Journal: PLoS ONE

Article Title: IGFBP3 Colocalizes with and Regulates Hypocretin (Orexin)

doi: 10.1371/journal.pone.0004254

Figure Lengend Snippet: Human narcolepsy susceptibility candidate genes analyzed by microarray, quantitative RT-PCR and distribution shown by in situ hybridization in mice.

Article Snippet: 236034_at , microcephaly, primary autosomal recessive 1 (MCPH1) , B , 83 , 2.6 , 0.030 , Hs00226253_m1 , , 0.8 , 0.361 , 6416651 , n.e..

Techniques: Microarray, Quantitative RT-PCR, In Situ Hybridization, Binding Assay

Journal: PloS one

Article Title: A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

doi: 10.1371/journal.pone.0040387

Figure Lengend Snippet: Figure 1. The human MCPH1 gene, its transcripts and predicted polypeptides. (A) Exon (filled boxes) and intron (open boxes) organization of the 241 906-bp encompassing MCPH1 gene locus. Red arrows indicate the positions of the regular and of the alternative (*) polyadenylation sites (polyA). (B) The full-length (FL) and the alternative transcripts De9–14, De1–3, and De8: numbered boxes indicate exons, black filled areas illustrate the entire coding regions (CDS), and colored areas show untranslated regions (UTR) as indicated. (C) Predicted polypeptides representing MCPH1 isoforms: blue boxes depict the positions of BRCT domains, while green boxes represent the site of the canonical nuclear localization signal sequence (NLS). Two additional amino acids, S and M, are included into MCPH1De9–14 prior to premature termination (#). (D) Expression of MCPH1 transcript variants. Columns represent the levels of MCPH1 transcripts in indicated adult and fetal tissues determined using quantitative real-time PCR. Data represent means 6 one S.D. of three independent experiments and are normalized to the geometric mean levels of UBC, GAPDH, B2M, and HPRT1 cDNA. doi:10.1371/journal.pone.0040387.g001

Article Snippet: Antibodies Rabbit antiserum (ab2612) and goat antiserum (AF3998) to MCPH1 were obtained from Abcam (Cambridge, UK) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Sequencing, Expressing, Real-time Polymerase Chain Reaction

Journal: PloS one

Article Title: A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

doi: 10.1371/journal.pone.0040387

Figure Lengend Snippet: Figure 2. Cell cycle-dependent regulation of MCPH1 transcripts. (A) HeLa cells were arrested in G1 phase by double thymidine block. Cultures harvested at various time points after release were analyzed using flow cytometry. (B) Plots represent numbers of cells as a function of their DNA content. A total of 90% of the cells synchronously progressed into S phase (0–4 h), entered G2 phase (4–6 h), started passing through mitosis after 7 h, and were completely in G1 phase after 12 h. (C) Levels of MCPH1-FL (diamonds, solid line), MCPH1De9–14 (squares, dotted line), and MCPH1De8 (circles, dashed line) mRNA. Data represent means 6 S.E.M. of three independent experiments and are normalized to the expression levels of GAPDH and B2M. doi:10.1371/journal.pone.0040387.g002

Article Snippet: Antibodies Rabbit antiserum (ab2612) and goat antiserum (AF3998) to MCPH1 were obtained from Abcam (Cambridge, UK) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Blocking Assay, Flow Cytometry, Expressing

Journal: PloS one

Article Title: A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

doi: 10.1371/journal.pone.0040387

Figure Lengend Snippet: Figure 3. Expression of GFP-tagged MCPH1 isoforms in MCPH1-deficient 562T fibroblasts. (A) Cells were transduced with GFP-tagged coding sequence of full-length MCPH1 cDNA in a conditional, doxycycline (DOX)-dependent construct with a second regulatory construct trKRAB. Cultures were exposed to increasing DOX concentrations as indicated. Whole-cell extracts were prepared 72 h later and analyzed for the expression of MCPH1 using immunoblotting with an antibody against GFP. (B) The graph shows MCPH1 band intensity relative to the loading control p84 plotted against DOX concentrations. Data represent means 6 one S.D. of three independent assays. (C) Immunoblot analysis of whole-cell extracts from non-transduced (NT) 562T cells (lane 1), 562T cells transduced with the regulatory construct only (lane 2), with GFP alone (30 kDa, lane 3) or with GFP fused to MCPH1-FL (120 kDa, lane 4), MCPH1De9–14 (94 kDa, lane 5), MCPH1De8 (78 kDa, lane 6), or MCPH1De1–7 (96 kDa, lane 7) with an antibody against GFP.. Nuclear matrix protein p84 served as the loading control. doi:10.1371/journal.pone.0040387.g003

