We report our experience with high resolution microarray analysis in infants and young children with developmental disability and/or aberrant behavior enrolled at the Centro Ann Sullivan del Peru in Lima, Peru, a low income country. Buccal cells were collected with cotton swabs from 233 participants for later DNA isolation and identification of copy number variation (deletions/duplications) and regions of homozygosity (ROH) for estimating consanguinity status in 15 infants and young children  randomly selected for microarray analysis. An adequate DNA yield was found in about one-half of the enrolled participants. Ten participants showed deletions or duplications containing candidate genes reported to impact behavior or cognitive development. Five children had ROHs which could have harbored recessive gene alleles contributing to their clinical presentation. The coefficient of inbreeding was calculated and three participants showed first-second cousin relationships, indicating consanguinity. Our preliminary study showed that DNA isolated from buccal cells using cotton swabs was suboptimal, but yet in a subset of participants the yield was adequate for high resolution microarray analysis and several genes were found that impact development and behavior and ROHs identified to determine consanguinity status. Research involving early recognition of developmental/intellectual disabilities with or without aberrant behaviors such as self-injury, aggression, stereotypic patterns, and autism is on the increase but the causation is poorly understood. Advances in genetic testing now allow for the identification of genetic disturbances including chromosomal abnormalities and gene mutations or variants . Most reF) based upon the level of homozygosity in the genome and assessed using SNP data from microarray analysis [F ≥ 0.0156) and their progeny account for an estimated 10% of the global population [Single nucleotide polymorphisms (SNPs) are also helpful in detecting regions of homozygosity [large genomic regions without SNPs which may indicate gene alleles identical by descent from a common ancestor with evidence of consanguinity or due to areas of loss of heterozygosity ] –5. Consaanalysis . CouplesNumerous studies have applied molecular karyotyping with microarray analysis in identifying subtle DNA changes to explain the causation of unexplained developmental/intellectual disabilities, autism spectrum disorders, and multiple congenital anomalies. Microarrays can identify submicroscopic deletions and duplications not found by routine G-banded chromosome studies. Chromosomal microarray analysis is now considered in the first tier of clinical genetic testing in those patients presenting with the above common reasons for referral . The tesHerein, we report our genetics experience with high resolution microarrays in individuals after 6 months of age from Peru, a low income country where these services are limited, and those at risk for developmental disability, autism, or aberrant behavior. The goals of our study consisted of the following: (1) to identify, screen, enroll, and assess Peruvian infants and children at a young age with atypical development or with behavioral concerns for high resolution microarray analysis to study copy number variation and consanguinity status and to generate clinical case reports and (2) to test whether an adequate DNA yield can be obtained from buccal cells collected with cotton swabs only and stored at room temperature until shipped to the USA for DNA isolation.Participants were recruited as part of an innovative national outreach effort to identify and treat children with developmental problems in urban, rural, underprivileged, or otherwise underserved regions of Peru. Participants were solicited by approved television, radio, and newspaper announcements throughout Peru, requesting parents to respond if they had a child between the ages of 6 to 36 months and concerns regarding their child's development, cognition, communication, behavioral problems especially self-injury (SIB), stereotypy and aggression, or a family history of disabilities, medical conditions, or issues related to environmental or other unexplained factors. The parents who responded to the announcements called the Telephone Triage Service at Centro Ann Sullivan del Peru (CASP), a developmental training center affiliated with the University of Kansas and located in Lima. After brief discussion of their appropriateness for the project, 341 respondents were invited to CASP for a screening interview by trained interviewers/examiners, using the Parental Concerns Questionnaire (PCQ), a checklist based upon risk factors related to behavioral problems among people with developmental disabilities (DD) . If pareParticipants were screened using the Parent Concerns Questionnaire, a 15-item yes/no checklist based on an extensive list of 53 developmental risk factors for severe behavioral problems in children across nine domains . The domEach participant received (1) anthropometric measures of height, weight, and head circumference; (2) developmental pediatric exam with consults by local Peruvian specialists from pediatric neurology, medical genetics, nutrition, and child psychiatry as needed. If the pediatricians suspected autism, the Child Autism Rating Scale (CARS) was completed by the parents ; (3) behBoth the BSID and CSBS are standardized measures with a standard deviation of 15. Both scales are designed for the age group under study. One standard deviation below the mean of 100 for these instruments is a commonly accepted cut-off considered to be at-risk for developmental delay. Two standard deviations are often defined as intellectual or communication disability along with a measure of social performance ability. A standard cut-off score for autism on the CARS is 35 but used as a screening measure only.μg) and quality  required for high resolution microarray analysis. Approximately 50% of the DNA samples obtained did not meet the laboratory criteria required for high resolution microarray hybridization, reflecting the buccal swab collection process, handling, and storage prior to DNA isolation. Participants were excluded from microarray analysis with an inadequate DNA yield or with a known cause of their atypical development and/or aberrant behavior  after review of medical, laboratory, and brain imaging records (if available) and clinical assessments with examinations performed by local medical experts and specialists. Of the approximately 50 DNA samples meeting laboratory criteria for microarray analysis, funding was available to analyze 15 representative children  and single nucleotide polymorphism (SNP) DNA probes. The remaining 10 samples were analyzed by iLife Discoveries  using the Affymetrix Cytoscan HD Array utilizing 2.3 million copy number variant (CNV) and single nucleotide polymorphism (SNP) DNA probes . The Affymetrix Chromosome Analysis Suite 1.2.2 software version was used for determination of the number, location, and size of the CNV  or regions of homozygosity (ROH) by downloading and analyzing the microarray genome-wide SNP and CNV probe data on each participant and following standards recommended by the manufacturer of the high resolution microarrays for determination of genetic lesions and copy number variation. Deletions (losses) and duplications (gains) in the genome were based on the number of DNA probes deleted (or duplicated) over and within an established genomic distance and the number of involved DNA markers. We chose 100 kb size gains and losses across the genome with a minimum of 50 markers to make a call for a genomic abnormality. For regions of known significance in the human genome, a minimal setting of 25 kb in size and 50 markers for gains and losses were needed. For ROH, a minimal setting of 3 to 5 Mb was utilized.F) was calculated based on ROHs for each participant using the model proposed by Kearney et al. [http://www.genome.ucsc.edu/index.html) [F = 0.25); 12.5% for second degree relatives  (F = 0.125); 6% for third-degree relatives (first cousins) (F = 0.0625)). Couples related as fifth degree relatives (second cousins or closer) with an F ≥ 0.0156 are considered consanguineous [In lieu of family history or pedigree analysis, we also estimated consanguinity and the coefficient of inbreeding   to estimClinical presentation of the 15 children studied included development delay or aberrant behavior as characterized by interdisciplinary assessments listed in Assessments for SIB, stereotyped behavior, and aggression were performed with the BPI-01 instrument. Normative data showed that 10 of the 15 children  had high scores according to Rojahn et al. [ COG5 gene which is located in the autism susceptibility 1 (AUTS1) locus known to cause atypical development and autism [The microarray genetic findings of the 15 children with atypical development and autism or aberrant behavior are summarized in d autism  supporti CDH9 gene, one of the cadherin gene members reported to play a role in autism and supported by genome-wide association studies indicating this genomic region to be highly associated with autism [ NDUFS7 and GAMT genes due to a chromosome 19p13.3 duplication (1043 kb in size). These genes may cause developmental delay, psychomotor retardation, hypotonia, regression, and metabolic problems [ NOTCH2 gene. Notch receptor defects can cause failure to thrive, renal abnormalities, heart defects, and learning disabilities [Participants 32 and 44 are both males at 41 months and 36 months of age, respectively, and presented with normal cognition. The CARS and BPI scores supported the diagnosis of autism. Participant 32 had a 5p14.1 deletion including theh autism . Participroblems , 21. He bilities  and may  ROR2 gene which encodes a receptor tyrosine kinase when defective causes Robinow syndrome. This syndrome is characterized by short stature, mesomelia, brachydactyly, a particular facial appearance, and developmental delay [ CACNA1H gene encoding a calcium ion channel subunit, causing susceptibility to epilepsy [ WDR45 gene was also found at Xp11.23, which regulates the assembly of multiprotein complexes and protein-protein interactions. Truncating and missense mutations of this gene have been implicated in neurodegeneration with brain iron accumulation leading to intellectual disability, dementia, dystonia, and tremors [ SYP gene was also duplicated at Xp11.23 and is an integral membrane protein that regulates synaptic vesicle endocytosis associated with X-linked intellectual disability and epilepsy [Participant 94 is a 19-month-old male with several microarray findings including a very small 9q22.31 partial duplication (52 kb in size) including theal delay . He showepilepsy . A parti tremors . In addiepilepsy . The coe PRODH gene encoding proline dehydrogenase. This enzyme catalyzes the conversion of proline to pyrroline-5-carboxylate with disturbances linked to the susceptibility of schizophrenia, autism, hyperactivity, aggression, and intellectual disability [Participant 102 is a 28-month-old male with tall stature and microcephaly with a very small gain of the 22q11.21 region containing thesability . He had  PCDH19 and SRPX2 genes. PCDH19 encodes protocadherin 19 known to play a role in autism while the SRPX2 gene is associated with Rolandic epilepsy, intellectual disability, and speech dyspraxia [ OPHN1) when defective are implicated in the development of intellectual disabilities. The OPHN1 geneencodes the Rho GTPase-activating protein associated with X-linked intellectual disabilities and cerebellar hypoplasia [Participant 113 is a 26-month-old female with an ROH at Xq21.33-q22.1 including theyspraxia . She hadpoplasia . The coe RTN4R gene for susceptibility to schizophrenia by encoding a protein component of myelin and preventing axonal regeneration [Participant 120 is a 36-month-old male who showed a very small gain at 22q11.21 which contains theneration . His BPI FOXP2 gene encoding forkhead box P2, a putative transcription factor and DNA binding domain. Mutations leading to haploinsufficiency of this gene can result in speech-language disorder-1 [Participant 146 is a 34-month-old male with growth retardation and showed an ROH identified at 7q31.1-q31.2 which contains the GNAS, MC3R, CDH6, TFAP2C, and approximately 50 other genes. GNAS beinga complex locus containing four genes with three of the genes  being imprinted or expressed differently depending on the parent of origin (reviewed by Butler [ MC3R (melanocortin 