Acceleration of the Meckel Syndrome by Near-Infrared Light Therapy

www.karger.com/nne This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License (www.karger.com/OA-license), applicable to the online version of the article only. Distribution for non-commercial purposes only

Design and construction of the MicroBooNE detector

This paper describes the design and construction of the MicroBooNE liquid argon time projection chamber and associated systems. MicroBooNE is the first phase of the Short Baseline Neutrino program, located at Fermilab, and will utilize the capabilities of liquid argon detectors to examine a rich assortment of physics topics. In this document details of design specifications, assembly procedures, and acceptance tests are reported

Measurement of the top-quark mass in tt¯ events with dilepton final states in pp collisions at √s = 7 TeV

Open Access: This article is distributed under the terms of the Creative Commons Attribution License.-- Chatrchyan, S. et al.The top-quark mass is measured in proton-proton collisions at s√=7 TeV using a data sample corresponding to an integrated luminosity of 5.0 fb−1 collected by the CMS experiment at the LHC. The measurement is performed in the dilepton decay channel tt¯→(ℓ+νℓb)(ℓ−ν¯¯ℓb¯), where ℓ=e,μ. Candidate top-quark decays are selected by requiring two leptons, at least two jets, and imbalance in transverse momentum. The mass is reconstructed with an analytical matrix weighting technique using distributions derived from simulated samples. Using a maximum-likelihood fit, the top-quark mass is determined to be 172.5±0.4 (stat.)±1.5 (syst.) GeV.Acknowledge support from BMWF and FWF (Austria); FNRS and FWO (Belgium); CNPq, CAPES, FAPERJ, and FAPESP (Brazil); MES (Bulgaria); CERN; CAS, MoST, and NSFC (China); COLCIENCIAS (Colombia); MSES (Croatia); RPF (Cyprus); MoER, SF0690030s09 and ERDF (Estonia); Academy of Finland, MEC, and HIP (Finland); CEA and CNRS/IN2P3 (France);BMBF, DFG, and HGF (Germany); GSRT (Greece); OTKA and NKTH (Hungary); DAE and DST (India); IPM (Iran); SFI (Ireland); INFN (Italy); NRF and WCU (Korea); LAS (Lithuania); CINVESTAV, CONACYT, SEP, and UASLP-FAI (Mexico); MSI (New Zealand); PAEC (Pakistan); MSHE and NSC (Poland); FCT (Portugal); JINR (Armenia, Belarus, Georgia, Ukraine, Uzbekistan); MON, RosAtom, RAS and RFBR (Russia); MSTD (Serbia); SEIDI and CPAN (Spain); Swiss Funding Agencies (Switzerland); NSC (Taipei); ThEP, IPST and NECTEC (Thailand); TUBITAK and TAEK (Turkey); NASU (Ukraine); STFC (United Kingdom); DOE and NSF (USA). Individuals have received support from the Marie-Curie program and the European Research Council (European Union); the Leventis Foundation; the A. P. Sloan Foundation; the Alexander von Humboldt Foundation; the Austrian Science Fund (FWF); the Belgian Federal Science Policy Office; the Fonds pour la Formation à la Recherche dans l’Industrie et dans l’Agriculture (FRIA-Belgium); the Agentschap voor Innovatie door Wetenschap en Technologie (IWTBelgium); the Ministry of Education, Youth and Sports (MEYS) of Czech Republic; the Council of Science and Industrial Research, India; the Compagnia di San Paolo (Torino); and the HOMING PLUS program of Foundation for Polish Science, cofinanced from European Union, Regional Development Fund.Peer Reviewe

Effects of low-level light therapy on hepatic antioxidant defense in acute and chronic diabetic rats