Article Snippet: Antibodies Rabbit antiserum (ab2612) and goat antiserum (AF3998) to MCPH1 were obtained from Abcam (Cambridge, UK) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Expressing, Transduction, Sequencing, Construct, Western Blot, Control

Journal: PloS one

Article Title: A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

doi: 10.1371/journal.pone.0040387

Figure Lengend Snippet: Figure 4. Complementation of PCC in patient fibroblasts. Cells are derived from patient with homozygous truncating mutation c.427dupA (p.T143NfsX5) in MCPH1. Chromosome preparations from (A) non-transduced cells and (B) cells expressing GFP only, or GFP fusions with (C) full- length, (D) De9–14, (E) De8, or (F) De1–7 MCPH1. Arrows indicate nuclei of prophase-like cells (PLCs). (G) Mean rates of PLCs (filled columns) of slides from A-F. Open columns represent mean mitotic indices. Error bars denote the S.D. of counts of approximately 1000 cells each from three independent experiments. doi:10.1371/journal.pone.0040387.g004

Article Snippet: Antibodies Rabbit antiserum (ab2612) and goat antiserum (AF3998) to MCPH1 were obtained from Abcam (Cambridge, UK) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Derivative Assay, Mutagenesis, Expressing

Journal: PloS one

Article Title: A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

doi: 10.1371/journal.pone.0040387

Figure Lengend Snippet: Figure 5. Only simultaneous downregulation of endogenous full-length (FL) and De9–14 MCPH1 induces PCC. (A) Immunoblots with an anti-MCPH1 antibody after transfection of HeLa cells with different siRNAs demonstrate efficient downregulation of either MCPH1-FL (93 kDa) or MCPH1De9–14 (70 kDa) or both isoforms (exon 8 and FL+ De9–14) but no downregulation in response to non-targeting siRNA or mock-transfected controls. Nuclear matrix protein p84 served as a loading control. (B) Columns represent PLC rates in HeLa cells transfected with MCPH1 siRNA as indicated in A. Error bars denote the S.D. doi:10.1371/journal.pone.0040387.g005

Article Snippet: Antibodies Rabbit antiserum (ab2612) and goat antiserum (AF3998) to MCPH1 were obtained from Abcam (Cambridge, UK) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Western Blot, Transfection, Control

Journal: PloS one

Article Title: A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

doi: 10.1371/journal.pone.0040387

Figure Lengend Snippet: Figure 6. Intracellular distribution of MCPH1 isoforms. (A) MCPH1-deficient fibroblasts expressing GFP alone or the specified GFP-MCPH1 fusion proteins were fractionated and cytoplasmic (Cyt) and nuclear (Nuc) protein extracts were analyzed using immunoblotting with an antibody against GFP. The nuclear matrix protein p84 and GAPDH were used as index proteins and loading controls. (B) Cells indicated in A stained with an anti-GFP antibody (green), counterstained with DAPI (blue) and analyzed using fluorescence microscopy. Arrows indicate the prophase-like nuclei. Scale bar = 10 mm. All MCPH1 isoforms exhibit unambiguous nuclear localization. doi:10.1371/journal.pone.0040387.g006

Article Snippet: Antibodies Rabbit antiserum (ab2612) and goat antiserum (AF3998) to MCPH1 were obtained from Abcam (Cambridge, UK) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Expressing, Western Blot, Staining, Fluorescence, Microscopy

Journal: PloS one

Article Title: A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

doi: 10.1371/journal.pone.0040387

Figure Lengend Snippet: Figure 7. Nuclear localization signals (NLSs) in human MCPH1. (A) The positions of the putative NLS motifs and their amino acid sequences are highlighted in the diagram of the full-length MCPH1. (B) Subcellular distribution of GFP-tagged wild-type (wt) MCPH1 and mutants with deleted NLSs as indicated were transiently expressed in HeLa cells. Cytoplasmic (Cyt) and nuclear (Nuc) protein extracts were immunoblotted with an antibody against GFP (left panel). GAPDH (center panel) and the nuclear matrix protein p84 (right panel) served as index proteins and loading controls. (C) Ratios of relative GFP band intensity in the cytoplasmic (Cyt) vs. nuclear (Nuc) fractions. Absolute numbers were assessed using densitometry and normalized to the loading controls. Columns designate means, and error bars represent the S.D. from three different experiments. Significant differences to wt MCPH1 are indicated by asterisks denoting p,0.05 (Student’s t-test). Scale bar = 10 mm. doi:10.1371/journal.pone.0040387.g007

