Intestinal failure is an uncommon but devastating condition whose natural history has dramatically improved over the past two decades. Infants with intestinal failure due to severe short bowel syndrome or other diagnoses previously considered incompatible with life are now routinely being saved and cared for in cutting edge, multidisciplinary programs. Enteral nutrition plays a central role in the management of children with intestinal failure. This review provides an overview of enteral nutrition in pediatric intestinal failure, with specific emphasis on recent advances in clinical management, patient outcomes, and emerging therapies.
Elevations in serum anti-flagellin and anti-LPS Igs are related to growth faltering in young Tanzanian children
BACKGROUND: Antibodies to LPS and flagellin have been described as indirect measures of increased gastrointestinal permeability and may be markers of environmental enteric dysfunction (EED), which is a condition associated with poor child growth.
OBJECTIVE: We assessed whether LPS- and flagellin-specific immunoglobulin (Ig) concentrations were associated with poor growth in young Tanzanian children at risk of EED. DESIGN: Blood samples were obtained from 590 childre
RESULTS: Anti-LPS and anti-flagellin IgA and IgG concentrations increased over the first year of life and were higher than concentrations (measured at 9 mo of age) in healthy controls. Children with anti-flagellin IgA, anti-LPS IgA, anti-flagellin IgG, and anti-LPS IgG concentrations in the highest quartile at 6 wk of age were 2.02 (95% CI: 1.11, 3.67), 1.84 (95% CI: 1.03, 3.27), 1.94 (95% CI: 1.04, 3.62), and 2.31 (95% CI: 1.25, 4.27) times, respectively, more likely to become underweight (weight-for-age z score < -2) after adjustment for covariates (P-trend < 0.05) than were children with Ig concentrations in the lowest quartile. Children with increased concentrations of anti-flagellin IgA were also more likely to become wasted; however, there was no association between any of the markers and subsequent stunting.
CONCLUSION: Serologic measures of increased intestinal permeability to bacterial components are associated with subsequent poor growth and could help identify children who may benefit most from preventive interventions. This trial was registered at clinicaltrials.gov as NCT00421668.
OBJECTIVE: Diarrheal diseases are a leading cause of morbidity and mortality worldwide, but the etiology of diarrhea and its relation to nutritional outcomes in resource-limited settings is poorly defined. We sought to determine the etiology of community-acquired diarrhea in Tanzanian infants and to assess the association with anthropometrics and novel intestinal biomarkers.
METHODS: A convenience sample of infants in a trial of zinc and/or multivitamin supplementation in Tanzania was selected. Subjects were enrolled at age 6 weeks and studied for 18 months. Stool samples were obtained from children with acute diarrhea. A novel, polymerase chain reaction-based TaqMan array was used to screen stool for 15 enteropathogens. A subset of subjects had serum gastrointestinal biomarkers measured.
RESULTS: One hundred twenty-three subjects with diarrhea were enrolled. The mean +/- SD age at stool sample collection was 12.4 +/- 3.9 months. Thirty-five enteropathogens were identified in 34 (27.6%) subjects: 11 rotavirus, 9 Cryptosporidium spp, 7 Shigella spp, 3 Campylobacter jejuni/coli, 3 heat stable-enterotoxigenic Escherichia coli, and 2 enteropathogenic E coli. Subjects with any identified enteropathogen had significantly lower weight-for-length z scores (-0.55 +/- 1.10 vs 0.03 +/- 1.30, P = 0.03) at the final clinic visit than those without an identified pathogen. Fifty of the 123 subjects (40.7%) had serum analyzed for antibodies to lipopolysaccharide (LPS) and flagellin. Subjects with any identified enteropathogen had lower immunoglobulin (IgA) antibodies to LPS (0.75 +/- 0.27 vs 1.13 +/- 0.77, P = 0.01) and flagellin (0.52 +/- 0.16 vs 0.73 +/- 0.47, P = 0.02) than those without an identified pathogen.
CONCLUSIONS: This quantitative polymerase chain reaction method may allow identification of enteropathogens that place children at higher risk for suboptimal growth. IgA anti-LPS and flagellin antibodies hold promise as emerging intestinal biomarkers.
