in 17 macaques after incubation periods ranging from about three to less than 8 years depending on the dose inoculated (100 mg to 50 μg), whereas 2.5 to 25 mg of brain tissue of L-type BSE intracerebrally inoculated into macaques resulted in an incubation period of 2 years. More than 10 years post-inoculation, an H-type BSE-exposed macaque also remains without neurological signs .We describe the transmission of spongiform encephalopathy in a non-human primate inoculated 10 years earlier with a strain of sheep c-scrapie. Because of this extended incubation period in a facility in which other prion diseases are under study, we are obliged to consider two alternative possibilities that might explain its occurrence. We first considered the possibility of a sporadic origin (like CJD in humans). Such an event is extremely improbable because the inoculated animal was 14 years old when the clinical signs appeared, i.e. about 40% through the expected natural lifetime of this species, compared to a peak age incidence of 60–65 years in human sporadic CJD, or about 80% through their expected lifetimes. Moreover, sporadic disease has never been observed in breeding colonies or primate research laboratories, most notably among hundreds of animals over several decades of study at the National Institutes of Healthres staining and reactive gliosisThe second possibility is a laboratory cross-contamination. Three facts make this possibility equally unlikely. First, handling of specimens in our laboratory is performed with fastidious attention to the avoidance of any such cross-contamination. Second, no laboratory cross-contamination has ever been documented in other primate laboratories, including the NIH, even between infected and uninfected animals housed in the same or adjacent cages with daily intimate contact . Third, the cerebral lesion profile is different from all the other prion diseases we have studied in this modelWe are therefore confident that the illness in this cynomolgus macaque represents a true transmission of a sheep c-scrapie isolate directly to an old-world monkey, which taxonomically resides in the primate subdivision (parvorder of catarrhini) that includes humans. With an homology of its PrP protein with humans of 96.4%Two earlier studies have reported the occurrence of clinical TSE in cynomolgus macaques after exposures to scrapie isolates. In the first study, the “Compton” scrapie isolate (derived from an English sheep) and serially propagated for 9 passages in goats did not transmit TSE in cynomolgus macaque, rhesus macaque or chimpanzee within 7 years following intracerebral challengeThe most striking observation in our study is the extended incubation period of scrapie in the macaque model, which has several implications. Firstly, our observations constitute experimental evidence in favor of the zoonotic potential of c-scrapie, at least for this isolate that has been extensively studied32333435in vitro conversion of PrP suggested that BSE and scrapie prions would have a similar conversion potential for human PrPSecondly, transmission of c-BSE to primates occurred within 8 years post exposure for the lowest doses able to transmit the disease . The occurrence of scrapie 10 years after exposure to a high dose (25 mg) of scrapie-infected sheep brain suggests that the macaque has a higher species barrier for sheep c-scrapie than c-BSE, although it is notable that previous studies based on Thirdly, prion diseases typically have longer incubation periods after oral exposure than after intracerebral inoculations: since humans can develop Kuru 47 years after oral exposureFourthly, epidemiologic evidence is necessary to confirm the zoonotic potential of an animal disease suggested by experimental studies. A relatively short incubation period and a peculiar epidemiological situation  led to a high degree of suspicion that c-BSE was the cause of vCJD. Sporadic CJD are considered spontaneous diseases with an almost stable and constant worldwide prevalence (0.5–2 cases per million inhabitants per year), and previous epidemiological studies were unable to draw a link between sCJD and classical scrapie67404243Fifthly, considering this 10 year-long incubation period, together with both laboratory and epidemiological evidence of decade or longer intervals between infection and clinical onset of disease, no premature conclusions should be drawn from negative transmission studies in cynomolgus macaques with less than a decade of observation, as in the aforementioned historical transmission studies of scrapie to primates18452147In addition to previous studies on scrapie transmission to primate18Primates were housed and handled in accordance with the European Directive 2010/63 related to animal protection and welfare in research, under constant internal surveillance of veterinarians. Animals were handled under anesthesia to limit stress, and euthanasia was performed for ethical reasons when animals lost autonomy. All the experimental protocols were approved by the French Atomic Energy Commission Ethical Committee .Macaca fascicularis) were provided by Noveprim (Mauritius), checked for the absence of common primate pathogens before importation, and handled in accordance to national guidelines. They were all homozygous for methionine at PRNP codon 129. Life expectancy of cynomolgus macaques in captivity is 35 to 39 years. Animals housed in level-3 animal care facilities  were daily controlled by animal caretaker and regularly examined at least once a week by veterinarians.Captive-bred 2–5 year-old male cynomolgus macaques  naturally infected with classical scrapie (21 kDa PrPres), and the atypical scrapie (Nor98) inoculum was derived from the cerebellum of a Norwegian sheep naturally infected with Nor98 (ARQ/ARR). The cattle-derived inocula were issued from a pool of 11 naturally infected UK cattle (c-BSE)54These samples were homogenized (10% weight/volume) in a 5% glucose solution, and the equivalent of 2.5 to 100 mg of brain inocula were intracerebrally (i.c.) inoculated to macaques under anesthesia. When specified, the equivalent of 8 mg of brain inocula was injected into tonsils  to model a peripheral exposure. Inoculation were performed according procedures established to avoid any cross-contamination .sc) were performed on brain sections as previously describedTissues were fixed in 10% formalin for processing for microscopic examination. Neuropathological methods and immunohistochemical detection of pathological prion protein (PrPres was purified according to the TeSeE purification protocol (Bio-Rad), in adapted conditions of proteolysis for strain discrimination as previously describedPrPHow to cite this article: Comoy, E. E. et al. Transmission of scrapie prions to primate after an extended silent incubation period. Sci. Rep. 5, 11573; doi: 10.1038/srep11573 (2015).