3 receptor) gene is known to play a role in obesity while the CDH4 is a member of the cadherin gene family mediating calcium-dependent cell-cell adhesion and plays a role in autism. TFAP2C modulates the transcriptional activity of vitamin A and target genes via the retinoic receptors and is expressed in migrating neural crest cells in the developing embryo [Participant 165 is a 32-month-old female with a 20q13.2-q13.3 deletion  involving POU1F1, RAI1, and EP300) associated with intellectual disability are found. If mutated and inherited in a recessive pattern then intellectual disability, hypotonia, speech delay, self-injury, microcephaly and behavioral problems can occur [Participant 176 is a 37-month-old male with ROHs noted on chromosomes 3, 17, and 22 within regions where several genes  which included 25 genes. Duplications of this chromosome region have been reported previously in individuals with mild to moderate global delay, abnormal head size, autism, self-injury, stereotypy, speech delay, and mild facial dysmorphism . In addiophrenia , 30. She NLGN1 gene which encodes a neuroligin. Neuroligins function as ligands for neurexins or cell-surface receptors. The neurexin/neuroligin complex is Ca(2+)-dependent and present at synapses in the central nervous system and required for efficient neurotransmission involved in the formation of synaptic contacts proposed to cause autism [NTRK2, MKS1) that may play a role in obesity and developmental or speech delay [Participant 218 is a 23-month-old male who showed a very small gain at 3q26.31 representing a partial duplication of thee autism , 43. In ch delay , 46. He  FAM20C gene associated with Raine syndrome. This syndrome is characterized by short stature, microcephaly, craniofacial dysplasia, renal abnormalities, developmental delay, and self-stimulating behavior [Participant 237 is a 21-month-old male who was large for age and showed a gain at 7p22.3 which includes thebehavior . In addiChromosomal microarray analysis is now considered in the first tier of clinical genetic testing when evaluating children presenting with unexplained developmental delay/intellectual disabilities, autism spectrum disorders, and/or multiple congenital anomalies. This method yields a much higher result than routine chromosome studies by detecting small submicroscopic deletions and duplications in the genome. Our study is the first to undertake advanced genetic testing using high resolution microarray technology at such a young age  systematically ascertained and assessed for atypical development and aberrant behavior from an underserved population in a low income country (Peru). We utilized locally available cotton swabs as salvia collection kits with DNA preservative were not accessible to collect buccal cells for later use in microarray hybridization. DNA collected from approximately one-half of the buccal cell samples was acceptable for microarray analysis indicating an overall suboptimal yield. Several previous studies using chromosomal microarrays have shown a diagnostic yield of 11% to 20% with clinically significant CNVs in those individuals presenting with unexplained intellectual disabilities in high income countries  .Detection of regions of homozygosity (ROH) with microarray technology in itself is not diagnostic for an underlying condition and may have no clinical outcome. However, when present in multiple areas throughout the genome versus long stretches of homozygosity confined to a single chromosome, then these regions may be due to identical by descent or from a common (or shared) ancestor raising the possibility that mutant recessive gene alleles if present in these regions could lead to genetic disorders. If these regions are large e.g., >3 Mb), then the likelihood increases for inheriting a genetic disorder with clinical consequences [ Mb, thenWhen parents of a child share a recent common ancestor then this relationship becomes consanguineous in nature with the closer relationship having a greater likelihood in producing a child with two copies of a deleterious recessive gene allele mutation received from the parents. Conversely, if one or more regions show extended homozygosity particularly longer than 10 Mb in size on a single chromosome, then the most likely cause is uniparental disomy with loss of heterozygosity of the entire chromosome (error in meiosis II) or as the segmental type . This eUnfortunately, no data were available from the parents. The detection of high levels of ROH without parental data limits accurate interpretation for consanguinity and one can only conclude that consanguinity is present in the community in which the participant lives. Any follow-up study should include parents and demographic survey with questions on family structure/consanguinity and socioeconomic status.Of the 15 children selected at random to study with microarray analysis, 10 had genomic duplications or deletions containing genes when defective are known to be associated with neurodevelopment and function including autism, behavior problems, schizophrenia, and developmental delay. Therefore, these rearrangements harboring genes become candidates for contributing to the clinical presentations observed in our participants. Two participants had identifiable microduplication/deletion syndromes including participant 165 with a 20q13.2-q13.33 deletion encompassing the complex GNAS locus and other genes within the region see . This de de novo CNV status or consanguinity. Limitations in our study included lack of resources and access of parental data and laboratory evaluations to analyze CNVs.Five of the children  in our study had ROHs which may have contributed to their clinical presentation if disturbed recessive candidate gene alleles were present in the extended stretches of homozygosity and thus these participants would have received two copies. The ROHs ranged from 3,018 to 6,237 kb in size. Three children did not have sufficient SNP data for calculation of the coefficient of inbreeding to determine inbreeding status (no ROHs seen). Six participants were found to be unrelated while three showed ROHs consistent with a first-second cousin relationship (consanguineous) but had recognized chromosomal structural anomalies as well including genes associated with developmental and/or behavioral problems. Three participants showed a second-third cousin relationship but limitations in our study prevented detailed family history and parental physical examinations with collection of specimens for parental laboratory evaluations in order to analyze CNVs or SNPs for determining familial versusIn summary, our study showed that in less developed countries high resolution microarray analysis can be done where blood collection and specimen processing may be limited using locally available cotton swabs for buccal cell collection without a DNA preservative and storage at room temperature. However, as expected, the capacity to provide adequate quantity and quality of DNA was suboptimal with about one-half of the specimens yielding DNA suitable for high resolution microarray analysis. Saliva is now becoming a more common specimen source for collection of DNA for genetic testing particularly for the pediatric age group and the use of a DNA preservative. Two-thirds of our participants showed DNA copy number variation consistent with deletions or duplications in genomic regions containing known or candidate genes for atypical development or behavioral problems. One-fifth of the study participants showed evidence of consanguinity but parental samples were not available. We conclude that advanced genetic testing can yield meaningful data even in suboptimal sample collection conditions with limited resources.


With up to 500,000 infections annually, Lassa virus (LASV), the cause of Lassa fever, is one of the most prevalent etiological agents of viral hemorrhagic fever (VHF) in humans. LASV is endemic in several West African countries with sporadic cases and prolonged outbreaks observed most commonly in Sierra Leone, Liberia, Guinea and Nigeria. Additionally several cases of Lassa fever have been imported into North America, Europe and Asia making LASV a global threat to public health. Despite this, currently no approved therapeutic or vaccine exists to treat or prevent LASV infections. Here, using a passaged strain of LASV that is uniformly lethal in Hartley guinea pigs, we demonstrate that favipiravir, a broad-spectrum antiviral agent and leading treatment option for influenza, has potent activity against LASV infection. In this model, once daily treatment with favipiravir significantly reduced viral titers in tissue samples and reduced mortality rates when compared with animals receiving vehicle-only or ribavirin, the current standard of care for Lassa fever. Favipiravir remained highly effective against lethal LASV infection when treatments were initiated nine days post-infection, a time when animals were demonstrating advanced signs of disease. These results support the further preclinical evaluation of favipiravir for Lassa fever and other VHFs. Bunyaviridae, Filoviridae, Arenaviridae and Flaviviridae) are known to cause VHFs, though more etiological agents are likely to exist. While some VHFs are associated with sporadic but potentially explosive outbreaks, others are essentially endemic in specific geographical regions and are often associated with high annual incidence rates. With an estimated 300,000–500,000 infections annually, Lassa virus  is a perfect example of the latter group24Viral hemorrhagic fevers (VHFs) are among the most deadly and feared group of diseases in humans and for most no approved vaccine or treatment existsArenaviridae; it has a bi-segmented RNA genome which produces five viral proteins using an ambi-sense coding strategy. In nature, LASV is maintained in the multi-mammate rat, Mastomys natalensisM. natalensis is found across sub-Saharan Africa, evidence of infected rodents has exclusively been noted in Western African countries. Due to this, cases of LF are most commonly observed in Sierra Leona, Liberia, Guinea and Nigeria. These four countries represent the regions historically considered endemic for LASV/LF; though it is becoming increasingly clear that other West African nations, including Mali, Cote d’Ivoire, Benin and Ghana are also at risk for sporadic cases and potentially explosive outbreaks of LF789LASV is a prototypical member of the The high annual incidence of LF in West Africa suggests a prophylactic vaccination strategy would be the most effective way of reducing the burden of LASV infection in this population. However, the lack of approved vaccines with no candidates currently in clinical trials necessitates the evaluation of therapeutic options, preferably those already approved for human use, for treating LASV infections and LF disease. To that end ribavirin, a broad spectrum antiviral agent which is licensed for the treatment of hepatitis C, is often used off-label to treat patients diagnosed with LF. Although ribavirin therapy has been shown to reduce the morbidity and mortality associated with LF, its limited efficacy is reliant on treatment initiation within 6 days of disease onsetin vitro and/or in vivo studies suggest that favipiravir exhibits broad-spectrum antiviral activity against a variety of RNA viruses, including alpha-, paramyxo-, picorna-, and caliciviruses, as well as etiological agents associated with VHFs including bunya-, flavi-, filo- and arenaviruses15171819202122232425262728in vitro studies have shown that the antiviral activity of favipiravir is similar to or better than that of ribavirin for most RNA virus15Favipiravir  is a novel antiviral agent recently approved in Japan as an anti-influenza drug and Phase 3 clinical studies have been completed in the United States for the same indication. Previous studies demonstrated that favipiravir inhibits the RNA-dependent RNA polymerase of influenza and it is effective against all strains and serotypes that it has been tested13141516in vivo using using a guinea pig-adapted strain of LASV-Josiah (GPA-LASV) which is uniformly lethal in Hartley outbred guinea pigs.Recently, favipiravir was shown to effectively prevent lethal disease associated with Pichindé virus infection in guinea pigs and hamsters which are surrogate disease models for VHFs of arenaviral etiology212650) in supernatants collected at day 3 and 5 post-infection from Vero cell cultures treated with varying concentrations of favipiravir resulted