A Model to Explain How the Bacille Calmette Guerin (BCG) Vaccine Drives Interleukin-12 Production in Neonates
The Bacille Calmette Guerin (BCG) vaccine is the only routine vaccination at birth that effectively induces neonatal T-helper 1 (Th1)-polarized immune responses. The primary cytokine that drives CD4+ T-cell Th1 differentiation is interleukin (IL)-12 p70, a heterodimeric cytokine composed of the IL-12 p35 and IL-12 p40 subunits. We therefore examined the mechanisms involved in BCG vaccine stimulation of IL-12 p35 and p40 production from human umbilical cord (neonatal) cells. We found that BCG bacilli did not upregulate IL-12 p35 mRNA production, but upregulated IL-12 p40 mRNA production in a Toll-like receptor (TLR)2-dependent manner, in human neonatal monocyte-derived dendritic cells (mdDCs). The combination of TLR2 signaling, Type I interferon (IFN), and Type II IFN induced maximal levels of IL-12 p35 and p40 mRNA production in human neonatal mdDCs. The cell-free supernatants of reconstituted BCG vaccine vials contained extracellular mycobacterial (BCG) DNA which could induce IFN-alpha (Type I IFN) production in human neonatal plasmacytoid dendritic cells (pDCs). BCG bacilli also stimulated human neonatal CD16lo natural killer (NK) cells to produce IFN-gamma (Type II IFN) in a TLR2-dependent manner. We have therefore proposed a model where BCG vaccine could stimulate the combination of neonatal conventional DCs (cDCs), pDCs, and CD16lo NK cells to produce optimal neonatal IL-12 p35 and p40 (IL-12 p70) production and subsequent CD4+ T-cell Th1 polarization. An adjuvant that emulates the mechanism by which the BCG vaccine stimulates neonatal IL-12 p35 and p40 production could improve vaccine strategies at birth for protection against intracellular pathogens and toxins.
The 18th annual international Targeted Therapies meeting brought together over 100 leading scientists and clinicians from around the world in the field of rheumatology. During the meeting, breakout sessions were held consisting of 5 disease-specific groups each with 20-40 experts assigned to each group based on clinical or scientific expertise. Specific groups included: rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis/spondyloarthritis, systemic lupus erythematous, and other connective tissue diseases (e.g. Sjogren's, Behcet's, others). In each group, experts were asked to identify unmet needs in 3 categorical areas: basic/translational science, clinical science and therapeutic development, and clinical care. Needs were prioritised as primary or secondary. Overall, similar primary unmet needs were identified within each disease foci. Within translational science, these included the need for better understanding the heterogeneity within each disease, such that predictive tools for therapeutic response could be developed. Within clinical science and therapeutic trials, the ability to prevent progression to disease onset in those at risk, and the ability to cure disease were identified. A further unmet need was to develop new and accessible therapeutics, as well as to conduct strategic trials of currently approved therapies. Within the clinical care realm, improved co-morbidity management and patient-centered care were identified as unmet needs. Lastly, it was strongly felt there was a need to develop a scientific infrastructure for well-characterised, longitudinal cohorts married with biobanks and mechanisms to support data-sharing. This infrastructure could facilitate many of the unmet needs identified within each disease area.
PD-L1 expression in EBV-negative diffuse large B-cell lymphoma: clinicopathologic features and prognostic implications
Programmed cell death ligand 1 (PD-L1) is a cell surface glycoprotein that regulates the cellular immune response and serves as a targetable immune checkpoint molecule. PD-L1 is expressed on tumor cells and the immune microenvironment of several human malignancies, including a subset of aggressive lymphomas. We sought to investigate further the clinical and pathologic features of EBV-negative diffuse large B-cell lymphoma (DLBCL) cases that express PD-L1. Immunohistochemical staining using an anti-PD-L1 monoclonal antibody was performed on DLBCL cases from 86 patients. These patients received standard chemotherapy treatment and were followed for up to 175 months. Overall, 14 cases (16%) were considered positive for PD-L1 in tumor cells. In comparison with PD-L1 negative cases, PD-L1 positive cases had a higher rate of non-GCB type (71% vs. 30%, P=0.0060), and higher Ann Arbor stage (II-IV) (100% vs. 73%, P=0.0327). No significant differences were seen in the immunohistochemical expression of BCL2, MYC, or Ki67. Patients with tumors expressing PD-L1 demonstrated inferior overall survival (OS) upon long term follow up (P=0.0447). Both age/sex-adjusted and multivariate analyses identified PD-L1 as an independent predictor for OS (P=0.0101 and P=0.0424). There was no significant difference, however, in terms of remission rates after first treatment, relapse rates, and progression free survival between the groups. Identification of DLBCL cases that express PD-L1 may serve to select a subset of patients that could further benefit from targeted immunotherapy.