Puma concolor) observations have increased in Midwest North America, with breeding populations re-establishing in several regions east of their contemporary range. The Cypress Hills Uplands, located in southwest Saskatchewan and southeast Alberta, was recently re-colonized by cougars and now supports the easternmost confirmed breeding population of cougars in Canada. A number of factors contribute to this cougar range expansion, but it is dispersal that provides the mechanism for re-colonization of historic range. We used GPS-collar data to examine space-use and movement behavior of sub-adult cougars, the age class associated with dispersal, in the Cypress Hills. Conditional logistic regression and a two-stage modeling approach were used to estimate resource selection functions (RSF) of sub-adult cougars during two distinct ranging behaviors: transient movements  and localizing movements . Linear regression was used to model movement rates, measured as the distance between consecutive 3-h GPS-relocations, of sub-adult cougars relative to different habitats, times of day and between transient and localizing behavior. All individual sub-adult cougars displayed bouts of transient and localizing behavior. All male cougars dispersed from their natal ranges and travelled considerably farther distances than female cougars. One male dispersed over 750 km eastward through the agricultural belt of northern Montana and southern Saskatchewan. Males occupied temporary home ranges in more open habitats on the fringes of the insular Cypress Hills, while females appeared to be recruited into the adult population, occupying treed habitat that provided more suitable cover. During both ranging behaviors, sub-adult cougars selected for rugged terrain and proximity to hydrological features (likely supporting riparian habitats) and avoided open cover types. Differences in habitat selection between ranging behaviors were observed in response to open water, roads and elevation. Although certain habitat characteristics were preferred, transient and localizing cougars used fast-paced nocturnal movements and shortened daytime movements when traversing open habitats to effectively limit their residency and exposure in less-suitable landscapes. Additionally, cougars moved greater distances at night during transient behavior compared to localizing behavior indicating cougars used cover of darkness to traverse novel terrain. In doing so, sub-adult cougars can successfully disperse several hundred kilometres across a matrix of open habitat in search of resources and mates.Cougar ( Puma concolor) populations in parts of North America, coupled with advances in GPS-tracking technology, provide an opportunity to examine the dispersal ecology of a solitary large carnivore which has applications for carnivore conservation around the globe.Carnivore populations around the world have experienced extensive reductions in their ranges from anthropogenic stressors such as habitat loss, habitat fragmentation and over exploitation . MaintaiCougars historically occurred across much of North America but were extirpated from large portions of their eastern range by direct persecution and reduced abundance of prey . A signin = 29) of known-sex carcasses were males  and that cougar confirmations declined as distance from western source populations increased , but also have been documented using fast-paced movements  and corrPast studies of dispersal in far ranging and cryptic species have often been based on coarse spatial and temporal data due largely to technological limitations. For cougars, data were often collected once per week  or opporAdvances in satellite collar telemetry have facilitated fine-scale analysis of movements and habitat selection of transient animals by collecting spatial data multiple times per day and transmitting these data to a remote receiver. Using this technology, our objective was to obtain a fine-scale quantitative assessment of the spatial ecology of sub-adult cougars in the Cypress Hills to examine factors influencing dispersal in an isolated population. More specifically, we quantify sex-biased differences in ranging behavior including cumulative distance travelled during transience, and the size, distribution and habitat composition of THRs used while localizing. In order to more thoroughly examine fine-scale habitat selection of sub-adult cougars, we used conditional logistic regression and a two-stage modelling approach to estimate population-level resource selection functions (RSF) for transient and localizing behaviors. Lastly, we estimated a linear regression model, using distances between consecutive GPS relocations as the response variable, to quantify factors influencing cougar movement rates during the sub-adult life stage.2; Latitude 49.7°N, Longitude 109.5°W; Pinus contorta), white spruce (Picea glauca) and trembling aspen (Populus tremuloides). The matrix surrounding the hills is an expanse of mixed grasslands, pasture lands and cultivated agricultural crops that dominate of much of midwestern North America (The Cypress Hills Uplands (∼2000 km America . The higOdocoileus virginianus), mule deer (O. hemionus), porcupines (Erethizon dorsatum) and elk (Cervus canadensis) (A resident population of cougars is believed to have re-colonized the Cypress Hills in the late 1990s and early 2000s coinciding with a marked increase in cougar sightings. Remote camera images of a family group in 2006 were the first evidence of breeding . Outsiden = 7) were captured in the Cypress Hills during 2010 and 2011 with the assistance of a professional houndsmen and trained tracking hounds. All animal handling was done by trained personnel in accordance with Animal Use Protocol 568-02-11 approved by the University of Alberta Animal Care Committee. We targeted mature juveniles (>12 months old) that were still dependent on their mothers. These criteria ensured the cougars could support a GPS collar  and that cougars would be monitored through the onset of independence. Male cougars (n = 4) were fitted with Argos- (Lotek 4400s) or Iridium-based (Advanced Telemetry Systems) satellite-GPS collars that transmitted data to an email receiver every 2–6 days. Females (n = 3) were fitted with either a satellite-GPS collar or a standard GPS collar (Lotek 4400s) that required remote downloading of data with a hand-held command unit. All collars were programmed to take a fix every 3 h.Cougars  the cougar established a home range evidenced by breeding or localizing behavior for more than 12 months; (2) the cougar died; (3) the GPS-radio collar quit transmitting ; or (4) the study period ended.GPS locations collected during the sub-adult monitoring period were categorized into two ranging behaviors  based on a visual assessment of the geographic point data . Transie2) of each THR and the straight-line and cumulative distances travelled from the center-point of the last THR to the center-point of that cougar’s natal home range. Percent overlap of each THR with known occupied adult range was calculated to examine the spatial distribution of sub-adult THRs in relation to adults. Adult range was based on the aggregated distribution of GPS locations from nine adult cougars  monitored between 2008 and 2013  to identify landscape attributes selected by sub-adult cougars during transient and localizing behaviors. RSFs were estimated using conditional logistic regression  or were masked from being sampled because they comprised a small fraction of habitat types available and were rarely, or never, encountered by cougars during this study. All covariates were standardized for statistical analysis.Candidate landscape variables were chosen based on landscape characteristics deemed important to the biology of cougars. This included topographic roughness , elevatiIn addition to assessing the habitat characteristics that influenced cougar space-use, we also examined how these landscape variables affected movement rates of sub-adult cougars. Step length, defined as the distance between consecutive GPS relocations , is a meCandidate explanatory variables in the step-length model included all the same covariates available in the RSF models but used linear unit measures. Specifically, for each step we calculated the length-weighted mean  to test whether cougars moved faster during transience while accounting for the effects of other habitat characteristics. Finally, we included interaction terms to test for diel shifts in activity associated with land cover or ranging behavior.In addition to habitat covariates, we also included day-period as a categorical variable by binning steps into either day , crepuscular (steps commencing at 600 and 1800 h), or night  period. Also, instead of estimating separate models for transience and localizing, we included ranging behavior as a categorical variable  during which an average of 1,481 GPS relocations  were collected per individual. Average fix success of sub-adult