in EC90 values of 1.7 and 11.1 μg/ml, respectively. As expected, no toxicity was observed at the highest concentration tested (100 μg/ml), translating to SI values of >59 and >9 for the day 3 and day 5 assays, respectively.Treatment of LASV infected Vero cells with favipiravir resulted in diminished infectious viral titers in a dose dependent manner . Analysi50 in these animals. All eight guinea pigs infected with GPA-LASV in the first experiment developed severe disease requiring euthanasia between days 14 and 19 post-infection with an average time to lethal disease of 15 days. In contrast, only a single animal infected with wild-type LASV Josiah developed severe disease and was euthanized 23 days post-infection (−1 to 101 TCID50) were refractory to lethal disease with essentially no signs of infection. A single animal infected with 102, two animals infected with 103, and all animals infected with 104 TCID50 of GPA-LASV developed severe disease  animals, the survival rates in favipiravir-treated animals were statistically significant with ectively . AlthougViral titrations conducted on serum and tissue samples from each group of animals provide corroborating support for the efficacy of favipiravir against LASV infection . Infectip-values of <0.01 for the day 5 and day 7 treatment onset groups and <0.05 for the day 9 treatment onset group.In the second efficacy experiment, we sought to determine if delayed treatment with favipiravir would remain effective in preventing lethal LASV infection. To that end, four groups of six guinea pigs were infected with GPA-LASV as in the first experiment and treatments for three groups with 300 mg/kg/d of favipiravir were initiated at 5, 7, and 9 days post-infection, while the forth control group received vehicle placebo beginning on day 5 post-infection. Once daily treatments for each group lasted for 14 consecutive days. The six control, placebo treated animals developed severe disease indicated by fever beginning on day 7 and significant weight loss (>20%) which first began on day 9 post-infection . The conAll surviving animals from both efficacy experiments demonstrated high titer (≥6400) anti-LASV nucleocapsid IgG antibodies in convalescent (day 42) serum samples; thereby confirming these animals were exposed to LASV.With an annual incidence rate of up to 500,000 infections, LASV is easily the second most prominent etiological agent of VHF, behind only dengue virus. In addition to a large endemic region comprising many countries in West Africa, dozens of imported cases of LF have been diagnosed in North America, Asia and Europe, which makes LASV/LF a global public health concern. Despite this, LF remains one of the world’s most neglected diseases with no licensed therapeutic or vaccine options currently available.A limitation to evaluating potential therapeutics against LASV infection or LF disease is lack of readily available rodent models which can be utilized in initial efficacy studies. Currently, the only immuno-competent small animal disease models for the study of LASV infection are strain 13 and less commonly strain 2 inbred guinea pigs4 TCID50 or greater (LD50 = 103 TCID50). Histological abnormalities in this model recapitulated those commonly observed in the inbred strain 13 modelIn order to overcome these hurdles we utilized a strain of LASV Josiah which had been passaged from terminally-ill outbred guinea pigs into healthy animals, four times. The resultant isolate  demonstrated a uniformly lethal phenotype at challenge doses of 1050 of GPA-LASV, placebo treated guinea pigs began to show signs of disease, including increased temperature and weight loss on days 6 and 8, respectively. Other signs of disease, including lethargy, ruffled fur, recumbency and reddening of ears and footpads appeared between days 9 and 11 post-infection. Animals in the placebo treatment group succumbed to infection or were euthanized between days 12 and 16 post-infection. Since ribavirin is currently the standard of care for LF patients, a comparison group was included in these studies. Animals treated with ribavirin appeared relatively normal throughout the treatment phase, although animals essentially only maintained their body weight with very minimal gains noted. Within two days of the end of treatments the four remaining animals in the ribavirin treatment group developed signs of disease and although treatment provided a prolonged time to death, it did not improve the overall survival rate. Similar results with ribavirin treatment  in a guinea pig model of LF have been previously notedFollowing infection with 100 × the LDSimilar to recent favipiravir efficacy studies against Pichindé virus infection in guinea pigsDue to the often non-descript prodrome associated with many etiological agents of VHFs, the development and characterization of broad spectrum antivirals is vital to increase life expectance as well as to curtail potential human-to-human spread of some of these agents. Our present findings suggest that in addition to other emerging VHF agents including Ebola, Crimean-Congo-hemorrhagic fever, Andes and Junin viruses, favipiravir is highly effective at preventing lethal disease associated with LASV infection, even when treatments are initiated several days after the onset of clinical disease. Combined with its excellent tolerability in humans, these studies further suggest that favipiravir should be a front line treatment option for patients demonstrating symptoms of VHF.In vivo efficacy studies were approved by the Institutional Animal Care and Use Committee of the Rocky Mountain Laboratories (RML). Animal work was conducted adhering to the institution’s guidelines for animal use, and followed the guidelines and basic principles in the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals by certified staff in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International accredited facility.All work with infectious LASV and potentially infectious materials derived from animals was conducted in a Biosafety Level 4 (BSL 4) laboratory in the Integrated Research Facility of the Rocky Mountain Laboratories (RML), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). Sample inactivation and removal was performed according to standard operating protocols approved by the local Institutional Biosafety Committee.in vivo efficacy studies animals were also subcutaneously implanted with IPTT-300 electronic transponders for temperature measurement with a DAS 6002 hand-held scanner . Animals were acclimated to BSL 4 environment for one week prior to challenge and, in the antiviral studies, sorted so that the average body weights of each treatment group was similar.Outbred male Hartley guinea pigs (300–350 g)  were ear-tagged for identification and group housed, 2 per box in an isolator caging unit. For the In vitro studies were conducted with LASV strain JosiahIn vivo experiments utilized a LASV strain Josiah that was passaged four times in Hartley guinea pigs  resulting in a uniformly lethal phenotype in these animals . In the first in vivo experiment a comparison group of animals infected with wild-type LASV Josiah was included. Both viruses were propagated in Vero cell culture and titered using standard 50% tissue culture infectious dose (TCID50) infectivity assays and the Reed-Muench formula.in vitro studies, compounds were dissolved directly in cell culture media (see below). For in vivo studies, the antiviral compounds were prepared in sterile water supplemented with 74.6 mg/ml meglumine excipient.Favipiravir was provided by the Toyama Chemical Company, Ltd.  and Ribavirin was purchased from R & D Systems . For 90) of favipiravir against LASV, nearly confluent (>95%) monolayers of Vero cells were infected with LASV Josiah at a multiplicity of infection (m.o.i.) of 0.01. After a 1 hour absorption, cells were washed and the inoculum replaced with culture media  containing varying concentrations  of favipiravir. On days 3 and 5 post-infection supernatants were collected and infectious viral titers were determined as above. Cell viability was assessed visually at the time of sample collection. The EC90, which indicates the dose at which the viral titer is reduced by 1 log10, was calculated using basic linear regression analysis on the points in the linear range (r-squared >0.9). The maximum concentration of favipiravir utilized in these in vitro studies (100 μg/ml) is well below the documented 50% cytotoxic concentration (CC50) in Vero cells (>500 μg/ml)50/EC90.In order to determine the 90% effective concentration (EC4 TCID50 of LASV or GPA-LASV (both strain Josiah) via intraperitoneal (i.p.) injection. Animals were monitored for disease progression for 42 days and were only euthanized if they reached a pre-determined clinical score indicative of advanced terminal disease . In a second experiment, six groups of four guinea pigs were challenged with ten-fold serial dilutions of GPA-LASV (dose range 1 × 104 to 1 × 10−1 TCID50) by i.p. injection. Animals were monitored for disease progression for 42 days and were euthanized when they demonstrated signs of advanced terminal disease.In order to determine an appropriate challenge dose with the GPA-LASV we first conducted a pilot experiment to ensure uniform lethality of GPA-LASV in outbred Hartley guinea pigs. To that end two groups of eight guinea pigs were infected with 1 × 10in vivo efficacy studies were conducted in two independent experiments. In the first study, three groups of nine guinea pigs were inoculated with 1 × 105 TCID50 GPA-LASV representing 100 × the 50% lethal dose (LD50) via i.p. injection. Beginning on day 2 post-infection, animals were treated for 14 consecutive days with favipiravir (150 or 300 mg/kg/d) or ribavirin (50 mg/kg/d). Since oral gavage is not recommended for guinea pigs, treatments were administered with once daily subcutaneous (s.c.) injections. A control group of six guinea pigs was inoculated with GPA-LASV as above and treated according to the same schedule with vehicle placebo. Additionally, three guinea pigs were mock-infected and included as normal controls. Animals were subjected to mild isoflurane anesthesia prior to each treatment. Weights and body temperatures were recorded every second day. At a time when control animals were showing signs of advanced, terminal disease three animals per treatment group were euthanized and tissue samples collected to assess the extent of viral replication. The remaining six animals per group were monitored for disease progression and survival for 42 days.The In the second experiment the efficacy of delayed treatment onset with favipiravir was assessed. Three groups of six guinea pigs were inoculated with GPA-LASV as above. Beginning on day 5, 7 and 9 post-infection, favipiravir treatments were initiated in one group of animals respectively with daily s.c. injections of 300 mg/kg/d for 14 consecutive days. A control group of six guinea pigs was inoculated as above and treated with vehicle placebo beginning on day 5 post-infection. Weights and temperatures were recorded every second day. Survivors were monitored for 42 days post-infection.50 methodologies. For IHC, tissue samples were inactivated and fixed with 10% formalin and processed according to standard methodologies. Thin sections were stained for viral antigen using a LASV GP2-specific monoclonal antibody Tissues collected in the first efficacy experiment were tested for the presence of LASV via infectious viral titration assays as well as immunohistochemistry (IHC). For infectious titers, serial ten-fold dilutions of clarified tissue homogenates (10% weight by volume) were analyzed for viral loads on Vero cells via standard TCIDSeroconversion to LASV infection in convalescent serum samples collected from surviving animals in the treatment studies was assessed using standard enzyme-linked immunosorbent serological assay (ELISA) methodologies with an in-house recombinant, bacterially expressed LASV nucleocapsid protein as the antigenStatistical differences between infectious titers in treatment groups were examined using one-way analysis of variance with Tukey-Kramer multiple-comparison posttest. Survival curves were compared using the log-rank (Mantel-Cox) test. Statistical analyses were accomplished using Prism 5 .How to cite this article: Safronetz, D. et al. The broad-spectrum antiviral favipiravir protects guinea pigs from lethal Lassa virus infection post-disease onset. Sci. Rep.5, 14775; doi: 10.1038/srep14775 (2015).