Article Snippet: Antibodies Rabbit antiserum (ab2612) and goat antiserum (AF3998) to MCPH1 were obtained from Abcam (Cambridge, UK) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques:

Journal: PloS one

Article Title: A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

doi: 10.1371/journal.pone.0040387

Figure Lengend Snippet: Figure 8. Colocalization of MCPH1 and cH2AX in ionizing irradiation-induced nuclear foci. (A) Non-transduced (NT) MCPH1-deficient 562T cells and 562T stably expressing GFP alone or the specified GFP-MCPH1 fusion proteins were fixed 2 h after irradiation with 10 Gy and co- stained with antibodies against cH2AX (red) and GFP (green). Nuclei were counterstained with DAPI (blue). Rectangles frame areas, which are shown enlarged in the bottom row. MCPH1 focus formation was observed for MCPH1 isoforms containing the C-terminal BRCT tandem. (B) Quantification of cells expressing foci containing cH2AX and/or (C) MCPH1. Error bars indicate the S.D. of three different measurements, counting approximately 300 nuclei. * p#0.05 vs. NT as calculated using the Student’s t-test. doi:10.1371/journal.pone.0040387.g008

Article Snippet: Antibodies Rabbit antiserum (ab2612) and goat antiserum (AF3998) to MCPH1 were obtained from Abcam (Cambridge, UK) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Irradiation, Stable Transfection, Expressing, Staining

Journal: PLoS Genetics

Article Title: Targeted Next-Generation Sequencing Identifies a Recurrent Mutation in MCPH1 Associating with Hereditary Breast Cancer Susceptibility

doi: 10.1371/journal.pgen.1005816

Figure Lengend Snippet: Frequency of the heterozygous MCPH1 c.904_916del mutation among familial, unselected and young breast cancer patients, and in population controls.

Article Snippet: The primary MCPH1 antibody (R&D systems, AF3998) was a goat polyclonal antibody raised against the amino acids 1–250 mapping to the N-terminus of MCPH1 of human origin.

Techniques: Mutagenesis

Journal: PLoS Genetics

Article Title: Targeted Next-Generation Sequencing Identifies a Recurrent Mutation in MCPH1 Associating with Hereditary Breast Cancer Susceptibility

doi: 10.1371/journal.pgen.1005816

Figure Lengend Snippet: Family history of cancers of MCPH1 c.904_916del positive index cases ^a .

Article Snippet: The primary MCPH1 antibody (R&D systems, AF3998) was a goat polyclonal antibody raised against the amino acids 1–250 mapping to the N-terminus of MCPH1 of human origin.

Techniques: Biomarker Discovery

Journal: PLoS Genetics

Article Title: Targeted Next-Generation Sequencing Identifies a Recurrent Mutation in MCPH1 Associating with Hereditary Breast Cancer Susceptibility

doi: 10.1371/journal.pgen.1005816

Figure Lengend Snippet: ( A ) Schematic presentation of the MCPH1 protein and the position of the observed truncating mutation. ( B ) Sequence chromatogram comparisons of genomic DNA and cDNA of heterozygous mutation carriers and a wild-type control. ( C ) Immunoblotting of 3 mutation carriers and 3 non-carriers with an antibody directed towards the amino-terminus of MCPH1. The representative image of altogether three independent experiments is shown.

Article Snippet: The primary MCPH1 antibody (R&D systems, AF3998) was a goat polyclonal antibody raised against the amino acids 1–250 mapping to the N-terminus of MCPH1 of human origin.