ABSTRACT: Diabetes causes oxidative stress in the liver and other tissues prone to complications. Photobiomodulation by near infrared light (670 nm) has been shown to accelerate diabetic wound healing, improve recovery from oxidative injury in the kidney, and attenuate degeneration in retina and optic nerve. The present study tested the hypothesis that 670 nm photobiomodulation, a low-level light therapy, would attenuate oxidative stress and enhance the antioxidant protection system in the liver of a model of type I diabetes. Male Wistar rats were made diabetic with streptozotocin (50 mg/kg, ip) then exposed to 670 nm light (9 J/cm 2 ) once per day for 18 days (acute) or 14 weeks (chronic). Livers were harvested, flash frozen, and then assayed for markers of oxidative stress. Light treatment was ineffective as an antioxidant therapy in chronic diabetes, but light treatment for 18 days in acutely diabetic rats resulted in the normalization of hepatic glutathione reductase and superoxide dismutase activities and a significant increase in glutathione peroxidase and glutathione-S transferase activities. The results of this study suggest that 670 nm photobiomodulation may reduce, at least in part, acute hepatic oxidative stress by enhancing the antioxidant defense system in the diabetic rat model.

Insulin secretion deficits in a Prader-Willi syndrome β-cell model are associated with a concerted downregulation of multiple endoplasmic reticulum chaperones

Prader-Willi syndrome (PWS) is a multisystem disorder with neurobehavioral, metabolic, and hormonal phenotypes, caused by loss of expression of a paternally-expressed imprinted gene cluster. Prior evidence from a PWS mouse model identified abnormal pancreatic islet development with retention of aged insulin and deficient insulin secretion. To determine the collective roles of PWS genes in β-cell biology, we used genome-editing to generate isogenic, clonal INS-1 insulinoma lines having 3.16 Mb deletions of the silent, maternal- (control) and active, paternal-allele (PWS). PWS β-cells demonstrated a significant cell autonomous reduction in basal and glucose-stimulated insulin secretion. Further, proteomic analyses revealed reduced levels of cellular and secreted hormones, including all insulin peptides and amylin, concomitant with reduction of at least ten endoplasmic reticulum (ER) chaperones, including GRP78 and GRP94. Critically, differentially expressed genes identified by whole transcriptome studies included reductions in levels of mRNAs encoding these secreted peptides and the group of ER chaperones. In contrast to the dosage compensation previously seen for ER chaperones in Grp78 or Grp94 gene knockouts or knockdown, compensation is precluded by the stress-independent deficiency of ER chaperones in PWS β-cells. Consistent with reduced ER chaperones levels, PWS INS-1 β-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. Combined, the findings suggest that a chronic shortage of ER chaperones in PWS β-cells leads to a deficiency of protein folding and/or delay in ER transit of insulin and other cargo. In summary, our results illuminate the pathophysiological basis of pancreatic β-cell hormone deficits in PWS, with evolutionary implications for the multigenic PWS-domain, and indicate that PWS-imprinted genes coordinate concerted regulation of ER chaperone biosynthesis and β-cell secretory pathway function

Sanger sequencing of genome editing events at sgRNA sites in derivation of maternal deletion INS-1 clonal lines.

(A-H) Sanger sequence traces are shown highlighting sgRNA1 (yellow), sgRNA3 (blue), SpCas9 NGG PAMs (grey), and insertion mutations (pink). (A-B) Maternal-deletion allele of clonal lines 5–9 (A) and 5–5 (B) are identical canonical deletions with 3.16 Mb deleted between sgRNA1 and sgRNA3 due to a breakpoint from DNA repair of DSBs occurring at each position 3-nt upstream of the PAM (PAM-3) nuclease sites (see S1A Fig). Deletion-PCR breakpoint fragments are from the Fig 1C gel. (C-E) Sequence of intact sgRNA1 site in parental INS-1 (C), and scarred alleles with a single T/A insertion at the canonical PAM-3 DSB position (pink highlight) in clonal lines 5–9 (D) and 5–5 (E). Sequenced PCR fragments spanning sgRNA1 are from the gel in S1I Fig (F-H) Sequence of intact sgRNA3 site in parental INS-1 (F), and scarred alleles with a G/C insertion or a C/G insertion at the PAM-3 site (pink highlight) in line 5–9 (G) or line 5–5 (H), respectively. Sequenced PCR fragments spanning sgRNA3 are from the gel in S1I Fig It may be noted that as the deletions for 5–9 and 5–5 are on the maternal allele, the sgRNA1 and sgRNA3 scarred alleles can be inferred to occur on the paternal allele for each cell line. Further, as each of lines 5–5 and 5–9 have different sgRNA3 scarred alleles (despite sharing deletion breakpoints and sgRNA1 scarred allele mutations) then these two cell lines clearly arose as independent genome editing events. (JPG)</p

Specificity of RT-PCR and RT-ddPCR assays for rat, mouse and human insulin genes in the INS-1 cell line panel.