Achromatium represent a significant proportion of bacterial communities near sediment-water interfaces at sites throughout the world. Our understanding of their potentially crucial roles in calcium, carbon, sulfur, nitrogen, and iron cycling is limited because they have not been cultured or sequenced using environmental genomics approaches to date. We utilized single-cell genomic sequencing to obtain one incomplete and two nearly complete draft genomes for Achromatium collected at Warm Mineral Springs (WMS), FL. Based on 16S rRNA gene sequences, the three cells represent distinct and relatively distant Achromatium populations (91–92% identity). The draft genomes encode key genes involved in sulfur and hydrogen oxidation; oxygen, nitrogen and polysulfide respiration; carbon and nitrogen fixation; organic carbon assimilation and storage; chemotaxis; twitching motility; antibiotic resistance; and membrane transport. Known genes for iron and manganese energy metabolism were not detected. The presence of pyrophosphatase and vacuolar (V)-type ATPases, which are generally rare in bacterial genomes, suggests a role for these enzymes in calcium transport, proton pumping, and/or energy generation in the membranes of calcite-containing inclusions.Large, sulfur-cycling, calcite-precipitating bacteria in the genus  Achromatium contains morphologically striking and unusually large bacteria distinguished by their abundant calcium carbonate inclusions. A single Achromatium cell may be shaped as a coccus or rod with a length between 10 and 100 μm, with large calcite inclusions (5–6 μm) and smaller sulfur inclusions (0.5–2 μm)  have recently been described in Achromatium populations from a salt marsh  indicated that physiological differences in Achromatium communities are not related to carbon metabolism  and sequenced, revealing the metabolic capacities of uncultured microorganisms  is a water-filled sinkhole located in the city of North Port, Florida, United States . The spring consists of a central basin and a single outflow with tidal fluctuations up to 20 cm Figure . The sprsolid) were dissolved in 1.2 N HCl mixed with 1:100 volume of Ti(III)-citrate as a reducing agent. Ti(III)-citrate was added to prevent the formation of S(0) during acid dissolution . Leakage was continuously monitored with the portable gas detector. Precipitated zinc sulfide minerals were subsequently dissolved and analyzed with the methylene blue method  for AVSsolid measurements.The top 2 cm of sediments at WMS were sampled for genetic, microbiological, chemical, and physical analyses. Samples were collected by scooping the sediments with pre-sterilized tubes and bottles. Sediments used for elemental analysis were frozen at −20°C after storage at 4°C for less than 1 month. After thawing, they were centrifuged at 8000x g for 10 min and the pore water was removed. Sediments were subsequently freeze-dried and powdered. Sediment organic matter content was measured with the Loss-On-Ignition (LOI) method by heating overnight at 750°C followed by further heating at 900°C for 3 h. Aliquots of the same sample were decarbonated in 1N HCl overnight and dried at 80°C prior to LOI as a control to determine the effect of possible carbonate particles on organic matter measurements. Solid-phase acid-volatile sulfides  or in the WMS outflow streams using sensors attached to a 50i multimeter  calibrated daily with fresh standards. Dissolved sulfide, sulfate, nitrate, iron and ammonium concentrations were measured in the field on a portable spectrophotometer using the methylene blue, SulfaVer 4, Cadmium Reduction, FerroVer and Salicylate methods respectively . Filtered (<0.2 μm) water samples were collected into acid-washed polypropylene bottles and tubes during sampling events in 2012 and 2013 to determine the concentrations of major cations and anions. Filtered water samples were immediately placed on ice and stored at 4°C until further processing. Cations were measured on acidified samples with Inductively Coupled Plasma-Atomic Emission Spectroscopy and anion concentrations were measured with a Dionex ICS-2500 Ion Chromatogram at Pennsylvania State University.Dissolved oxygen (DO), pH, conductivity and temperature of the spring water were determined Achromatium populations decline after 2 weeks in natural sediments stored at temperatures between 4 and 30°C. Thus, fresh sediments from the basin were regularly collected as described above and shipped on ice overnight to Pennsylvania State University for further processing.Based on our observations, Achromatium. The cells were concentrated using the “gold panning” method that takes advantage of their fast sedimentation rate  at pH 7 to remove residual free DNA. Single cells of Achromatium were carefully hand-picked with a pre-sterilized syringe needle  attached to a micromanipulator, transferred to 3 μl of UV-treated PBS, and immediately frozen at −20°C.Fresh sediments were sequentially filtered through pre-sterilized 100, 63, and 40 μm sieves to remove particles larger than Achromatium cells in PBS were thawed at room temperature and lysed following the addition of alkaline Buffer D2 solution at 65°C for 10 min. Stop Solution was added to stop the lysis reaction and to neutralize the solution. Genomic DNA was then amplified with Φ DNA polymerase at 30°C for 8 h followed by deactivation at 65°C for 3 min. Results of the amplification were checked by NanoDrop . Amplified genomic products were then screened for the presence of 16S rRNA gene sequences using 1 uL of 1:25 dilutions of amplified gDNA in a polymerase chain reaction (PCR) with 27f and 1492r primers .Genomic amplification was carried out using the REPLI-g Single Cell Kit according to manufacturer's protocol . Briefly, single rs Lane, . PCR cycrs Lane, . UnpurifThe three amplified genomic products were subjected to whole genome sequencing at the Genomics Core Facility at Pennsylvania State University. Genomic libraries were prepared using a TruSeq DNA PCR-Free kit  to reduce bias and sequenced using the Illumina MiSeq platform  . Quality of the reads was checked with FastQC (bioinformatics.babraham.ac. uk/projects/fastqc). Based on the quality check, the first 10 and last 15 base pairs of the reads were trimmed with Trimmomatic  and type 1 [NiFe]-hydrogenases from the single cell genomes were further grouped into their respective homologs based on phylogenetic analyses. The sequences were aligned with ClustalX. Alignments were populated with closely related sequences and manually curated. Phylogenetic trees were prepared using MEGA as described above. The Sqr sequences were classified according to Gregersen et al.  based on a motif list prepared by Santamaría-Hernando et al.  and can be accessed under the accession numbers Geochemistry of the WMS spring water was analyzed during sampling trips in 2012 and 2013. In 2012, dissolved oxygen (DO) in the basin was depleted (<20 μM) below a water depth of 2 m, while total sulfide concentration varied between 20 and 30 μM throughout the water column Figure . In contThree locations along the outflow stream were chosen as sample sites in addition to the basin Figure . All of n = 2), high organic matter  and low AVSsolid . Decarbonation prior to LOI analysis did not yield notably different estimates of sedimentary organic matter content, suggesting that inorganic carbon in carbonate minerals was not present in the sediment in significant amounts.Surface sediments from all sampling sites were composed of organic-rich peat mixed with silicate sand. Surface sediments from the littoral zone of the basin were characterized by high porosity  with 99% 16S rRNA gene identity, while WMS1 and WMS2 were only weakly affiliated with existing clades  and penicillin (outer membrane protein gene tolC).Each of the three draft genomes contains a partial set of the genes required for glycolysis, the tricarboxylic acid cycle and the synthesis of key amino acids and vitamins. Collectively however, the draft genomes contain the complete set of genes needed for glycolysis and the tricarboxylic acid cycle as well as for the synthesis of the 20 common amino acids and key vitamins. WMS3 also contained genes encoding for resistance to the antibiotics vancomycin . The WMS3 draft genome contained the complete pil gene set (pilGHIJTU) while the WMS1 and WMS2 draft genomes contained at least one gene involved in twitching motility. Several genes encoding chemotaxis response proteins were identified including the aerotaxis receptor (aer), the cheABRWY complex, motility protein B (motB) and genes encoding for various methyl-accepting chemotaxis proteins. The regulators of these genes/proteins are not known  through the activity of sulfide:quinone reductase (Sqr). A gene encoding for Sqr type C was identified in WMS2 while a gene encoding Sqr type A was identified in WMS3 , adenosine phosphosulfate reductase (aprAB) and heterodisulfide reductase (hdrABC)  in each single-cell genome, suggesting that all three populations have the potential to utilize oxygen as an electron acceptor. We did not identify any genes that encode typical nitrate reductase enzymes . In WMS3, we found a gene encoding for hydroxylamine reductase, which may function in dissimilatory nitrate reduction to ammonium  was identified in WMS2. No genes encoding proteins implicated in Fe reduction were identified. Siderophore biosynthesis genes were similarly not identified, even though genes encoding for TonB-dependent receptors and siderophore-specific ABC transporters were present in the draft genomes.In addition to oxygen, nitrite and nitric oxide, several other electron acceptors are potentially utilized by Achromatium. Genes encoding for sulfur globule protein B (SgpB)  and may store them for energy reserves. However, PPK alone does not indicate the presence of polyP granules. Finally, we identified genes encoding for polyhydroalkanoate synthase (PHA) in WMS1 and WMS3 and for glycogen synthase in WMS3. Acetate may be converted