BACKGROUND: Numerous human genes encode potentially active DNA transposases or recombinases, but our understanding of their functions remains limited due to shortage of methods to profile their activities on endogenous genomic substrates.
RESULTS: To enable functional analysis of human transposase-derived genes, we combined forward chemical genetic hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) screening with massively parallel paired-end DNA sequencing and structural variant genome assembly and analysis. Here, we report the HPRT1 mutational spectrum induced by the human transposase PGBD5, including PGBD5-specific signal sequences (PSS) that serve as potential genomic rearrangement substrates.
CONCLUSIONS: The discovered PSS motifs and high-throughput forward chemical genomic screening approach should prove useful for the elucidation of endogenous genome remodeling activities of PGBD5 and other domesticated human DNA transposases and recombinases.
Insulin-like Signaling Promotes Glial Phagocytic Clearance of Degenerating Axons through Regulation of Draper
Neuronal injury triggers robust responses from glial cells, including altered gene expression and enhanced phagocytic activity to ensure prompt removal of damaged neurons. The molecular underpinnings of glial responses to trauma remain unclear. Here, we find that the evolutionarily conserved insulin-like signaling (ILS) pathway promotes glial phagocytic clearance of degenerating axons in adult Drosophila. We find that the insulin-like receptor (InR) and downstream effector Akt1 are acutely activated in local ensheathing glia after axotomy and are required for proper clearance of axonal debris. InR/Akt1 activity, it is also essential for injury-induced activation of STAT92E and its transcriptional target draper, which encodes a conserved receptor essential for glial engulfment of degenerating axons. Increasing Draper levels in adult glia partially rescues delayed clearance of severed axons in glial InR-inhibited flies. We propose that ILS functions as a key post-injury communication relay to activate glial responses, including phagocytic activity.
External validation of the DHAKA score and comparison with the current IMCI algorithm for the assessment of dehydration in children with diarrhoea: a prospective cohort study
BACKGROUND: Dehydration due to diarrhoea is a leading cause of child death worldwide, yet no clinical tools for assessing dehydration have been validated in resource-limited settings. The Dehydration: Assessing Kids Accurately (DHAKA) score was derived for assessing dehydration in children with diarrhoea in a low-income country setting. In this study, we aimed to externally validate the DHAKA score in a new population of children and compare its accuracy and reliability to the current Integrated Management of Childhood Illness (IMCI) algorithm.
METHODS: DHAKA was a prospective cohort study done in children younger than 60 months presenting to the International Centre for Diarrhoeal Disease Research, Bangladesh, with acute diarrhoea (defined by WHO as three or more loose stools per day for less than 14 days). Local nurses assessed children and classified their dehydration status using both the DHAKA score and the IMCI algorithm. Serial weights were obtained and dehydration status was established by percentage weight change with rehydration. We did regression analyses to validate the DHAKA score and compared the accuracy and reliability of the DHAKA score and IMCI algorithm with receiver operator characteristic (ROC) curves and the weighted kappa statistic. This study was registered with ClinicalTrials.gov, number NCT02007733.
FINDINGS: Between March 22, 2015, and May 15, 2015, 496 patients were included in our primary analyses. On the basis of our criterion standard, 242 (49%) of 496 children had no dehydration, 184 (37%) of 496 had some dehydration, and 70 (14%) of 496 had severe dehydration. In multivariable regression analyses, each 1-point increase in the DHAKA score predicted an increase of 0.6% in the percentage dehydration of the child and increased the odds of both some and severe dehydration by a factor of 1.4. Both the accuracy and reliability of the DHAKA score were significantly greater than those of the IMCI algorithm.
INTERPRETATION: The DHAKA score is the first clinical tool for assessing dehydration in children with acute diarrhoea to be externally validated in a low-income country. Further validation studies in a diverse range of settings and paediatric populations are warranted.
FUNDING: National Institutes of Health Fogarty International Center.
BACKGROUND: Early recognition and treatment of circulatory volume loss is essential in the clinical management of dengue viral infection. We hypothesized that a novel computational algorithm, originally developed for noninvasive monitoring of blood loss in combat casualties, could: (1) indicate the central volume status of children with dengue during the early stages of "shock"; and (2) track fluid resuscitation status.
METHODS: Continuous noninvasive photoplethysmographic waveforms were collected over a 5-month period from three children of Thai ethnicity with clinical suspicion of dengue. Waveform data were processed by the algorithm to calculate each child's Compensatory Reserve Index, where 1 represents supine normovolemia and 0 represents the circulatory volume at which hemodynamic decompensation occurs. Values between 1 and 0 indicate the proportion of reserve remaining before hemodynamic decompensation.