(A) RT-PCR with gel analysis for insulin genes. Abbreviations are: (1), (2), represent amplicons 1 and 2 for a given gene; mCherry, INS-1(832/13)::mCherry cell line; 832/13, INS-1(832/13) cell line; E, INS1-E cell line; Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; *, non-specific band in the indicated RT-PCR assay (note that this amplified product is not present using the same PCR primer pair in the RT-ddPCR assay shown in S16J Fig, likely due to different chemistry or annealing temperature in the two assays). Note that control line 16 has greatly reduced expression of the Hs INS (amplicons 1 and 2) and NeoR segments of the INS-NeoR transgene, as also seen in the RT-ddPCR data [see S16–S16K Fig] and RNA-seq data [see S16M Fig], likely reflecting epigenetic inactivation of the INS-NeoR transgene in a majority of cells for line 16 (as DNA analysis indicated the transgene remained present). (B-L) RT-ddPCR assays for the listed amplicons, with ddPCR performed using EvaGreen. All abbreviations are as for S16A. (B) RT-ddPCR assay for Rn Ins1 amplicon 1. (C) RT-ddPCR assay for Rn Ins1 amplicon 2. (D) RT-ddPCR assay for Rn Ins2 amplicon 1. (E) RT-ddPCR assay for Rn Ins2 amplicon 2. (F) RT-ddPCR assay for Mm Ins2. (G) RT-ddPCR assay for mCherry amplicon 1. (H) RT-ddPCR assay for mCherry amplicon 2. (I) RT-ddPCR assay for Hs INS amplicon 1. (J) RT-ddPCR assay for Hs INS amplicon 2. (K) RT-ddPCR assay for NeoR. (L) RT-ddPCR assay for control gene Gpi. (M) RNA-seq analysis of insulin genes expressed in the INS-1 lines. In addition to the endogenous rat (r) Ins1 and Ins2 genes, a custom rat genome build identified mouse (m) Ins2-mCherry and Hs INS-NeoR transgene mRNA levels. Red numbers indicate that control line 16 is an outlier with drastically reduced expression of the Hs INS-NeoR transgene (see S16A Fig legend). (JPG)</p

Reductions in insulins and ER chaperone levels in PWS-deletion <i>vs</i>. control INS-1 lines.

Western blots of whole cell lysates from PWS (3, 19–1, 19–4) and control (5–9, 2, 16) INS-1 β-cell lines grown under control (DMSO) conditions for 5 h were assessed using a panel of antibodies. (A) Anti-insulin, detecting all cellular forms of insulin (Pre-Pro-, Pro-, and fully processed rat INS) as well as mouse proinsulin2 (Pro-mINS2)-mCherry. (B) Quantitation of Pro-mINS2-mCherry, Pre-Pro-INS and Pro-INS detected with anti-insulin in the PWS and control INS-1 lines (n = 6 each genotype). (C) Anti-mCherry, detecting mouse proinsulin2 (Pro-mINS2)-mCherry and C-peptide (CP)-mCherry. (D) Quantitation of Pro-mINS2-mCherry and CP-mCherry detected with anti-mCherry in the PWS and control INS-1 lines (n = 6 each). (E) Anti-KDEL, detecting the two major ER chaperones GRP94 (endoplasmin; HSP90B1) and GRP78 (BiP; HSPA5). (F) Quantitation of GRP78, GRP94, and total KDEL detected with anti-KDEL in the PWS and control INS-1 lines (n = 9 each). Anti-α-Tubulin, anti-GAPDH, and anti-GPI were used as controls for protein loading levels in (A), (C), and (E), respectively. For (A) and (C), control cell lines included INS-1::mCherry (mCh), INS-1(832/13) parental, and INS1-E. In (A), unlabeled bands likely are Pro-mINS2-mCherry proteolytic fragments not containing the anti-mCherry epitope, as not detected in (C). For (B,D,F), statistical comparison by Welch’s t-test: *, P P P < 0.0005; ns, not significant.</p