cougars was 82.1% .2 and overlapped with observed adult range by 79.8%. Male THRs averaged 172.4 km2 with 16.1% overlap of adult range. Average land-cover composition of THRs used by males included significantly more grassland (P < 0.05) and significantly less forested cover (P < 0.05) than sub-adult females. Otherwise there was no significant difference (P > 0.05) observed between male and female THRs in the remaining land cover categories . The final population-level model included all candidate covariates but not all appeared to have a great effect on step length . SpecifiProportion of shrubland and wetland did not influence cougar step length in relation to proportion of tree cover . Cougar Sub-adult cougars in the Cypress Hills demonstrated male-biased dispersal behavior which is common among polygynous mammals  and repoOnly one marked cougar emigrated from the Cypress Hills Uplands during our study period. The remaining six sub-adult cougars made long distance exploratory forays into the surrounding grassland-dominated matrix, but eventually returned to the vicinity of the Cypress Hills. In contrast with sub-adult males, sub-adult females used THRs that overlapped considerably with known adult ranges with two of the three females eventually showing evidence of breeding. These data indicate sub-adult females were successfully recruited into the Cypress Hills population. True range expansion is dictated by the presence of females . As a reCanis lupus; Lynx canadensis; Panthera pardus; The most notable dispersal event we documented was that of a male cougar (M7) who successfully traversed 749 km over 100 days, from 13 February 2012 to 22 May 2012 . Cougar By examining habitat selection of sub-adult cougars during different ranging behaviors, it is apparent that certain habitat characteristics have variable importance to cougars depending on whether they are localizing in a THR or traversing relatively novel landscapes. For example, cougars selected for lower elevations during both ranging behaviors but the slope of the response curves differed dramatically. Localizing cougars demonstrated a gradual response to elevation which could be explained by the sub-adults’ tendencies, especially males, to avoid the forested uplands dominated by adult cougars. During transience, the relative probability of use dropped quickly with increases in elevation. This could indicate low lying features are important travel corridors for cougars during dispersal, perhaps providing some form of lateral cover to offset the lack of vegetative cover in more open habitat types. In California, cougars were observed using canyon bottoms as preferred travel routes .Differences in habitat selection between ranging behaviors was also observed in response to open water and roads. Localizing sub-adult cougars had a strong  response to distance to open water whereas open water was not included in the most-supported transient models for any individual cougar and thus had no effect at the population-level. This difference could result from open water representing a localized resource that might be less important to a travelling cougar. During localizing behavior cougars avoided areas in proximity to both paved and unpaved roads but appeared indifferent to these features during transience. Proximity to hydrological features was a selected habitat characteristic during both ranging behaviors which is consistent with other findings that cougars select for riparian areas . HistoriIn addition to selecting habitat characteristics that likely facilitated movement through novel terrain, sub-adult cougars moved considerably farther at night during transience than while localizing, indicating cougars were also relying on the cover of darkness while traversing unfamiliar landscapes. Also, sub-adult cougars generally shifted to faster-paced nocturnal movements while traversing less-suitable habitats during both ranging behaviors. This was particularly evident when traversing agriculture, pasture and grasslands, but was less pronounced when crossing wetlands and shrublands, which likely afforded greater vegetative cover. Likewise, dispersing cougars in a range and basin landscape in New Mexico also used fast-paced directional movements to cross matrices of unsuitable habitat . In contThe technological and analytical methods we utilized have applications for any species of wildlife that is prone to long distance dispersal or is far ranging by nature, and is capable of supporting a satellite-GPS collar. Although we were limited in the number of sub-adult cougars we sampled, our study focused on a relatively small and isolated population of cougars; therefore our sample likely represents a reasonable proportion of the sub-adult cougars present during this study. We also collected a large sample of GPS relocations and step segments for individual cougars which contributed to our population-level estimates using a two-stage modelling approach. Thus, the results of this study provide valuable insights into the fine-scale spatial ecology of a polygnynous carnivore which have typically been examined at much coarser scales due to technological limitations.If cougars continue to re-colonize the prairie-dominated landscape of the Midwest, their distribution will likely be spatially structured as geographically isolated sub-populations separated by a matrix of less-suitable habitats. Dispersal is fundamental for re-colonizing former habitat patches/range and maintaining the long-term presence and genetic viability of metapopulations . As suchBy co-examining space-use and movement of sub-adult cougars in an isolated population, our results provide insight into how cougar range expansion is progressing in the North American Midwest. Managers should realize that although certain habitat characteristics are preferred, cougars will not restrict their movements to these features. Instead, cougars will adopt faster, nocturnal movements to effectively limit their residency and exposure in these less-suitable landscapes. In doing so, cougars can successfully disperse several hundred kilometres across prairie-dominated landscapes in search of resources and mates.10.7717/peerj.1118/supp-1Supplemental Information 1coug_ID); local date (lmt_date); local time (hour); geographic coordinates ; valid_step ; step_length (meters) and ranging behavior . These raw data formed the basis for all spatial analyses presented in the manuscript.Global positioning system (GPS) data for sub-adult cougars collected between 2010–2012 in the Cypress Hills region of Alberta and Saskatchewan, Canada. Data provide are: individual cougar identification (Click here for additional data file.


Atherosclerosis is considered to be an inflammatory disease in which monocytes and monocyte-derived macrophages play a key role. Circulating monocytes can be divided into three distinct subtypes, namely in classical monocytes , intermediate monocytes  and non-classical monocytes . Low density lipoprotein particles are heterogeneous in size and density, with small, dense LDL (sdLDL) crucially implicated in atherogenesis. The aim of this study was to examine whether monocyte subsets are associated with sdLDL serum levels.We included 90 patients with angiographically documented stable coronary artery disease and determined monocyte subtypes by flow cytometry. sdLDL was measured by an electrophoresis method on polyacrylamide gel.Patients with sdLDL levels in the highest tertile  showed the highest levels of pro-inflammatory NCM  when compared with patients in the middle  and lowest tertile . Furthermore, patients in the highest sdLDL tertile showed lower CM levels than patients in the middle and lowest tertile . Levels of IM were not related to sdLDL levels . In contrast to monocyte subset distribution, levels of circulating pro- and anti-inflammatory markers were not associated with sdLDL levels.The atherogenic lipoprotein fraction sdLDL is associated with an increase of NCM and a decrease of CM. This could be a new link between lipid metabolism dysregulation, innate immunity and atherosclerosis. Atherosclerosis is considered to be an inflammatory process in which monocytes and monocyte-derived macrophages play a key role in both initiation and progression of the disease., 2 CircuFurthermore, the CD16+ monocyte population was shown to be expanded in patients suffering from stable coronary artery disease (CAD) and correlated with intima-media thickness and BMI in apparently healthy adults., 9 The pElevated total cholesterol and low density lipoprotein (LDL)-cholesterol levels have long been identified as potent risk factors in atherogenesis., 15 HoweThe mechanism leading to elevated levels of inflammatory monocyte subpopulations in patients with atherosclerotic vascular disease is poorly understood. Therefore, the aim of this study was to examine whether monocyte subsets are associated with sdLDL in patients with stable, coronary artery disease. In addition, we