to either PHA or glycogen for carbon and energy storage using energy gained from polyP degradation, similar to Thiomargarita  Brune,  were ideaq)2 and a predicted Na/H+-Ca exchanger (yrbG) were identified in WMS2 and WMS3. We also identified several genes encoding subunits of vacuolar(V)-type ATPases and for pyrophosphatase (PPase), which can generate a transmembrane H+ electrochemical gradient that can be used to drive transport of substrates across biological membranes  were missing. Achromatium proteins containing hemolysin motifs were very distantly related (<30% identity) to Ca-binding proteins in the genomes of C. thermalis PCC 7203 and Cyanothece sp. PCC 7425 and had no homology to proteins in the genome of T. elongatus BP-1. Proteins containing cadherin motifs in Achromatium did not share any homology to proteins in the three cyanobacteria.We compared the Achromatium phylotypes have been grouped into two clusters based on phylogenetic analysis of the 16S rRNA gene and the presence/absence of a deletion affecting helix 38 in the V6 region , and in salt marsh sediments where sulfide concentrations are highly variable (0–2500 μM). The freshwater sediments are characterized by a weak sulfide generation capacity (low sulfate reduction rates) and/or continuous sulfide removal through iron sulfide precipitation  at the time of sampling, and in the littoral zone of the basin, where sulfide concentrations were highly variable with a maximum of 30 μM over a 24 h period.es Gray, . PreviouAchromatium in the WMS basin sediments could benefit by accessing sulfide minerals for reducing power, we compared the amount of total dissolved sulfide available in the sediment pore water (assuming 30 μM concentration) vs. the amount of sulfide trapped in sulfide minerals (AVSsolid) over the same sediment depth interval. Accounting for porosity, 1 cm3 of sediment contains approximately 0.03 μmol of dissolved sulfide and 0.05–0.10 μmol of sulfide trapped in sulfide minerals. Therefore, Achromatium would gain an advantage relative to other sulfur-oxidizing bacteria in the surface sediments by being able to access sulfide from AVSsolid.To evaluate whether Achromatium  do not appear to contain genes for RuBisCO, but can assimilate organic carbon , we observed little turbulence and a lack of conspicuous mat-forming sulfur oxidizers. Achromatium was not detected at sample sites in between or further downstream, where water turbulence was consistently higher. Our observations are consistent with the idea that water flow characteristics play an important role in controlling the distributions of Achromatium.Water flow characteristics including turbulence have been shown to be important in predicting the outcome of competition among sulfur-oxidizing Achromatium populations at WMS are likely carbon mixotrophs with highly versatile respiratory metabolisms. WMS populations can probably generate energy from the oxidation of sulfide, thiosulfate, S(0), and hydrogen. Interestingly, phylogenetically distinct Achromatium cells had significant differences in their genetic potential for carrying out energy generation reactions , fumarate or CO2, and also recycle H2 produced by the enzyme nitrogenase . The WMS2 draft genome contains a gene encoding a copper-containing nitrite reductase (EC 1.7.2.1). Genes encoding typical nitrate reductase enzymes  were not detected even after an extensive BLASTP/BLASTX search of the Achromatium draft genomes (assembled and unassembled reads) against sequences of well-characterized nitrogen respiration enzymes. Although nitrate profiles within sediments showed no clear relationship with Achromatium cell numbers in one previous study 2, Na/H+-Ca exchangers, V-type ATPases, and PPase. V-type ATPases are common in eukaryotes and archaea but are rarely present in bacteria  and all three cyanobacterial genomes. Interestingly, V-type ATPases and PPase were not present in the genomes of these cyanobacteria. This suggests that either different calcification pathways are utilized in these different lineages or that the genes discussed here are not essential for intracellular calcification. Further studies are required to resolve this matter.We compared the draft genomes of Achromatium as well as several species of cyanobacteria. Whereas cyanobacteria are known to induce the extracellular precipitation of calcium carbonate minerals as a result of rapid uptake of inorganic carbon during photosynthesis, bacteria such as Achromatium oxidize reduced sulfur compounds completely to sulfate, resulting in a net production of protons that should have a negative influence on carbonate precipitation. The function of intracellular carbonate inclusions in prokaryotes is not well understood, and may differ even among cyanobacterial species . Mechanisms by which Achromatium could enhance AVS mineral dissolution include the production of siderophores  oxidation to sulfate. Given the significant variations in the number of protons produced or consumed during each phase of respiration depending on whether O2 or oxidized nitrogen species are used as electron acceptors  oxidation :Achromatium generates ATP across calcite-containing vacuole membranes containing V-type ATPases and PPase, by analogy to ATP generation across vacuolar membranes containing nitrate in “Candidatus Allobeggiatoa”  by two orders of magnitude under anaerobic or aerobic growth , a typical cell gains between 0.08 and 375 μmol of ATP from the reduction of stored nitrate. This yields at least four orders of magnitude more energy than calcite precipitation. No nitrate vacuoles have been observed in Achromatium populations. If calcite precipitation serves as an alternative energy generation scheme mediated by V-type ATPases and PPase in vacuolar membranes, calcite inclusions should accumulate when redox energy substrate availability is low. If calcite inclusions function as a reservoir of buffer to offset sulfuric acid production, then they should accumulate in parallel with S(0) inclusions. Given the spatially and temporally dynamic habitats favored by Achromatium populations, these two predictions are not mutually exclusive. A third hypothesis, that calcite serves as ballast to anchor Achromatium cells vulnerable to being swept away at the sediment water interface, is consistent with existing observations and should also be considered.Further work is required to resolve the striking and enigmatic calcite precipitation behavior of 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.


In this study, Wurtz and colleagues investigated to what extent elevated body mass index (BMI) within the normal weight range has causal influences on the detailed systemic metabolite profile in early adulthood using Mendelian randomization analysis.Please see later in the article for the Editors' Summary Increased adiposity is linked with higher risk for cardiometabolic diseases. We aimed to determine to what extent elevated body mass index (BMI) within the normal weight range has causal effects on the detailed systemic metabolite profile in early adulthood.2). Circulating metabolites were quantified by high-throughput nuclear magnetic resonance metabolomics and biochemical assays. In cross-sectional analyses, elevated BMI was adversely associated with cardiometabolic risk markers throughout the systemic metabolite profile, including lipoprotein subclasses, fatty acid composition, amino acids, inflammatory markers, and various hormones (p<0.0005 for 68 measures). Metabolite associations with BMI were generally stronger for men than for women . A gene score for predisposition to elevated BMI, composed of 32 established genetic correlates, was used as the instrument to assess causality. Causal effects of elevated BMI closely matched observational estimates , suggesting causative influences of adiposity on the levels of numerous metabolites (p<0.0005 for 24 measures), including lipoprotein lipid subclasses and particle size, branched-chain and aromatic amino acids, and inflammation-related glycoprotein acetyls. Causal analyses of certain metabolites and potential sex differences warrant stronger statistical power. Metabolite changes associated with change in BMI during 6 y of follow-up were examined for 1,488 individuals. Change in BMI was accompanied by widespread metabolite changes, which had an association pattern similar to that of the cross-sectional observations, yet with greater metabolic effects .We used Mendelian randomization to estimate causal effects of BMI on 82 metabolic measures in 12,664 adolescents and young adults from four population-based cohorts in Finland , individuals with higher BMI have an elevated risk of developing life-shortening cardiometabolic diseases—cardiovascular diseases that affect the heart and/or the blood vessels  and metabolic diseases that affect the cellular chemical reactions that sustain life . People become unhealthily fat by consuming food and drink that contains more energy  than they need for their daily activities. So adiposity can be prevented and reversed by eating less and exercising more.Adiposity—having excessive body fat—is a growing global threat to public health. Body mass index  is a coarse indicator of excess body weight, but the measure is useful in large population studies. Compared to people with a lean body weight  metabolic profile because individuals with high BMI may share other characteristics (confounding factors) that are the actual causes of both adiposity and metabolic abnormalities. Moreover, having a change in some aspect of metabolism could also lead to adiposity, rather than vice versa (reverse causation). Importantly, if there is a causal effect of adiposity on cardiometabolic risk factor levels, it might be possible to prevent the progression towards cardiometabolic diseases by weight loss. Here, the researchers use “Mendelian randomization” to examine whether increased BMI within the normal and overweight range is causally influencing the metabolic risk factors