Techniques: Mutagenesis, Sequencing, Control, Western Blot

Journal: PLoS Genetics

Article Title: Targeted Next-Generation Sequencing Identifies a Recurrent Mutation in MCPH1 Associating with Hereditary Breast Cancer Susceptibility

doi: 10.1371/journal.pgen.1005816

Figure Lengend Snippet: Patients with breast cancer are marked with black half circles. Other cancer types are marked with grey squares. The age at diagnosis, when known, is marked below the cancer type. Individuals genotyped for MCPH1 c.904_916del are marked with either a plus (mutation positive) or a minus sign (mutation negative). A slashed pedigree symbol indicates a deceased individual. Triangle indicates the initially studied index patient (BR-0653, BR-0887 and BR-0154, respectively). Abbreviations: Bas: basalioma, Bt: brain tumor, Br: breast cancer, Col: colon cancer, Csu: cancer site unknown, Lar: laryngeal cancer, Ov: ovarian tumor, Pro: prostate cancer, Ut: uterine cancer, Vul: vulvar cancer.

Article Snippet: The primary MCPH1 antibody (R&D systems, AF3998) was a goat polyclonal antibody raised against the amino acids 1–250 mapping to the N-terminus of MCPH1 of human origin.

Techniques: Biomarker Discovery, Mutagenesis

Journal: PLoS Genetics

Article Title: Targeted Next-Generation Sequencing Identifies a Recurrent Mutation in MCPH1 Associating with Hereditary Breast Cancer Susceptibility

doi: 10.1371/journal.pgen.1005816

Figure Lengend Snippet: Occurrence of chromosomal aberrations in blood lymphocyte metaphases of heterozygous MCPH1 c.904_916del mutation carriers and healthy controls.

Article Snippet: The primary MCPH1 antibody (R&D systems, AF3998) was a goat polyclonal antibody raised against the amino acids 1–250 mapping to the N-terminus of MCPH1 of human origin.

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: (A) Schematic of knockout strategy for Mcph1 gene based on knockout-first design. A promoterless cassette including LacZ and neo genes was inserted in the third intron of Mcph1 gene flanked by FRT sites. LoxP sites flank the critical exon (exon4 of Mcph1 gene in knockout-first design). See http://www.knockoutmouse.org/martsearch/project/41705 for more details. (B) Short range PCR for genotyping. Wild type allele produces one band of 366bp. Due to the insertion of the cassette, primers designed for the wild type allele do not have product for the mutant allele using the short range PCR (illustrated as the left panel, schematic illustration is not in scale). The homozygous allele produces only one band of 185 bp. The heterozygotes produce two bands of 185 bp and 366 bp. (C) Quantitative real-time PCR showed largely reduced transcript of Mcph1 in Mcph1 tm1a / tm1a (n = 3) mice compared to wild type mice (n = 3) and the residual levels vary in different organs.

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques: Knock-Out, Mutagenesis, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: (A) ABR measurement results of 14 week old Mcph1 tm1a/tm1a mice (n = 11) in MGP showed mild to moderate hearing impairment, or normal hearing compared to control mice. The green baseline area shows a reference range for the control wild type mice with the same genetic background, plotting the median and 2.5 to 97.5 percentile of the population (n = 440). (B) Input-output function (IOF) analysis. The peak-peak amplitude of wave 1 (P1-N1 amplitude) of click-evoked ABRs is plotted as a function of dB SL (Sensation Level, dB above threshold) for wild type (green) and Mcph1 tm1a/tm1a (red) mice. There was no significant difference of IOF slopes of Mcph1 tm1a/tm1a (n = 24, slope = 0.144+/−0.066; mean +/− SD) and wild type mice (n = 36, slope = 0.133+/−0.048) (t-test, p = 0.444).

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques: Control

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: (A) Results of recurrent ABR measurement (click thresholds) with age. Hearing impairment can be detected as early as 3 weeks old in Mcph1 tm1a/tm1a mice (n = 13). Hearing profile of the Mcph1 tm1a/tm1a mice showed either a stable, progressive, or fluctuating pattern with age (three of them marked dark). All the wild type (n = 13) and heterozygous (n = 17) mice displayed normal click thresholds with age. (B) Auditory chain (incus-stapes joint) and oval window sound transduction was severely impeded. Normal incus-stapes joint of auditory chain in a Mcph1 + / + mouse, and a clear oval window is necessary for sound vibration conduction. After removing some of the amorphous material in the middle ear cavity of a Mcph1 tm1a / tm1a mouse, the incus-stapes joint (arrow head) and the oval window (arrow) is present but embedded in the amorphous material. Scale bar, 1 mm. (C–F) Correlation between middle ear defects and hearing sensitivity change with time. (C) Normal ABR thresholds and middle ear structure in a wild type mouse: normal middle ear cavity is full of air, tympanic membrane is transparent and normal morphology of ossicles. (D) Progressively elevated ABR thresholds with age in a Mcph1 tm1a / tm1a mouse. Amorphous mass filled the middle ear cavity and outgrew into external ear canal. Ossicles were embedded in the amorphous mass and appeared to have thinner bony structure. (E) Fluctuating ABR thresholds in a Mcph1 tm1a / tm1a mouse. Watery effusion with bubbles was seen in the middle ear cavity and normal gross morphology of ossicles. (F) Stable and moderate hearing impairment in a Mcph1 tm1a / tm1a mouse. The middle cavity was filled with pus-like secretion. Normal gross morphology of ossicles but with rough surface. Scale bar, 1 mm.