tested whether sdLDL serum levels correlate with pro- and anti-inflammatory cytokines.This is a single-center, cross-sectional study. Between September 2009 and April 2010, we recruited ninety consecutive patients with stable CAD undergoing elective coronary angiography. Patients gave written, informed consent for this study, which was approved by the ethical committee of the Medical University of Vienna and complies with the Declaration of Helsinki. Inclusion criteria comprised male and female patients aged > 18 years with stable CAD undergoing elective coronary angiography. Exclusion criteria consisted of recent acute coronary syndrome, defined as ST-elevating myocardial infarction (STEMI), non-STEMI or unstable angina with or without percutaneous coronary intervention (PCI) within the last three months, heart failure, valvular disease, malignant disease, liver, kidney or other acute or chronic inflammatory diseases. Arterial hypertension was defined as systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg in at least two measurements or the current use of antihypertensive drugs. Subjects were defined as being diabetic if treated for insulin or non-insulin-dependent diabetes mellitus or plasma fasting glucose ≥ 126 mg/dL in at least two measurements. Extent of coronary artery disease is given as the number of epicardial coronary arteries with a ≥70% stenosis. High-dose statin treatment was defined as treatment with atorvastatin with a dosage of at least 40mg or rosuvastatin at a dosage of at least 10mg daily.Blood was drawn in the morning prior to elective coronary angiography after venipuncture from an antecubital vein using a 21-gauge butterfly needle . After the initial 3 mL of blood were discarded, blood was drawn into an EDTA tube (Greiner Bio-One) for immediate analysis by flow cytometry. Furthermore, a 3.8% sodium citrate Vacuette tube , a serum separator tube (Greiner Bio-One) and an EDTA tube (Greiner Bio-One) were collected, immediately centrifuged  and stored at—80°C for later analysis.Standard laboratory measurements including high-sensitive C-reactive protein (hsCRP) were analyzed in the central laboratory of the General Hospital of Vienna. For the measurement of plasma interleukin-6 (IL-6), a specific enzyme-linked immunosorbent assay (ELISA) was used . Plasma levels of interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) were determined using a customized multiplex assay  according to manufacturer's instructions. In 37% of patients, TNF-α levels were undetectable and thus were set to 15 pg/mL, representing the lowest detection limit.Whole blood flow cytometry for determination of leukocyte and monocyte subset distribution was performed using a FACS Canto II with the FACS Diva Software (both Becton Dickinson). The staining and gating strategy is outlined in Krankenanstalten Dr. Dostal using enzymatic methods. For lipid subfraction quantification, the Quantimetrix LDL Lipoprint System , was used according to manufacturer's instructions as described elsewhere.[For all lipid measurements, serum samples were used immediately after thawing from -80°C. Levels of total cholesterol, HDL, LDL and triglycerides were measured by the general laboratory of lsewhere., 23 In blsewhere.2 or by Fisher’s exact test where appropriate. Continuous variables are given as mean ± standard deviation or as median (interquartile range). Parametric data was compared using the unpaired Student's t-test or by ANOVA, while skewed data (assessed by the Kolmogorov–Smirnov test) was compared by the unpaired Student's t-test or by ANOVA after log-transformation. Correlations were calculated using Pearson’s correlation coefficient. A value of p<0.05 (two-tailed) was considered statistically significant. All statistical analyses were performed with the Predictive Analysis SoftWare PASW Statistics 18.0 .Categorical variables are expressed as counts or percentages and were compared by the χWe included 90 patients with angiographically proven stable coronary artery disease. Clinical characteristics of the patient population are given in Mean number of CM as determined by flow cytometry  were 270Patients were divided into three groups according to sdLDL tertiles . As outlined in Although sdLDL only demonstrated a moderate correlation with the proportion of circulating NCM in the total study population, patients with sdLDL levels in the highest tertile showed strikingly increased levels of the pro-inflammatory NCM when compared to patients in the middle and lowest tertile.  in the total patient cohort was 0.2 (IQR: 0.09–0.5) mg/dL and did not differ according to sdLDL tertiles  and reduced levels of classical monocytes (CD14++CD16-). This association was independent of BMI, statin dose and hsCRP-levels. The small proportion of intermediate monocytes (CD14++CD16+) did not differ according to sdLDL tertiles.Monocytes and monocyte-derived macrophages have been implicated in all stages of atherogenesis, from initiation and progression, to destabilization and rupture of atherosclerotic lesions with possible detrimental outcome.–26 MonocHypercholesterolemia is considered a major risk factor for the development of atherosclerosis. As a response to the accumulation and modification of LDL within the vessel wall, monocytes migrate into the intima taking up modified LDL-particles thereby initiating plaque growth. Subset-specific interaction with lipoprotein metabolism has been suggested by several in vitro and in vivo studies, indicating specific expression of scavenger receptors and binding of oxidized and enzymatically modified lipoproteins.–35 In a In our study, including only patients with angiographically proven stable CAD, we could show a statistically significant inverse correlation between percentage levels of CM and both total cholesterol and LDL, while NCM did not correlate with either LDL, HDL or total cholesterol, which is in contrast to the above described findings in the literature. Important discrepancies between the populations studied might explain these diverging findings, as in our population about 85% of patients were treated with a statin, in contrast to the two populations discussed above. FurthermDespite the obvious link between hypercholesterolemia and atherogenesis, many individuals with LDL-levels within the normal range, still develop atherosclerotic disease. This sugIn our study, sdLDL levels showed a weak correlation with NCM. However, when patients were stratified according to tertiles of sdLDL levels, patients in the highest sdLDL tertile show a more pro-inflammatory distribution of monocyte subsets. Additionally, CM levels were lowest in patients in within the highest sdLDL tertile. These findings were independent of BMI, statin dose and hsCRP levels. The latter findings are of importance, as in another study including 166 overweight patients, the correlation between LDL and monocyte subsets was diminished after adjusting for BMI, which remained the only significant regressor for monocyte subset distribution, as detected by multivariate regression analysis. In the IHere we could demonstrate that monocyte subsets were not associated with statin treatment and that the association between sdLDL and monocyte subset distribution was independent of statin treatment dose.In addition, we demonstrated that sdLDL plasma levels exhibit no association with circulating pro- and anti-inflammatory markers, namely hsCRP, IL-6, IL-10 and TNF-α. This is in line with previously published literature, as in the Quebec Cardiovascular Study cohort including 2025 men free of CAD at baseline, sdLDL levels only marginally correlated with markers of inflammation such as hsCRP. In the lSome limitations of the present study have to be acknowledged. First, this is a single center study with a rather small number of patients. Furthermore, the cross-sectional study design only allows us to outline associations between monocyte subset distribution and LDL subfractions, while we cannot draw functional insights into monocyte subset plasticity in atherosclerotic disease. In addition, we did not include a control group with absence of coronary stenosis at coronary angiography. This would be of particular interest, as the absence of low-grade vascular inflammation would help to assess possible direct effects of sdLDL on monocytes. However, our results indicate a link between innate immunity and lipid metabolism in stable atherosclerosis.In conclusion, this study provides evidence for the first time for an association between plasma levels of atherogenic sdLDL particles and an increased proportion of non-classical monocytes and a smaller classical monocyte population. These results might represent a new link between an atherogenic lipoprotein phenotype and innate immunity in stable atherosclerotic disease. Further studies are required to gain functional insight into the mechanistic relationship between monocyte subsets and small dense LDL and their specific roles in atherogenesis.