RESULTS: This case report describes a 7-year-old Thai boy, another 7-year-old Thai boy, and a 9-year-old Thai boy who exhibited signs and symptoms of dengue shock syndrome; all the children had secondary dengue virus infections, documented by serology and reverse transcriptase polymerase chain reaction. The three boys experienced substantial plasma leakage demonstrated by pleural effusion index > 25, ascites, and > 20 % hemoconcentration. They received fluid administered intravenously; one received a blood transfusion. All three boys showed a significantly low initial Compensatory Reserve Index ( > /=0.20), indicating a clinical diagnosis of "near shock". Following 5 days with fluid resuscitation treatment, their Compensatory Reserve Index increased towards "normovolemia" (that is, Compensatory Reserve Index > 0.75).
CONCLUSIONS: The results from these cases demonstrate a new variation in the diagnostic capability to manage patients with dengue shock syndrome. The findings shed new light on a method that can avoid possible adverse effects of shock by noninvasive measurement of a patient's compensatory reserve rather than standard vital signs or invasive diagnostic methods.
Nutrient deprivation strategies have been proposed as an adjuvant therapy for cancer cells due to their increased metabolic demand. We examined the specific inhibitory effects of amino acid deprivation on the metastatic phenotypes of the human triple-negative breast cancer (TNBC) cell lines MDA-MB-231 and Hs 578T, as well as the orthotopic 4T1 mouse TNBC tumor model. Among the 10 essential amino acids tested, methionine deprivation elicited the strongest inhibitory effects on the migration and invasion of these cancer cells. Methionine deprivation reduced the phosphorylation of focal adhesion kinase, as well as the activity and mRNA expression of matrix metalloproteinases MMP-2 and MMP-9, two major markers of metastasis, while increasing the mRNA expression of tissue inhibitor of metalloproteinase 1 in MDA-MB-231 cells. Furthermore, methionine restriction downregulated the metastasis-related factor urokinase plasminogen activatior and upregulated plasminogen activator inhibitor 1 mRNA expression. Animals on the methionine-deprived diet showed lower lung metastasis rates compared to mice on the control diet. Taken together, these results suggest that methionine restriction could provide a potential nutritional strategy for more effective cancer therapy.
Adoption of a streamlined version of the bacterial clustered regular interspersed short palindromic repeat (CRISPR)/Cas9 defense system has accelerated targeted genome engineering. The Streptococcus pyogenes Cas9 protein, directed by a simplified, CRISPR-like single-guide RNA, catalyzes a double-stranded DNA break at a specific genomic site; subsequent repair by end joining can introduce mutagenic insertions or deletions, while repair by homologous recombination using an exogenous DNA template can incorporate new sequences at the target locus. However, the efficiency of Cas9-directed mutagenesis is low in Drosophila melanogaster Here, we describe a strategy that reduces the time and effort required to identify flies with targeted genomic changes. The strategy uses editing of the white gene, evidenced by altered eye color, to predict successful editing of an unrelated gene-of-interest. The red eyes of wild-type flies are readily distinguished from white-eyed (end-joining-mediated loss of White function) or brown-eyed (recombination-mediated conversion to the whitecoffee allele) mutant flies. When single injected G0 flies produce individual G1 broods, flies carrying edits at a gene-of-interest were readily found in broods in which all G1 offspring carried white mutations. Thus, visual assessment of eye color substitutes for wholesale PCR screening of large numbers of G1 offspring. We find that end-joining-mediated mutations often show signatures of microhomology-mediated repair and that recombination-based mutations frequently involve donor plasmid integration at the target locus. Finally, we show that gap repair induced by two guide RNAs more reliably converts the intervening target sequence, whereas the use of Lig4169 mutants to suppress end joining does not improve recombination efficacy.
Prevention of sexual transmission of Ebola in Liberia through a national semen testing and counselling programme for survivors: an analysis of Ebola virus RNA results and behavioural data
BACKGROUND: Ebola virus has been detected in semen of Ebola virus disease survivors after recovery. Liberia's Men's Health Screening Program (MHSP) offers Ebola virus disease survivors semen testing for Ebola virus. We present preliminary results and behavioural outcomes from the first national semen testing programme for Ebola virus.