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques: Transduction, Membrane

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: A & B illustrate click-evoked ABR waveforms recorded in a wildtype (green) and a Mcph1 tm1a / tm1a (red) mouse, respectively. The dB SPL of the click stimulus for each response is indicated on the abscissa. The scale bar indicates 5 µV amplitude of response. The heavy lines indicate the click-ABR threshold allocated to each mouse. C. (inset) Mean click-evoked ABR waveforms recorded at 21 dB sensation level for wildtype (n = 8, green) and Mcph1 tm1a / tm1a (n = 8, red).

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques:

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: Clear middle ear cavity (MEC) and thin mucoperiosteum in wild type mice (A,C). MECs of Mcph1 tm1a / tm1a mice (B,D) were filled with exudate and lined with thickened mucoperiosteum. High magnification for mucoperiosteum of MEC framed in A and B (C,D). Inflammatory cells (E,F) in MECs. Scale bar, 200 µm (A,B), 20 µm (C–F).

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques:

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: (A) Scanning electron microscope (SEM) showed normal development of hair cells at P4 in Mcph1 tm1a / tm1a mice ( Mcph1 + / + , n = 3; Mcph1 tm1a / tm1a , n = 3. scale bar, 10 µm). (B) HE slides displayed comparable structure of inner ears (basal turn) in Mcph1 + / + and Mcph1 tm1a / tm1a mice at 4–5 weeks old ( Mcph1 + / + , n = 3; Mcph1 tm1a / tm1a , n = 3. scale bar, 50 µm).

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques: Microscopy

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: Immunochemistry using an antibody shows that Mcph1 (brown labelling) is expressed in epithelial cells of the middle ear cavity at P7 (A,B) and P28 (C,D) wild type mice. Mcph1 is expressed in both ciliated (B,D) cells close to orifice of Eustachian tube and non-ciliated cells (A,C). Scale bar, 20 µm.

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques:

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: (A) X-ray assay showed comparable structure of craniofacial skeleton between wild type and Mcph1 tm1a / tm1a mice at 14 weeks old. (B) Measurement showed that skull width and length in female Mcph1 tm1a / tm1a mice (n = 6) are significantly smaller than those of wild type mice (n = 7) (Rank Sum test). The weight of the brain was measured at 16 weeks of age showed that female and male Mcph1 tm1a / tm1a mice (n = 3, each sex) had lighter brain than the local control mice (n = 3, each sex) and MGP wild type mouse baseline (female, n = 10; male n = 187).

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques: Control

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: Adult Mcph1 tm1a / tm1a (n = 7) mice showed increased genomic instability when compared to wild type mice (n = 34) as determined by an increased prevalence of micronucleated normochromatic erythrocytes (MN-NCE). Data is presented as the Mean ± SEM.

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques:

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: (A) No difference of weight change (Mean ± SEM) between Mcph1 tm1a/tm1a (n = 4) and Mchp1 +/+ (n = 8) mice infected by Salmonella Typhimurium and monitored by weight loss for 21 days. (B) Viable bacterial counts from the spleen (i) and the liver (ii) from the same mice infected by Salmonella Typhimurium at 21 days post infection. There was only one Mcph1 tm1a/tm1a mouse out of 4 tested with any bacteria present in the liver. Mann Whitney U tests were used, p (two-tailed) is indicated in the figure. (C) Viable bacterial counts (Mean ± SEM) being shed in the stool from Mcph1 tm1a/tm1a (n = 5) and Mchp1 +/+ (n = 5) mice were similar over a 28 day infection of Citrobacter rodentium .