Emergency department (ED) settings have gained interest as venues for illegal drug misuse prevention and intervention, with researchers and practitioners attempting to capitalize on the intersection of need and opportunity within these settings. This study of 686 adult patients visiting two EDs for various reasons who admitted drug use compared daily cannabis-only users, nondaily cannabis-only users, and other drug users on sociodemographic and drug-related severity outcomes. The three drug use groups did not differ on most sociodemographic factors or medical problem severity scores. Forty-five percent of the sample was identified as having a drug use problem. ED patients who used drugs other than cannabis were at particular risk for high drug use severity indicators and concomitant problems such as psychiatric problems and alcohol use severity. However, 19–29% of cannabis-only users were identified as having problematic drug use. Furthermore, daily cannabis-only users fared less well than nondaily cannabis users with regard to drug use severity indicators and self-efficacy for avoiding drug use. Results may assist emergency medicine providers and medical social workers in matching patients to appropriate intervention. For example, users of drugs other than cannabis  may need referral to specialty services for further assessment. Enhancement of motivation and self-efficacy beliefs could be an important target of prevention and treatment for cannabis-only users screened in the ED. Such efforts may shed light on differences in drug use patterns and associated health effects, and provide important information for future intervention efforts. This study examined the sociodemographic characteristics, drug use severity, and problems related to drug use among ED patients who were daily versus nondaily cannabis-only users, as well as those who reported noncannabis drug use.With the passage of less punitive measures or outright legalization of cannabis use for medical and/or recreational use in 23 states, as well as mounting evidence of dose-dependent psychocognitive health effects of cannabis,14 Over a 1-year period, interviewers working during peak hours attempted to approach all capable adult patients, regardless of the reason for the patient's visit. Patients under the age of 18, those with severely altered mental status, those physically incapable of participating due to severe illness or injury, and those unable to speak English or Spanish were excluded from participation. After providing consent, as approved by our university and hospital IRBs, these patients were interviewed further to collect sociodemographic information, additional drug use data, and measures of problem areas related to drug use. Refusals to be screened by trained interviewers were rare.14Participants were 686 consecutively enrolled adult patients visiting the EDs and trauma units of two large urban safety net hospitals in Southern California who reported to trained paraprofessional health interviewers that they had used illicit drugs, or legal drugs in a nonprescribed manner, during the past 30 days .Sociodemographic variables included participants' age; gender; race/ethnicity ; marital status ; years of education; annual household income ; and whether or not the participant had been employed in the past 30 days.16 The ASI-Lite gathers quantitative information  about the participant's recent use of several types of drugs . Fifty-seven percent of participants (n=394) were cannabis-only users. Three drug use groups were created: (a) cannabis-only users who reported using cannabis less than every day during the past 30 days (n=253), (b) cannabis-only users who reported using cannabis every day during the past 30 days (n=141), and (c) other drug users (n=292) who reported using other illegal or nonprescribed drugs either alone or in combination with cannabis. The ASI-Lite also collects information about the number of days in the past 30 days that one used alcohol, although alcohol did not factor into creation of the three drug use groups. The ASI-Lite yields mathematically derived composite severity scores of problem areas in the participant's life commonly affected by substance use, including a drug use severity score, a medical problem score, a psychiatric problem score, and an alcohol problem score.16 ASI-Lite composite scores range from 0 to 1, with higher scores indicating greater severity of the problem.Participants completed the ASI-Lite, a condensed version of the Addiction Severity Index.18 Problematic drug use (yes/no) was computed based on a DAST score greater than or equal to 3, a cut-point that has demonstrated accuracy in classifying patients according to Diagnostic and Statistical Manual of Mental Disorders (DSM) classification. Self-efficacy for avoiding drug use was measured using a four-item scale assessing confidence in avoiding drug use in various situations, with scores ranging from 1 (low confidence) to 5 (high confidence).19Participants also completed the 10-item drug abuse screening test (DAST).After ruling out problems related to multicollinearity among the correlates, ANOVA and chi-square bivariate analyses were conducted to assess differences among the three drug use groups' sociodemographic characteristics, drug use severity measures, and potential problems related to drug use.14 Three-fourths of the 686 participants were male. The average age was 36.9 years (SD=13.2). The sample was ethnically diverse, with a third being Latino. Sixty-five percent reported an annual income of less than $10,000 per year. Cannabis was by far the most common drug used (84%). Fifty-seven percent of participants were cannabis-only users. The most common noncannabis drugs of use among other drug users were amphetamines (46.7%), cocaine (21.5%), heroin (18.8%), and opiates (17.9%).Overall participant sociodemographic characteristics were reported in a previous article.Results suggest that ED patients who use drugs other than cannabis are at particular risk for high drug use severity and concomitant problems such as psychiatric problems and alcohol use severity. However, 19–29% of cannabis-only users were identified as having problematic drug use. Furthermore, daily cannabis-only users fared less well than nondaily cannabis users with regard to drug use severity indicators and self-efficacy for avoiding drug use, even though they were younger as a group.20 Cannabis dependence does occur, and individuals often perceive themselves as unable to stop.21 Daily cannabis-only users in our study were found to have particularly low confidence in avoiding drug use (compared with nondaily cannabis users and even other drug users). However, there is a distinction between self-efficacy and motivation, perceived importance, and desire to avoid drug use—daily cannabis users may not be motivated or see the importance of avoiding use. Connor et al.22 found low cannabis refusal self-efficacy was related to problematic use. Enhancement of motivation and self-efficacy beliefs could be an important target of prevention and treatment. As with other drug use, behavioral interventions and motivational enhancement appear to be useful with problematic cannabis use.21 Screening, brief intervention, and referral to treatment (SBIRT) is a promising public health strategy that has been implemented in EDs to reduce risks associated with substance use.23–25 Although SBIRT has been shown to be effective for alcohol misuse, its impact on drug use, including cannabis, is less conclusive. However, SBIRT's emphasis on motivation and harm reduction could fit well with cannabis use reduction goals to the degree that many cannabis users express interest in reducing use rather than abstinence.21Although cannabis may be less harmful to individual health relative to other forms of drug use, heavier cannabis use is a concern because it may be associated with mental and physical health problems.26 The quantity used and the frequency of use per day were not collected. In addition, reliance on self-reports may have resulted in underestimates of cannabis use and other drug use.Possible limitations of the study include not having information about the reason for the ED visit and uncertainty of the degree to which respondents are representative of the overall ED population. Use of cannabis in terms of medical or recreational use was not analyzed, an important distinction since there is some evidence that medical and recreational cannabis users differ in their clinical characteristics.5 suggested a clinical decision rule for performing a detailed assessment of ED patients for possible problematic drug use that is based on the type of drug. Because the ED represents an important “opportunistic” venue for harm reduction and intervention efforts with drug users, hospital staff and health educators screening for substance use should be attentive to differences in use patterns among patients, as some adverse consequences may depend on the type and frequency of substances being used.Across the drug use groups, 45% of the ED participants screened positive for problematic drug use, a percentage that varied considerably by type of drug (cannabis vs. other) and frequency of use (daily vs. nondaily). This information may assist emergency medicine providers and social work professionals in matching patients to appropriate intervention. For example, users of drugs other than cannabis  may need referral to specialty services for further assessment. Recently, Konstantopoulos et al.