METHODS: The MHSP operates out of three locations in Liberia: Redemption Hospital in Montserrado County, Phebe Hospital in Bong County, and Tellewoyan Hospital in Lofa County. Men aged 15 years and older who had an Ebola treatment unit discharge certificate are eligible for inclusion. Participants' semen samples were tested for Ebola virus RNA by real-time RT-PCR and participants received counselling on safe sexual practices. Participants graduated after receiving two consecutive negative semen tests. Counsellors collected information on sociodemographics and sexual behaviours using questionnaires administered at enrolment, follow up, and graduation visits. Because the programme is ongoing, data analysis was restricted to data obtained from July 7, 2015, to May 6, 2016.
FINDINGS: As of May 6, 2016, 466 Ebola virus disease survivors had enrolled in the programme; real-time RT-PCR results were available from 429 participants. 38 participants (9%) produced at least one semen specimen that tested positive for Ebola virus RNA. Of these, 24 (63%) provided semen specimens that tested positive 12 months or longer after Ebola virus disease recovery. The longest interval between discharge from an Ebola treatment unit and collection of a positive semen sample was 565 days. Among participants who enrolled and provided specimens more than 90 days since their Ebola treatment unit discharge, men older than 40 years were more likely to have a semen sample test positive than were men aged 40 years or younger (p=0.0004). 84 (74%) of 113 participants who reported not using a condom at enrolment reported using condoms at their first follow-up visit (p < 0.0001). 176 (46%) of 385 participants who reported being sexually active at enrolment reported abstinence at their follow-up visit (p < 0.0001).
INTERPRETATION: Duration of detection of Ebola virus RNA by real-time RT-PCR varies by individual and might be associated with age. By combining behavioural counselling and laboratory testing, the Men's Health Screening Program helps male Ebola virus disease survivors understand their individual risk and take appropriate measures to protect their sexual partners.
FUNDING: World Health Organization and the US Centers for Disease Control and Prevention.
BACKGROUND: Cells respond to numerous internal and external stresses, such as heat, cold, oxidative stress, DNA damage, and osmotic pressure changes. In most cases, the primary response to stress is transcriptional induction of genes that assist the cells in tolerating the stress and facilitate the repair of the cellular damage. However, when the transcription machinery itself is stressed, responding by such standard mechanisms may not be possible.
RESULTS: In this study, we demonstrate that depletion or inactivation of RNA polymerase II (RNAPII) changes the preferred polyadenylation site usage for several transcripts, and leads to increased transcription of a specific subset of genes. Surprisingly, depletion of RNA polymerase I (RNAPI) also promotes altered polyadenylation site usage, while depletion of RNA polymerase III (RNAPIII) does not appear to have an impact.
CONCLUSIONS: Our results demonstrate that stressing the transcription machinery by depleting either RNAPI or RNAPII leads to a novel transcriptional response that results in induction of specific mRNAs and altered polyadenylation of many of the induced transcripts.
The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins
BACKGROUND: The Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action.
RESULTS: The two forms of the toxin were resolved to 2.7 A and 2.0 A respectively and showed very similar structures. Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin (pfam05791). Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin. Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated. A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action.
CONCLUSIONS: Cry6 proteins are members of the alpha helical pore-forming toxins - a structural class not previously recognized among the Cry toxins of B. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities.
Care transitions from the hospital to home remain a vulnerable time for many patients, especially for those with heart failure (CHF) and chronic obstructive pulmonary disease (COPD). Despite regular use in chronic disease management, it remains unclear how technology can best support patients during their transition from the hospital. We sought to evaluate the impact of a technology-supported care transition support program on hospitalizations, days out of the community and mortality. Using a pragmatic randomized trial, we enrolled patients (511 enrolled, 478 analyzed) hospitalized with CHF/COPD to "E-Coach," an intervention with condition-specific customization and in-hospital and post-discharge support by a care transition nurse (CTN), interactive voice response post-discharge calls, and CTN follow-up versus usual post-discharge care (UC). The primary outcome was 30-day rehospitalization. Secondary outcomes included (1) rehospitalization and death and (2) days in the hospital and out of the community. E-Coach and UC groups were similar at baseline except for gender imbalance (p = 0.02). After adjustment for gender, our primary outcome, 30-day rehospitalization rates did not differ between the E-Coach and UC groups (15.0 vs. 16.3 %, adjusted hazard ratio [95 % confidence interval]: 0.94 [0.60, 1.49]). However, in the COPD subgroup, E-Coach was associated with significantly fewer days in the hospital (0.5 vs. 1.6, p = 0.03). E-Coach, an IVR-augmented care transition intervention did not reduce rehospitalization. The positive impact on our secondary outcome (days in hospital) among COPD patients, but not in CHF, may suggest that E-Coach may be more beneficial among patients with COPD.NIH trial registry number: NCT01135381Trial Protocol: http://dx.doi.org/ 10.1016/j.cct.2012.08.007.