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques: Infection, Bacteria, MANN-WHITNEY, Two Tailed Test

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: Mcph1 tm1a/tm1a and Mchp1 +/+ mice were immunised intranasally with fragment C of tetanus toxin on day 0, 7 and 21. Presence of antigen-specific Ig in serum isolated at day 28 was showed no difference in Mcph1 tm1a/tm1a (n = 6) and Mchp1 +/+ (n = 5). The solid bar represents the Mean ± SEM. Mann Whitney U tests were used, p (two-tailed) is indicated in the figure.

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques: Isolation, MANN-WHITNEY, Two Tailed Test

Journal: PLoS ONE

Article Title: Mcph1 -Deficient Mice Reveal a Role for MCPH1 in Otitis Media

doi: 10.1371/journal.pone.0058156

Figure Lengend Snippet: (A) Slit lamp images (12× magnification) revealed corneal (center) opacity and vascularisation (right) in Mcph1 tm1a / tm1a mice. The difference of ocular abnormality portion is significant between wild type (n = 23) and Mcph1 tm1/tm1a (n = 14) mice (Fischer's exact test: p = 0.002). (B) Wild type eye shows normal lens and retina. The anterior and posterior chamber spaces are well defined. ac = anterior chamber; pc = posterior chamber. (C) Mcph1 tm1a / tm1a eye shows cataractous lens and thin retina. The anterior and posterior chambers are collapsed. Scale bar, 500 µm (B,C). (D) Inset from (C) with solid line shows cataractous lens and loss of retinal cell layers. Scale bar, 100 µm.

Article Snippet: Real-time PCR was performed in quadruplicate for each sample using the probe (Applied Biosystem, Mm00557495_m1, covering exon 3–4 boundary) in an ABI Prism 7000 (Applied Biosystem).

Techniques:

Journal: Open Medicine

Article Title: The analyses of human MCPH1 DNA repair machinery and genetic variations

doi: 10.1515/med-2024-0917

Figure Lengend Snippet: Annotation of Exon Expression of MCPH1 derived from the GTEx Analysis V8. The functional annotations of the rare variants p.Val10SerfsTer5 andp.Ser571Ter to the exons and tissue specific expression values.

Article Snippet: More recently, mutations in the MCPH1 were associated with congenital hearing impairment (CHI) [ , ], and previously in otitis media in the mice model [ ].

Techniques: Expressing, Derivative Assay, Functional Assay

Journal: Open Medicine

Article Title: The analyses of human MCPH1 DNA repair machinery and genetic variations

doi: 10.1515/med-2024-0917

Figure Lengend Snippet: Overview of MCPH1 disease-associated variants identified in different domains of the canonical transcript. (ENST00000344683.9)

Article Snippet: More recently, mutations in the MCPH1 were associated with congenital hearing impairment (CHI) [ , ], and previously in otitis media in the mice model [ ].

Techniques:

Journal: Open Medicine

Article Title: The analyses of human MCPH1 DNA repair machinery and genetic variations

doi: 10.1515/med-2024-0917

Figure Lengend Snippet: Protein altering variants in identified in MCPH1 in the gnomAD data, and their VEP and ClinVar annotations considered pathogenic, likely pathogenic or uncertain significance

Article Snippet: More recently, mutations in the MCPH1 were associated with congenital hearing impairment (CHI) [ , ], and previously in otitis media in the mice model [ ].

Techniques:

Journal: Open Medicine

Article Title: The analyses of human MCPH1 DNA repair machinery and genetic variations

doi: 10.1515/med-2024-0917

Figure Lengend Snippet: The two highlighted regions in purple show where MCPH1 functions. The MCPH1 forms parts of the MRN complex, in conjunction with SET, condensin II, SMC2, SMC4, NCAPD3 , NCAPH2 , and NCAPG2 . The NHEJ pathway is initiated in response to the formation of DNA DSBs induced by DNA-damaging agents, such as ionizing radiation. The DNA DSBs are recognized by the MRN complex, leading to ATM activation and ATM-dependent recruitment of several DNA damage checkpoints and repair proteins to DNA DSB sites.

Article Snippet: More recently, mutations in the MCPH1 were associated with congenital hearing impairment (CHI) [ , ], and previously in otitis media in the mice model [ ].

Techniques: Activation Assay