Haemophilus influenzae type b (Hib) vaccine, both recommended for inclusion in routine childhood immunization programs worldwide –coordinated Global Invasive Bacterial Vaccine-Preventable Diseases (IB-VPD) sentinel hospital surveillance network provides data for decision making regarding use of pneumococcal conjugate vaccine and orldwide ,2. WHO rorldwide . Surveilorldwide . To use WHO-coordinated IB-VPD sentinel hospital surveillance for meningitis is established at large  referral hospitals. A limitation of sentinel surveillance has been the inability to calculate meningitis incidence rates because of the absence of a known denominator of children at-risk for meningitis. In 2012, WHO, under the guidance of a technical advisory group, developed a method for estimating the denominator of at-risk children to calculate hospitalization rates for meningitis among children in the catchment area of a sentinel hospital . The metEstimation of the denominator begins with determining whether population estimates for children aged <5 years are available by districtCases are spot-mapped by district of residence to visualize geographical dispersion and outliers. Districts are then rank-ordered according to the number of cases, and district-specific hospitalization rates for children aged <5 years are calculated using administrative population estimates. Using this rank-order, the sentinel hospital geographical catchment area is defined as those districts where 80% of suspected meningitis patients reside; the catchment area may be adjusted to fewer or more districts based on district-specific rates or expert local opinion. Expert national opinion is then sought to identify nonsentinel hospitals likely to admit at least 10% as many children aged <5 years with suspected meningitis as the sentinel hospital among children residing in the sentinel hospital’s geographical catchment area. These nonsentinel hospitals are visited and the number of children with suspected meningitis residing in the sentinel hospital catchment area is determined by reviewing clinical, laboratory, and residence data, using the same methods as at the sentinel hospital. The denominator of at-risk children is estimated by multiplying the population aged <5 years in the identified districts by the percentage of suspected meningitis patients aged <5 years who are admitted to sentinel hospitals. For example, if 90 of 100 patients among catchment area residents are admitted to the sentinel hospital and 10 cases are admitted to nonsentinel hospitals, the total at-risk population aged <5 years would be 90% of the children residing in the geographic catchment area. Annual meningitis hospitalization rates and 95% confidence intervals (CIs) were calculated using the mid-P exact test in OpenEpi software.Pilot testing of the method was conducted in The Gambia and Senegal during 2012 to assess its feasibility. Before the assessment, Hib vaccines were introduced in the routine childhood immunization programs in both countries , and oneSentinel hospitals conducting IB-VPD surveillance need to maintain consistent practices, including case identification, specimen collection, and laboratory procedures, among a stable at-risk population of children to monitor meningitis trends over time. Time trends are used to assess changes in admissions for suspected meningitis, laboratory-confirmed infections, and serotype or strain of causative organisms after vaccine introductions. This report describes a method for estimating denominators of at-risk children to better understand sentinel hospital surveillance data. Use of this method is limited to geographic areas with existing administrative population estimates, large sentinel hospitals that admit ≥100 children aged <5 years with suspected meningitis over a 2-year period , and hospitals with easily accessed records that include residence information. Accuracy depends on quality of population estimates and hospital records. Ideally, the denominator should be reassessed every 2 years, because population migration and changes in health-care–seeking can occur over time.Because most children with meningitis are treated as inpatients, with admissions generally limited to a small number of health facilities that have the capacity to care for severely ill patients, this method may also be used to estimate meningitis incidence. However, the hospitalization rate might underestimate or overestimate actual meningitis incidence rates because children with meningitis might die at home, and some might be misdiagnosed on admission or diagnosed differently at sentinel versus nonsentinel hospitals. Thus, rates derived using this method should be interpreted cautiously; in developing countries with poor population access to health care, these estimated incidence rates likely represent minimum estimates. In sentinel hospitals with reliable and consistent laboratory diagnostic practices, IB-VPD surveillance can be used to estimate hospitalization rates for probable bacterial meningitisWHO provides support to countries to strengthen national capacity to better monitor and evaluate vaccination programs by using cased-based surveillance with laboratory confirmation, a goal of the Global Vaccine Action Plan.


We report a patient with bilateral choroidal metastasis from disseminated cervical squamous cell carcinoma. A 52-year-old woman presented with progressive bilateral visual loss due to choroidal masses in both eyes. The fundus examination revealed posterior serous retinal detachment in both eyes associated with creamy choroidal lesions. A thorough systemic work-up revealed choroidal metastasis from a squamous cell carcinoma of the cervix. This case highlights the importance of a thorough systemic evaluation in patients with choroidal tumours. Choroidal metastasis is the most common intraocular malignancy in adults . The posA 52-year-old Caucasian woman presented with a three-month history of progressive, painless, bilateral visual loss. The only significant medical history was that of regular cigarette smoking. Visual acuity was “counting fingers” (at one metre) in each eye. Anterior segment examination was unremarkable. Fundus examination demonstrated posterior exudative retinal detachment in the right eye associated with creamy, ill-defined choroidal lesions and retinal pigment epithelial mottling . FundoscThe breast and lung are the primary sites of malignancy in approximately 70% of cases of choroidal metastasis . A literThe current case demonstrates a rare and devastating cause of bilateral, progressive vision loss due to metastatic squamous cell carcinoma of the cervix without prior diagnosis of the primary cancer. SCC is a particularly aggressive form of cervical cancer with five-year survival rate of 20–25% [External beam radiotherapy can be used to improve visual function in this setting, as with other cases of choroidal metastasis . CombineIn summary, SCC can, in rare situations, metastasize to the choroid; however, the fundus features noted in this case should trigger the differential diagnosis of choroidal metastasis with appropriate systemic investigation, including pelvic imaging.