from many biological pathways during early adulthood. Because gene variants are inherited randomly, they are not prone to confounding and are free from reverse causation. Several gene variants are known to lead to modestly increased BMI. Thus, an investigation of the associations between these gene variants and risk factors across the systemic metabolite profile in a population of healthy individuals can indicate whether higher BMI is causally related to known and novel metabolic risk factors and higher cardiometabolic disease risk.2) living in Finland and the blood levels of 82 metabolites in these young individuals at a single time point. Statistical analysis of these data indicated that elevated BMI was adversely associated with numerous cardiometabolic risk factors. For example, elevated BMI was associated with raised levels of low-density lipoprotein, “bad” cholesterol that increases cardiovascular disease risk. Next, the researchers used a gene score for predisposition to increased BMI, composed of 32 gene variants correlated with increased BMI, as an “instrumental variable” to assess whether adiposity causes metabolite abnormalities. The effects on the systemic metabolite profile of a 1-kg/m2 increment in BMI due to genetic predisposition closely matched the effects of an observed 1-kg/m2 increment in adulthood BMI on the metabolic profile. That is, higher levels of adiposity had causal effects on the levels of numerous blood-based metabolic risk factors, including higher levels of low-density lipoprotein cholesterol and triglyceride-carrying lipoproteins, protein markers of chronic inflammation and adverse liver function, impaired insulin sensitivity, and elevated concentrations of several amino acids that have recently been linked with the risk for developing diabetes. Elevated BMI also causally led to lower levels of certain high-density lipoprotein lipids in the blood, a marker for the risk of future cardiovascular disease. Finally, an examination of the metabolic changes associated with changes in BMI in 1,488 young adults after a period of six years showed that those metabolic measures that were most strongly associated with BMI at a single time point likewise displayed the highest responsiveness to weight change over time.The researchers measured the BMI of 12,664 adolescents and young adults The World Health Organization provides information on Global Burden of Disease Study website provides the latest details about global obesity trendsThe obesity, cardiovascular disease, and type 2 diabetes The UK National Health Service Choices website provides information about American Heart Association provides information on all aspects of cardiovascular disease and diabetes and on keeping healthy; its website includes personal stories about heart attacks, stroke, and diabetesThe overweight and obesity and information about heart disease, stroke, and diabetesThe US Centers for Disease Control and Prevention has information on all aspects of heart disease, vascular disease, and obesity (in English and Spanish)MedlinePlus provides links to other sources of information on Mendelian randomization Wikipedia has a page on  To2 in the three cohorts of young adults reflects the population average in Finland The study comprised 12,664 adolescents and young adults from four general population cohorts who all had detailed metabolite profiles measured and information on gene score for predisposition to elevated BMI. In addition, 1,488 individuals also had metabolite profiling data at 6-y follow-up. Clinical characteristics of the four cohorts are shown in p<0.0005 in meta-analyses). Metabolite associations tended to be stronger for men than for women ; however, the association patterns were generally in the same direction, towards a more risk-prone systemic metabolite profile for those with higher BMI. Lipoprotein lipids displayed a characteristic association pattern with BMI: the most pronounced associations were observed for VLDL lipids, whereas associations with LDL lipids were weaker, albeit increased for small LDL lipid concentration. HDL lipids displayed heterogeneous associations, with strong inverse associations for large HDL lipid concentration and HDL particle size. Prominent direct associations with BMI were observed for monounsaturated and saturated fatty acids. The ratio of polyunsaturated fatty acids to total fatty acids was inversely associated with BMI. Fatty acid associations were about twice as strong for men as for women. Only weak associations were observed for glycolysis-related metabolites for both sexes in this young study population. In contrast, branched-chain and aromatic amino acids were strongly positively associated with BMI, with magnitudes comparable to those of total cholesterol and triglycerides. Further, sizeable associations were observed between BMI and metabolic risk factors such as markers of inflammation and liver function, as well as between BMI and adiposity-related hormones.Cross-sectional associations of BMI with 82 metabolic measures are illustrated in 2), as illustrated in 2) are also indicated in n = 3,676, aged 40–74 y) and the Pieksämäki Study , and were similar when adjusting for smoking status, alcohol intake, and physical activity index  and explained 1.3%–2.6% of the variation in BMI in the study populations  for any of the metabolic measures analyzed. The cross-sectional associations and causal effect estimates of each metabolite in absolute concentration units and with exact p-values are listed in Causal effect estimates of BMI on the 82 metabolic measures are shown by the orange bars in p<0.0005 (multiple testing corrected), and for a further 19 metabolic measures at p<0.05 . The strongest causal effect estimates were observed for VLDL and LDL lipids, branched-chain and aromatic amino acids, inflammatory markers, leptin, insulin, and blood pressure. Prominent inverse causal effects were found for large HDL lipid concentration, HDL particle size, and sex hormone–binding globulin.In terms of individual metabolic risk factors, the causal estimates were significant for 24 of the metabolic measures at R2 = 0.85 [95% CI 0.79–0.91]), as well as when omitting the widely studied FTO locus  or any other individual variant from the gene score (R2 = 0.86 [95% CI 0.81–0.91]). None of the metabolites were associated with the gene score when the model was adjusted for observed BMI (p<0.05 for interaction) for the causal estimates are listed in Causal effect estimates were similar when using an unweighted gene score as the instrument for Mendelian randomization analyses  with a slope of 1.60±0.05 . The longitudinal associations further replicated in the Pieksämäki cohort of 456 middle-aged adults with 6-y follow-up (R2 = 0.79 [95% CI 0.71–0.87]).Larger metabolic changes than expected based on the cross-sectional associations were observed for numerous lipoprotein lipid and cholesterol measures, fatty acids, and branched-chain amino acids, as well as inflammatory markers, adiponectin, and insulin. The magnitudes of the longitudinal associations in absolute concentration units are listed in Changes in the metabolite profile with weight loss and weight gain at 6-y follow-up are illustrated in In this study of 12,664 healthy adolescents and young adults, elevated BMI was associated with adverse effects on numerous known and novel risk markers for cardiovascular disease and type 2 diabetes throughout the systemic metabolite profile FTO locus on standard metabolic risk factors and cardiovascular disease The causal effects of adiposity across multiple metabolic measures corroborate prior Mendelian randomization studies, which have examined the role of the 2 increment in BMI were modest, the combined effects across the metabolite profile may have considerable implications. With the increasing trends in BMI worldwide, the adverse metabolic effects of adiposity observed in adolescents and young adults starting within the lean range of BMI may translate into direct consequences for cardiometabolic risk in the general population Despite the heritable component of adiposity, BMI is a modifiable risk factor. Changes in BMI were paralleled by changes throughout the metabolite profile  and 7, wOur study has both strengths and limitations. BMI is a heterogeneous marker of adiposity; however, it predicts the risk of related complications and is relevant for large population studies The ideal body weight that healthy adults should strive to attain remains controversial Figure S1Correlations of the assayed metabolic measures.(PDF)Click here for additional data file.Figure S2Metabolite concentrations as a function of BMI on a continuous scale in young women and men.(PDF)Click here for additional data file.Figure S3Correspondence between gene score associations and cross-sectional associations of metabolic measures when the gene score associations are adjusted for observed BMI.(PDF)Click here for additional data file.Figure S4Associations of metabolite changes with change in BMI at 6-y and 10-y follow-up in the Cardiovascular Risk in Young Finns Study, and at 6-y follow-up in the Pieksämäki Study.(PDF)Click here for additional data file.Table S1Mean (SD) metabolite concentrations in each cohort and conversion factors to absolute units.(PDF)Click here for additional data file.Table S2Correlations between the gene score for elevated BMI and potential confounders.(PDF)Click here for additional data file.Table S3Cross-sectional associations, causal effect estimates, and longitudinal associations of BMI with systemic metabolites in absolute concentration units.(PDF)Click here for additional data file.Table S4Suggestive sex interactions in causal effect estimates of BMI on metabolites.(PDF)Click here for additional data file.Table S5Metabolite changes paralleled by weight loss and weight gain during 6-y follow-up in absolute concentration units.(PDF)Click here for additional data file.Text S1Study populations.(PDF)Click here for additional data file.