Blogging is a form of online journaling that has been increasingly used to document an attempt in weight loss. Despite the prevalence of weight loss bloggers, few studies have examined this population. We examined characteristics of weight loss bloggers and their blogs, including blogging habits, reasons for blogging, like and dislikes of blogging, and associations between blogging activity and weight loss. Participants (N = 194, 92.3 % female, mean age = 35) were recruited from Twitter and Facebook to complete an online survey. Participants reported an average weight loss of 42.3 pounds since starting to blog about their weight loss attempt. Blogging duration significantly predicted greater weight loss during blogging (beta = -3.65, t(185) = -2.97, p = .003). Findings suggest that bloggers are generally successful with their weight loss attempt. Future research should explore what determines weight loss success/failure in bloggers and whether individuals desiring to lose weight would benefit from blogging.
Human iPSC-Derived Neuronal Model of Tau-A152T Frontotemporal Dementia Reveals Tau-Mediated Mechanisms of Neuronal Vulnerability
Frontotemporal dementia (FTD) and other tauopathies characterized by focal brain neurodegeneration and pathological accumulation of proteins are commonly associated with tau mutations. However, the mechanism of neuronal loss is not fully understood. To identify molecular events associated with tauopathy, we studied induced pluripotent stem cell (iPSC)-derived neurons from individuals carrying the tau-A152T variant. We highlight the potential of in-depth phenotyping of human neuronal cell models for pre-clinical studies and identification of modulators of endogenous tau toxicity. Through a panel of biochemical and cellular assays, A152T neurons showed accumulation, redistribution, and decreased solubility of tau. Upregulation of tau was coupled to enhanced stress-inducible markers and cell vulnerability to proteotoxic, excitotoxic, and mitochondrial stressors, which was rescued upon CRISPR/Cas9-mediated targeting of tau or by pharmacological activation of autophagy. Our findings unmask tau-mediated perturbations of specific pathways associated with neuronal vulnerability, revealing potential early disease biomarkers and therapeutic targets for FTD and other tauopathies.
MMP-9 and MMP-2 Contribute to Neuronal Cell Death in iPSC Models of Frontotemporal Dementia with MAPT Mutations
How mutations in the microtubule-associated protein tau (MAPT) gene cause frontotemporal dementia (FTD) remains poorly understood. We generated and characterized multiple induced pluripotent stem cell (iPSC) lines from patients with MAPT IVS10+16 and tau-A152T mutations and a control subject. In cortical neurons differentiated from these and other published iPSC lines, we found that MAPT mutations do not affect neuronal differentiation but increase the 4R/3R tau ratio. Patient neurons had significantly higher levels of MMP-9 and MMP-2 and were more sensitive to stress-induced cell death. Inhibitors of MMP-9/MMP-2 protected patient neurons from stress-induced cell death and recombinant MMP-9/MMP-2 were sufficient to decrease neuronal survival. In tau-A152T neurons, inhibition of the ERK pathway decreased MMP-9 expression. Moreover, ectopic expression of 4R but not 3R tau-A152T in HEK293 cells increased MMP-9 expression and ERK phosphorylation. These findings provide insights into the molecular pathogenesis of FTD and suggest a potential therapeutic target for FTD with MAPT mutations.
Chromatin is thought to carry epigenetic information from one generation to the next, although it is unclear how such information survives the disruptions of nucleosomal architecture occurring during genomic replication. Here, we measure a key aspect of chromatin structure dynamics during replication-how rapidly nucleosome positions are established on the newly replicated daughter genomes. By isolating newly synthesized DNA marked with 5-ethynyl-2'-deoxyuridine (EdU), we characterize nucleosome positions on both daughter genomes of S. cerevisiae during chromatin maturation. We find that nucleosomes rapidly adopt their mid-log positions at highly transcribed genes, which is consistent with a role for transcription in positioning nucleosomes in vivo. Additionally, experiments in hir1Delta mutants reveal a role for HIR in nucleosome spacing. We also characterized nucleosome positions on the leading and lagging strands, uncovering differences in chromatin maturation dynamics at hundreds of genes. Our data define the maturation dynamics of newly replicated chromatin and support a role for transcription in sculpting the chromatin template.