Cancer cells reprogram metabolism to provide the energy, nucleotides, lipids, amino acids and other building blocks required to proliferate and survive in the stressful tumor microenvironment . ChangesGLS (kidneytype glutaminase) and GLS2 (liver-type glutaminase). Glutamate can then be converted to the tricarboxylic acid (TCA) cycle intermediate α-ketoglutarate by glutamate dehydrogenase or transaminases. While glucose-derived pyruvate is shunted away from the mitochondria in tumors, glutamine-derived α-ketoglutarate can fuel the TCA cycle. Although Warburg's observations led to the assumption that the TCA cycle was not critical in tumors, mitochondrial TCA metabolism provides important precursors for the synthesis of cellular building blocks [Glutamine, the most abundant amino acid in the blood, can be taken up by transporters (including SLC1A5/ASCT2) and utilized as a source of carbon and nitrogen for energy production and biosynthesis [MYC, one of the most frequently deregulated genes in cancer, is a transcription factor that promotes cell proliferation, induces the Warburg effect, and drives ribosome biosynthesis and translation to increase cell mass . MYC-driin vitro and in xenografts models [Gls expression and decreased Gls2 expression compared to surrounding tissue, and confirmed that the upregulation of GLS and downregulation of GLS2 is also found in human HCC. We showed that treatment with BPTES, specific to the GLS isoform, prolonged survival of LAP/MYC mice compared to vehicle treated controls. BPTES-treated mice showed smaller tumors with decreased staining of the proliferation marker KI-67. Consistent with GLS inhibition, tumors treated with BPTES showed increased glutamine levels and decreased glutamate levels compared to controls. BPTES treatment was well tolerated in mice. Then, using a MYC-driven cell line as a model to study the effects of GLS inhibition, we demonstrated that BPTES treatment blocked DNA replication, resulting in cell death. Further, we confirmed the in vivo specificity of BPTES by rescuing xenograft growth with the expression of a BPTES resistant GLS mutant.As GLS is broadly expressed in many cancer types and catalyzes the first step of glutamine catabolism, it represents a potential anti-cancer therapy target. While initial attempts to target glutamine metabolism with glutamine analogs led to wide spread toxicity, the development of an allosteric GLS inhibitor ethyl sulfide) showed promise s models . We founin vitro studies show that cell lines of many cancer types depend on glutamine and GLS activity, some recent studies indicate that tumors may not be as commonly glutamine dependent as cells grown in a dish [While BPTES shows encouraging preclinical efficacy, a BPTES related compound (CB-839) with improved pharmacological properties has entered phase I clinical trials . Many chn a dish . Howeverin vitro and reprogram glutamine metabolism in various transgenic models in vivo, the tumor tissue of origin can impact how glutamine metabolism is affected by MYC expression. For example, while transgenic MYC expression in the LAP/MYC model reprograms glutamine metabolism and promotes glutaminase dependence, a MYC-driven lung tumor model does not exhibit reprogrammed glutamine metabolism and shows increased expression of glutamine synthetase [Prediction of therapeutic response to GLS inhibition will require the identification of biomarkers, development of new tools, and a detailed understanding of how mutational status interacts with the tissue type of origin to control tumor metabolism. While MYC has been shown to induce glutamine dependence nthetase . Studiesnthetase , 7. Siminthetase . It remaGlutamine metabolism plays a diverse role in metabolism, controlling cellular energetics, redox state, amino acid production, cell signaling and nucleotide synthesis. The centrality of GLS in these diverse cellular functions makes GLS inhibition an ideal candidate for combination therapies. In addition to reports already in the literature of GLS showing promise in combination therapy in preclinical studies, we speculate that GLS inhibition will show synthetic lethality with drugs that perturb cellular metabolism, nucleotide synthesis, redox state or DNA repair among others.


Iatrogenic coronary artery dissection during coronary angiography with or without rupture is a rare but feared complication. We herein report a case of iatrogenic left main coronary artery dissection in a 49-year-old female. Admitted to our hospital with a recent history of severe hypotension, she develpled apnea during angiography. She was intubated and resuscitated with an Epinephrine infusion in the Cath-Lab. The diagnosis was iatrogenic left main coronary artery dissection based on angiography. Immediately, the patient was transferred to the operating room in a lethargic state with an Epinephrine infusion and prepared for emergency coronary artery bypass graft surgery. In the ICU, she was completely alert with no hemodynamic complications and finally was discharged in a good overall condition. At 18 months' follow-up, the patient was in a stable situation with good daily function. The LMCA dissection often leads to abrupt vessel closure and cessation of the blood flow toward a large portion of the myocardium, resulting in acute pump failure and hemodynamic collapse.2 Conservative treatment may be adequate for limited dissections, but extensive dissections with pericardial extravasation require immediate treatment.3 Both emergency coronary artery bypass graft surgery (CABG) and use of covered stents consitute effective in treatment for the LMCA dissection. Iatrogenic left main coronary artery (LMCA) dissection, induced by catheter insertion, is one of the most feared complications, albeit with a reported incidence rate of less than 0.1%.Here we present a case of iatrogenic LMCA dissection following angiography, which was treated successfully with emergency CABG.A 49-year-old obese opium-addicted female patient with a history of mild hypertension, asthma, and chest pain of 3 months' duration was admitted for coronary angiography in July 2012. In the Cath-Lab during angiography, she developed severe hypotension and apnea. Therefore, she was intubated and resuscitated with an Epinephrine infusion without delay. The intubated patient was subsequently referred to our hospital for emergency coronary artery bypass graft surgery (CABG). Coronary angiography reported the dissection of the left main coronary artery (LMCA) (Type B), extending to the immediate distal portion of the left anterior descending artery (LAD) and the mid portion of the left circumflex artery (LCX)  and 2. EOn admission, the patient was still intubated and was in a stable hemodynamic state with an Epinephrine infusion. Without dealy, she was transported to the operating room in a lethargic state and was prepared for the induction of anesthesia. In the operating room, the patient was monitored via standard electrocardiography and pulse oximeter. The veins on both arms were cannulated with 16-G catheters under local anesthesia with Lidocaine (1%). The catheter was inserted in the left radial artery to monitor blood pressure. The patient was induced with Sufentanil (50 μg), Etomidate (14 mg), and Cisatracurium (14 mg). Anesthesia was maintained using an infusion of Propofol (50-75 μg/kg/min), Sufentanil (2 μg/kg/h), Cisatracurium (2 μg/kg/min), and Dexamethasone (8 mg). Additionally, a vitamin C infusion (500 mg) was administered. Subsequently, a tri-lumen catheter was inserted in the right subclavian vein.Under general anesthesia, median sternotomy was performed, followed by the harvesting of the left internal mammary artery (LIMA) and the greater saphenous vein from left lower extremity. Operative findings confirmed the dissection and that it extended up to the immediate distal portion of the LAD and the mid portion of the LCX . SystemiThe operation was uneventful, and the patient was transferred to the Open Heart Intensive Care Unit, where she was extubated after 6 hours in complete alertness and without any hemodynamic complications. She was finally discharged home in a good overall condition. On the last follow-up visit (January 2014), she was in a stable situation with good daily function.3 Complications, although uncommon, still occur during coronary angiography and can prove life-threatening in some instances. Catheter-induced dissection of a coronary artery is a rare but well-recognized complication of coronary angiography with a high mortality rate if left untreated.3, 4 The exact mechanism of iatrogenic coronary artery dissection is not clear. It has been related to vigorous hand-injection of contrast medium, subintimal passage of the guide-wire, or inappropriate handling of the guide-wire catheter.3 Most dissections are focal and limmited to the site of the injury; they rarely extend and propagate from the original site of the injury, affecting much more of the artery. The term "spiral dissection" is often used to discribe a dissection that spreads from the site of the original intimal tear and extends the length of the artery. Usually, dissections propagate in the direction of the arterial blood flow. Thus, they typically extend distal to the site of the initial injury; it is very unusual for a dissection to propagate proximally. The development of an aortic