Exposure of 4 days post fertilization Ntr larvae to the prodrug metronidazole (Met), which Ntr metabolizes into cytotoxic metabolites, resulted in dose-dependent cell death and axon degeneration. This was limited to the Ntr-expressing sensory neurons, as neighboring glia and motor axons were unaffected. Cell death was rapid, becoming apparent 3–4 hours after Met treatment, and was followed by phagocytosis of soma and axon debris by cells within the nerves and ganglia beginning at 4–5 hours of exposure. Although neutrophils appear to be activated in response to the degenerating neurons, they did not accumulate at the sites of degeneration. In contrast, macrophages were found to be attracted to the sites of the degenerating axons, where they phagocytosed debris. We demonstrated that peripheral glia are critical for both the phagocytosis and inflammatory response to degenerating neurons: mutants that lack all peripheral glia  exhibit a much reduced reaction to axonal degeneration, resulting in a dramatic decrease in the clearance of debris, and impaired macrophage recruitment. Overall, these results show that this zebrafish model of peripheral sensory axon degeneration exhibits many aspects common to peripheral neuropathies and that peripheral glia play an important role in the initial response to this process.Axon degeneration is a feature of many peripheral neuropathies. Understanding the organismal response to this degeneration may aid in identifying new therapeutic targets for treatment. Using a transgenic zebrafish line expressing a bacterial nitroreductase (Ntr)/mCherry fusion protein in the peripheral sensory neurons of the V, VII, IX, and X cranial nerves, we were able to induce and visualize the pathology of axon degeneration  Currently, millions of people suffer from peripheral neuropathies (PN) that arise from numerous causes, including chemotherapeutics, alcohol, toxins, viruses, physical trauma, genetics, and diabetes Axon degeneration is a feature common to many PNs in vivo visualization of cellular processes in real time; transgenic lines allow selective targeting of a specific cell type; and the availability of a drug-based suicide gene technology that permits conditional and selective ablation of targeted cell populations. This suicide system utilizes a combination of the bacterial enzyme nitroreductase (Ntr) and the prodrug metronidazole (Met) that has been used to model other diseases in zebrafish Axon degeneration has been studied in several animal models that use physical trauma  and all efforts were made to minimize the number of animals used and their suffering.Tg(isl:gfp)Tg(p2rx3.2(−4.0):gal4vp16)Tg(UAS:nsfB-mcherry)zdf10foxd3panther (gift from Steve Johnson) Tg(mpx:gfp)  gl22Tg) Tg(p2rx3.2(−4.0):gal4vp16);Tg(UAS:nsfB-mcherry) embryos/larvae, in addition to expression in sensory neurons, there was also variable ectopic expression between individuals within a single clutch. Therefore, larvae were put into separate groups based on Ntr expression, so that individuals with equivalent expression could be compared for a given experiment.All animal husbandry was carried out as described by Westerfield Larvae at 4 dpf were treated with metronidazole  at a final concentration of 1, 5, or 10 mM in fish water. We found 10 mM to give the maximum degree of ablation, so we used this concentration for our studies. Control fish were treated with fish water alone.Larvae were anesthetized with 0.02% tricaine in fish water and transferred to a 96-well plate. Epifluorescent images were taken using a Nikon TE200 inverted microscope equipped with a CoolSNAP HQ digital camera. MetaMorph software  was used to acquire and process images. Cropping and rotating of images was carried out using Adobe Photoshop. For time lapse imaging, larvae were embedded in 0.5% low-melting point agarose containing 0.02% tricaine, and images were taken every 5 minutes.Live embryos/larvae were anesthetized with tricaine (0.01%) and placed in low-melting point agarose (0.5–1.0%) wells present on specially designed tissue culture dishes: these were manufactured by creating a 10 mm hole in the bottom dish and then covering this hole by gluing a glass cover slip on the external surface of the dish. Imaging was performed using a 20x water immersion objective mounted on an Olympus FV-1000 MPE confocal microscope. All images were handled using either Olympus Fluoview or ImageJ software, with final adjustments to contrast and brightness made using Adobe Photoshop.Larvae at 4 or 5 dpf were anesthetized with 0.02% tricaine in fish water and heads were removed with a small scalpel under a dissecting microscope. Microwave assisted larval fixation and embedding were performed as described Larvae at 4 dpf were treated with control PTU water or 10 mM Met for the indicated times, then live larvae were incubated in 10 ug/mL acridine orange in fish water for 20 min in the dark at RT. Larvae were then rinsed three times with fish water, anesthetized with tricaine, and put in a 96-well plate for imaging Larvae at 4 dpf were treated with control PTU water or 10 mM Met for the indicated times, fixed in 4% paraformaldehyde for either 2 hr RT or overnight at 4°C, washed twice for 5 min in PBS, then stained with Sudan Black  for 20 min. Larvae were washed in 70% EtOH until non-specific staining disappears, then rehydrated with a series of EtOH dilutions in PBS plus 0.1% Tween 20 Larvae at 4 dpf were treated with control PTU water or 10 mM Met for the indicated times, stained by incubating in 2.5 ug/mL Neutral Red solution  in PTU water for 6–8 hr in the dark, then rinsed with PTU water and kept in PTU water ON to wash out background staining Tg(mpx:gfp)/Tg(p2rx3.2(−4.0):gal4vp16/UAS:nsfB-mcherry) larvae treated with control PTU water or 10 mM Met were taken every 30 sec for 30 min. Together, these videos spanned a treatment time of 6 hr. The ImageJ plugin MTrackJ Multiple time lapse videos of 4 dpf Sudan Black and Neutral Red stained larvae were embedded on their side in 1% low melt point agarose on an imaging plate and the focus was adjusted to obtain serial images from consecutive planes in the larvae. Using Adobe Photoshop, stained cells were marked in each image and the markings were overlaid to ensure that each cell was only counted once. Marked cells were counted manually in a region that measured a half ear-length from all sides of the ear. Students t test was used to compare means.p2rx3.2:gal4vp16;UAS:nsfB-mcherry) (from here on abbreviated as Ntr) coli nitroreductase (nsfB) protein, fused to mCherry, in the peripheral sensory neurons contributing to the V, VII, IX, and X cranial nerves. It is important to note that due to the variegated expression pattern, only a subpopulation of sensory neurons are targeted. This lack of complete ablation mimics many PNs in which only a subpopulation of sensory axons is affected We have developed a zebrafish model of peripheral sensory axon degeneration, exhibiting characteristics similar to those seen in many sensory PNs, utilizing the nitroreductase/metronidazole (Ntr/Met) suicide system + axons after overnight exposure, demonstrating the high efficiency of this drug in ablating peripheral sensory neurons expressing the Ntr protein )Tg(islet:gfp);Ntr larvae were treated with 10 mM Met overnight, the Ntr-expressing sensory axons appeared punctate, red motile cells were seen in the surrounding tissue, and the ganglia showed signs of degeneration , we crossed Ntr fish to a transgenic line that expresses GFP in branchiomotor neurons ) was used to label dead and dying cells Together, these experiments confirmed that selective ablation of peripheral sensory neurons and their axons could be visualized in real time using the Ntr/Met system in zebrafish.Epifluorescence imaging and AO staining provided strong evidence that axon degeneration and cell death were the result of Met treatment in Ntr larvae. However, these techniques are indirect indicators. Therefore, we wanted to directly verify that these processes did occur by examining tissues at an ultrastructural level using transmission electron microscopy (TEM). 4 dpf Ntr larvae were treated with fish water or 10 mM Met for 4 or 18 hours, then fixed and processed for TEM. Transverse sections of larvae were taken posterior to the ear  and staiTEM images of the transition zone showed a cross section of the vagal nerve bundle in untreated control , 4 h MetTEM images of the vagal ganglia allowed detection of both neuron and satellite glia cell bodies . In contIn addition to the changes in neuronal integrity, the TEM images also revealed phagocytic cells wrapped around presumed dead or dying neurons (electron-dense cell bodies) in the ganglia of Met-treated Ntr fish ) One drawback of SB staining is that it only provides a static snapshot of the number and positions of neutrophils at any given time. To determine if neutrophils were transiently moving through the region of the ganglia and axons, we sought to carry out time-lapse videos of neutrophil movement in response to degenerating axons. To visualize neutrophils, we crossed Ntr fish to a transgenic line that expresses GFP under the control of the promoter of the myeloperoxidase gene gl22Tg ). Overnight treatment with 10 mM Met resulted in the appearance of mCherry-labeled motile cells in both the ganglia and nerve, and confocal imaging revealed that these were co-labeled with GFP  protein panther;Ntr larvae were treated with Met, time lapse imaging showed fluorescent puncta beginning to form at 2–3 hours along the axons and in the plexus . Together, the mpeg:gfp imaging, the NR staining and panther; Ntr data indicated that macrophage recruitment is one response to axon injury in this model.To verify that macrophages were recruited in response to axon degeneration, we examined Met-induced degeneration in a mutant with abnormal macrophage behavior, e plexus , similare plexus ’. Howevepanther;Ntr), Met-treatment still resulted in the appearance of puncta and the degenerating axons/neurons gradually lost their fluorescence  for our experiments. A caveat in the use of this mutant line is that the description of the foxD3 phenotype is based on the absence of transcription factor mRNAs in peripheral glia −/−foxD3; Ntr , indicating that the absence of peripheral glia was not interfering with Met-induced neuronal cell death. Indeed, it appeared that cell death in the mutants occurred to a greater degree, as there were a higher number of dead and dying cells in Met-treated mutants than in Met-treated wild type clutch mates.To determine if this delayed appearance of red motile cells and lack of puncta was perhaps due to postponed or decreased neuronal cell death in the mutants, AO staining was performed on 4 dpf  ganglia . Similar−/−;foxD3 Ntr larvae were treated with 10 mM Met for 4 hours or left untreated, then fixed and processed for TEM. In a nerve cross section of a 4 hour Met-treated −/−;foxD3 Ntr larva   . These fFrom our TEM analysis, we found that mutant larvae had damaged somas and axons with poor membrane integrity after a 4 hour Met treatment; however, in time-lapse videos of these mutants, engulfment of cell debris did not appear until 8–10 hours into Met treatment. In Met-treated wild type clutch mates, damage was apparent by 4 hours and this was concurrent with the appearance of phagocytic cells. These findings suggest that satellite and ensheathing glia are necessary for debris phagocytosis.