dissection complicating a coronary dissection is very rare.6Coronary angiography has become a routine diagnostic procedure in current clinical practice.7 In rare cases, a coronary artery dissection may propagate retrogradely and involve the ascending aorta.8The National Heart Lung and Blood Institute (NHLBI) classification system for coronary artery dissection types are as follows: Type A dissections represent minor radiolucent areas within the coronary lumen during contrast injection with little or no persistence of contrast after the dye has cleared. Type B dissections are parallel tracts or a double lumen separated by a radiolucent area during contrast injection, with minimal or no persistence after dye clearance. Type C dissections appear as contrast outside the coronary lumen  with the persistence of contrast after dye has cleared from the lumen. Type D dissections represent spiral (barber shop pole) luminal filling defects, frequently with excessive contrast staining of the dissected false lumen. Type E dissections appear as new, persistent filling defects within the coronary lumen. And, Type F dissections represent those that lead to the total occlusion of the coronary lumen without distal antegrade flow. Types A and B are generally clinically benign, whereas Types C through F portend significant morbidity and mortality if untreated.,9Iatrogenic LMCA dissection results from mechanical injury to the arterial wall during catheter manipulation or the passage or deployment of an interventional device. Extensive catheter manipulation, catheter type , stiffer and less manageable guide-wires (e.g. pressure wire), unusual LMCA anatomy or location, operator's experience, and presence of the LMCA atherosclerosis have all been associated with an increased risk of dissection.2amputated". Nevertheless, even in case of hemodynamic stability, rapid deterioration may occur shortly afterward because of abrupt flow compromise due to progressive dissection or superimposed thrombus formation, which is why it seems imperative to always envisage a "revascularization plan" via percutaneous coronary intervention (PCI) or CABG.2Once dissection occurs, the clinical picture differs, depending on the remaining antegrade coronary flow, and can range from an asymptomatic patient to a patient in refractory cardiogenic shock whose LMCA is "1 reported iatrogenic LMCA dissection with an incidence of less than 0.1%.1 Eshtehardi et al.10 stated that the overall incidence of iatrogenic LMCA dissection during their study period was 0.07% and almost twice as common with PCI than coronary angiography. From their 38 patients, 1 (3%) patient died before any therapeutic attempt was performed, 6 (16%) patients were treated conservatively, and 31 (82%) patients underwent stent implantation and/or CABG. In their study, the in-hospital outcome was favorable irrespective of the therapeutic strategy. During their 5-year follow-up, the authors reported that among the 31 patients who underwent revascularization treatment by stenting or CABG, 1 patient died in each group from a cardiac cause, denoting that there were no significant differences between the different revascularization treatment strategies.10 In contrast to Demetrios Antoniades et al.,3 who believe emergency CABG is a time-consuming procedure that entails the risk of irreversible myocardial damage, our case showed that in some situations, it can prove a life-saving method without any complications. In case of acute LMCA dissection, the technique of surgery and myocardial protection is extremely important. Retrograde coronary perfusion should be strongly considered in these cases along with controlled warm reperfusion. In our patient, however, after the infusion of an antegrade cold cardioplegic solution into the aortic root, complete cardiac arrest occurred. Therefore, it was not necessary to use retrograde coronary perfusion. Although we did not employ this method, it merits consideration it in this section. Awadalla et al.Iatrogenic LMCA dissection is a rare complication of coronary angiography procedures with favorable early and long-term outcomes when recognized timely and managed properly. Furthermore, emergency CABG is still an acceptable option for the treatment of this complication.


The objective of the study was to characterize alterations of structural and functional connectivity within the developing sensori-motor system in infants with focal perinatal brain injury and at high risk of cerebral palsy.Functional magnetic resonance imaging (fMRI) and diffusion tensor imaging (DTI) data were used to study the developing functional and structural connectivity framework in six infants born prematurely at term equivalent age. This was first characterised in three infants without focal pathology, which was then compared to that derived from three infants with unilateral haemorrhagic parenchymal infarction and a subsequent focal periventricular white matter lesion who developed later haemiparesis.Functional responses to passive hand movement were in the contralateral perirolandic cortex, regardless of focal pathology. In infants with unilateral periventricular injury, afferent thalamo-cortical tracts appeared to have developed compensatory trajectories which circumvented areas of damage. In contrast, efferent corticospinal tracts showed marked asymmetry at term equivalent age following focal brain injury. Sensori-motor network analysis suggested that inter-hemispheric functional connectivity is largely preserved despite pathology and that impairment may be associated with adverse neurodevelopmental outcome.Following focal perinatal brain injury, altered structural and functional connectivity is already present and can be characterized with MRI at term equivalent age. The results of this small case series suggest that these techniques may provide valuable new information about prognosis and the pathophysiology underlying cerebral palsy.The online version of this article (doi:10.1007/s00234-014-1412-5) contains supplementary material, which is available to authorized users. In the third trimester of gestation, the brain’s connectivity framework is established through a complex but highly programmed sequence of maturation, mediated through key transient developmental structures such as the subcortical subplate . The impPreterm infants are at risk of intra-ventricular haemorrhage (IVH) and periventricular haemorrhagic parenchymal infarction (HPI) . The devThe work was approved by the NHS Research Ethics Committee, and written parental consent was obtained prior to all data acquisition. Data were collected at the Queen Charlotte and Chelsea Hospital, London, UK between 2009 and 2012.The study population consisted of six prematurely born infants: three infants known not to have focal brain pathology (controls) (selected from a previously described cohort ) and thrInfants were clinically assessed by a paediatrician, who was in attendance throughout the MRI scan. All infants were given chloral hydrate sedation  20 min prior to image acquisition. During scanning, temperature, oxygen saturations and heart rate were monitored, hearing protection was applied , and the head was immobilized with a vacuum-evacuated polystyrene-bead-filled pillow .b value of 750 s/mm2 using a spin echo EPI sequence . Passive sensori-motor stimulation for the fMRI experiment was performed using a simple block paradigm  and a fully automated hand interface in the form of a custom-made inflatable balloon which was placed in the palm contralateral to the known periventricular lesion in case infants (or right hand in controls) . To. To19]. p < 0.05 .Control infants had no focal brain lesions on conventional MR images, had no evidence of asymmetrical neurological signs and had a neurodevelopmental outcome within the normal range at 1-year corrected age  and given that MR-derived markers of white matter microstructural integrity are known to change during normal human development in addition to following injury . To provIn summary, in this study, we demonstrate the feasibility of applying fMRI and diffusion tractography to offer new insights into the early effects of acquired perinatal brain injury in a small case series of infants with a clearly defined pattern of injury. We found that at TEA, functional responses to passive sensori-motor stimulation are in the contralateral perirolandic cortex, with established thalamo-cortical and symmetrical CST structural connectivity and a consistent pattern of inter-hemispheric functional connectivity. In the presence of unilateral HPI, passive sensori-motor functional responses remain in the lesional hemisphere, with an apparently altered but intact pattern of thalamo-cortical structural connectivity. In contrast, unilateral deficit of CST integrity at TEA is predictive of later contralateral motor impairment, and altered inter-hemispheric functional connectivity is associated with poor neurodevelopmental outcome. This small data series suggests that MRI in early infancy has a marked potential to provide new information regarding the pathophysiology of cerebral palsy, could be used as a possible biomarker for testing future therapeutic interventions and may provide new clinical prognostic information.ESM 1(GIF 64 kb)High Resolution Image (TIFF 9482 kb)Below is the link to the electronic supplementary material.