−/−;foxD3 Ntr larvae to examine macrophage recruitment, and again there was no significant difference in NR stained cells after 2.5 hr between untreated  and Met treated mutants , or 18 hr between untreated  or Met treated mutants . These findings were surprising given the fact that time lapse videos of Met-treated −/−;foxD3 Ntr showed that motile red cells were eventually recruited  and 4 hr-Met treated mutants  (data not shown), indicating that the number of neutrophils did not increase. We also carried out NR staining on ecruited . This deecruited . TherefoDegeneration is a hallmark feature of most PNs. These disorders have a variety of causes and symptoms, affect millions of people, and have no cures. Insults leading to a PN can arise in any type of nerve, can include both myelinated and unmyelinated axons, and often result in axon degeneration and death of the neuron To investigate unmyelinated axon degeneration, we generated a novel zebrafish model. The advantages of this model are that degeneration is targeted specifically to the peripheral sensory neurons, it is conditional, it can be visualized in vivo, the affected nerves are unmyelinated, and many aspects of the organismal response to this degeneration mimic what is seen in the clinical setting. We also demonstrate that the initial response to neuron degeneration is dependent upon the peripheral non-myelinating glia that ensheath both the axons and the cell bodies.While the initial insults that lead to PN in humans are varied, common outcomes include cell death, DNA damage, disruption of microtubules, defects in proteins required for fission and fusion of mitochondria, high levels of reactive oxygen species (ROS) causing oxidative stress, and induction of an inflammatory response Neuronal degeneration and death via apoptosis in human PNs is linked to an increase in neuronal ROS concentrations and mitochondrial dysfunction e.g., chemotherapy-induced sensory PNs Vulnerability of select axons is seen in many human PNs, where the sensory neurons are affected while the motor neurons intact remain intact; Met treatment resulted in neuronal death slightly before, or contemporaneously with, axonal degeneration. Axon damage first appeared as a loss of definition of individual axons within a nerve, followed by the appearance of puncta (phagocytosed debris) and recruitment of immune cells. This is similar to what has been reported for degeneration in other animal models of PN. However, we did not observe a significant increase in the number of neutrophils responding to axon degeneration, which is in contrast to studies in rats and mice that have shown significant neutrophil recruitment in dorsal root ganglia after sciatic chronic constriction injury Neutrophils are not the only immune cell that can be recruited to damaged or dying axon/neurons. Our data showed that macrophages were recruited to degenerating axons, which agrees with the well-established characteristics of Wallerian degeneration and the recruitment of macrophages in models using nerve transection, ligation, and crush + mutants lacking peripheral glia (foxd3−/−) were exposed to Met, no discernible puncta were observed along the axons during treatment, and the appearance of immune cells was dramatically delayed. This was not due to decreased degeneration of sensory neurons/axons, and in fact the opposite occurred: axon degeneration advanced more rapidly in the −/−;foxD3Ntr than in wild type. TEM analysis revealed that axons in mutants treated with Met for only 4 hours contained the large vacuoles and broken membranes that more closely resembled axons from wild types treated overnight with Met. Also, dark puncta located in the axons of Met-treated wild type larvae were not seen in mutants, suggesting that these inclusions were formed due to the presence of peripheral glia. In the ganglia of mutants exposed to Met for 4 hours, degenerating cell bodies had more apparent membrane damage and nuclear defects than equivalently-treated wild type larvae.When Ntr−/−foxd3 larvae, recruitment of immune cells still occurred, although considerably delayed. In wild type larvae, immune cells were recruited starting at 4–5 hours of Met treatment. In the mutants, this initial response has been delayed to more than 8–10 hours of Met exposure. In addition, the response in the mutants is less robust than that seen in their wild-type clutch mates. Since the red motile cells seen in both −/−foxd3 mutants and wild types had similar sizes and movements, such as the speed they migrated and the extension of philopodia, it is assumed that they are macrophages and/or microglia. In addition, no phagocytic cells were detected in TEM images in the ganglia of Met-treated mutants, as was seen in Met-treated wild types. This suggests that these non-myelinating peripheral glial cells have a pivotal role in immune cell recruitment.While puncta were not observed in the axons of treated −/−sox10 zebrafish mutants that lack those glia These findings are supported by previous studies demonstrating that non-myelinating glia express cytokines and chemokines that are known to recruit immune cells in a capsaicin-induced degeneration model One of the major differences between our study and those carried out in mammals is that the nerves we targeted contain only unmyelinated axons. Studies have shown that myelin plays a significant role in immune cell recruitment and in hindering regeneration of axons. Degrading myelin expresses complement C3, which is important in opsonization and phagocytosis of myelin by macrophages −/−;foxD3 Ntr mutant that lacks peripheral glia, we highlight the importance of non-myelinating satellite and ensheathing glia in initiating the inflammatory response and in phagocytosing cell debris. Without satellite and ensheathing glia, neutrophil and macrophage recruitment is postponed, causing a delay in the clearance of harmful debris. This debris can potentially prevent successful regeneration and functional recovery of affected neurons.In this paper, we describe a new model to investigate drug-induced peripheral sensory axon degeneration and hypothesized that non-myelinating glia have a crucial role in the inflammatory response to axon degeneration and phagocytosis of debris. We approached this using the Ntr/Met system to ablate peripheral sensory neurons and assess the inflammatory response, cell death, and the ultrastructure of degenerating axons and cell bodies in both wild type and mutant larvae lacking peripheral glia. Using this method, we found that administering Met caused axon degeneration and neuronal cell death in Ntr-expressing cells, and also elicited an inflammatory response. Using the Some of our findings involving the inflammatory response differed from other studies, such as the lack of significant neutrophil contribution Figure S1Complete ablation of Ntr-expressing peripheral sensory neurons after Met treatment. 4 dpf Ntr larvae treated with A) PTU water (control) or B) 10 mM Met for 18 hours. Confocal microscopy at 5 dpf revealed that vagal nerve axons  remain intact in control animals, but lose all structural integrity when treated overnight with Met (B). Anterior to left, dorsal at top.(TIF)Click here for additional data file.Figure S2Met treatment increases mitochondrial damage in axons. TEM images show examples of healthy (A) and unhealthy (B) mitochondria (arrowheads) present in EM sections of control or treated vagal nerve bundles. Mitochondria were scored and the percentage of mitochondria found as healthy or unhealthy is shown in panel C .(TIF)Click here for additional data file.Figure S3Neutrophil response to degenerating neurons in 4dpf Ntr larvae. A) Sudan Black staining of Ntr treated with fish water (control) or 10 mM Met for 4 hr, (neutrophils labeled with arrowheads). B) SB-stained cell counts in larvae treated with control or 10 mM Met for various times. Average number of SB-stained cells/fish per treatment group shown for incubation times of 3 h , 4 h , 5 h , 6 h , and 18 h . None were statistically significant. C) Neutrophil movement in Mpx:gfp;Ntr larvae treated with fish water or 10 mM Met. Graph shows average velocity (pixels/sec) of neutrophils over time: 0–0.5 h , 0.5–1 h , 1–1.5 h , 1.5–2 h , 2–2.5 h , 2.5–3 h , 3–3.5 h , 3.5–4 h , 4–4.5 h , 4.5–5 h , 5–5.5 h , and 5.5–6 h . Anterior is to the left; dorsal is at the top in all panels. Eye and otic vesicle (OV) are outlined.(TIF)Click here for additional data file.Figure S4TEM images of posterior lateral line nerve in wild type and −/−;foxD3Ntr larvae. A) Cross section of the posterior lateral line nerve (pLL) in 4 dpf wild type Ntr  shows axons surrounded by myelin (arrowheads). B) pLL in 4 dpf −/−;foxD3Ntr  shows axons with adjacent axonal membranes (arrowheads). No myelin is seen.(TIF)Click here for additional data file.Figure S5Loss of foxD3 does not result in an impaired immune response. Tail transections were performed on 4 dpf −/−;mpxfoxD3:gfp or WT clutch mates. Larvae were anesthetized with tricaine and tails were transected with a scalpel at the junction of the body and tail fin. 2 hr after the tail transection, larvae were either imaged for GFP+ cells  or stained with NR to visualize macrophages. A) Uncut tail of WT clutch mate shows the location of the tail transection . A’) Transection site of WT clutch mate showed an increase in neutrophils at the location of the cut. B) Uncut tail of −/−;mpxfoxD3:gfp shows location of tail transection. B’) Transected tail of −/−;mpxfoxD3:gfp also showed an increase in neutrophils at the location of the cut (arrow), similar to that seen in WT clutch mates. C) Neutral Red staining in uncut tail of WT clutch mate. C’) NR staining at transection of WT clutch mate (arrow) showed an increase in macrophages at the location of the cut. D) NR staining at the transection site in a −/−;mpxfoxD3:gfp larva. D’) NR staining of transection site in a −/−;mpxfoxD3:gfp showed an increase in macrophages at the location of the transection (arrow), similar to that seen in WT clutch mates.(TIF)Click here for additional data file.Movie S1Timelapse video of Met-treated Ntr larva. 4 dpf Ntr larva treated with 10 mM Met for 18 hours. Images taken every 12 min. Anterior to left, dorsal at top.(MP4)Click here for additional data file.Movie S2Timelapse video of untreated mpx:gfp;Ntr larva. 4 dpf mpx:gfp;Ntr treated with fish water. Images taken every 30 sec. Video spans a time frame of 30 min taken 4 hours after addition of fish water. Anterior to left, dorsal at top.(MP4)Click here for additional data file.Movie S3Timelapse video of Met-treated mpx:gfp;Ntr larva. 4 dpf mpx;gfp;Ntr treated with 10 mM Met. Images taken every 30 sec. Video spans a time frame of 30 min taken 1.5 hours after the start of Met treatment. Anterior to left, dorsal at top.(MP4)Click here for additional data file.Movie S4Timelapse video of Met-treated panther;Ntr larva. 4 dpf panther;Ntr larva treated with 10 mM Met for 18 hours. Images taken every 5 min. Anterior to left, dorsal at top.(MP4)Click here for additional data file.Movie S5Timelapse video of Met-treated −/−;foxD3Ntr larva. 4 dpf −/−;foxD3Ntr larva treated with 10 mM Met for 23 hours. Images taken every 5 min. Anterior to left, dorsal at top.(MP4)Click here for additional data file.


Transcytosis of proteins and hydrodynamic flow of cytoplasm is a major mechanism to sustain physiology in all cells, observable from gametes  to maturde novo synthesized NO by neuronal nitric oxide synthase (nNOS) to maintain a pool of glucokinase in association with insulin secretory granules  synthesis within the enteric nerve terminals in response to an action potential –15 and dgranules . Glucoki release , 27, and release . Incipiefunction .en passant nerve terminal junctions in the gastrointestinal tract, inhibitory neurotransmission involves release of vesicular ATP and instantaneously synthesized gaseous NO  in the N-terminal portion of the tail region of myosin Va that facilitates cargo binding . For exal-arginine transporter SLC7A3, which may result in defective NO synthesis. The hypersecretor phenotype seen in the early stages of DBA/2J mice (In DBA/2J mice, prejunctional nitrergic synthesis during enteric nerve-smooth muscle neurotransmission has been demonstrated to be significantly reduced , 12. It /2J mice  may repr/2J mice –46. It iMyosin Va has been shown to facilitate both the first phase of insulin release, as well as during sustained phase when storage pool vesicles are recruited to a readily releasable pool in a non-linear dynamics , 46. ThiThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.