Isolation of equine herpesvirus 3 (EHV-3) from equine coital exanthema of two stallions and sero-epidemiology of EHV-3 infection in Japan

In the spring of 2015, two stallions reared in Farms A and B in Hokkaido in Japan showed symptoms of equine coital exanthema. Equine herpesvirus 3 (EHV-3) was isolated from penis swab samples of both stallions, and the isolates from each stallion in Farms A and B were designated as SS-1 and YS-1 strains, respectively. BamHI restriction profiles of SS-1 and Japanese reference strain Iwate-1 were indistinguishable, but the BamHI-A fragment of YS-1 was larger than those of SS-1 and Iwate-1 by 1.9 kbp because of the lack of two BamHI sites. Nucleotide sequence analyses of glycoprotein G (gG), gB, gC and VP13/14 coding regions revealed that SS-1 and YS-1 had 99.77% to 100% identities to each other. These results suggested that the origins of SS-1 and YS-1 were different. For a sero-epidemiological survey, serum neutralizing tests using SS-1 against 319 sera of horses from eight farms in Hokkaido were conducted. Six of the eight farms were EHV-3 antibody-positive, and positive rates ranged from 2.6% to 17.6%. To determine the infection time of four EHV-3 antibody-positive horses, a retrospective study was conducted. Infection time of the four horses was in the breeding season, and re-infection or reactivation of latently infected EHV-3 might have occurred in one horse. However, these four horses had never shown any clinical symptoms. The results suggested that several EHV-3 strains are distributed in Japan and that infection is maintained widely in horses without clinical symptoms

Relationship between Tissue Gliding of the Lateral Thigh and Gait Parameters after Trochanteric Fractures

Trochanteric fractures lead to severe functional deficits and gait disorders compared to femoral neck fractures. This study aims to investigate gait parameters related to gliding between tissues (gliding) after trochanteric fracture (TF) surgery. This study implemented a cross-sectional design and was conducted amongst patients who underwent TF surgery (n = 94) approximately three weeks post-trochanteric fracture surgery. The following parameters were evaluated: (1) gliding between tissues; (2) lateral femoral pain during loading; (3) maximum gait speed; (4) stride time variability and step time asymmetry as measures of gait cycle variability; (5) double stance ratio and single stance ratio for assessment of stance phase, (6) jerk; and (7) Locomotor rehabilitation index as a measure of force changes during gait. The gliding coefficient was significantly correlated with lateral femoral pain (r = 0.517), jerk root mean square (r = −0.433), and initial contact-loading response jerk (r = −0.459). The jerk of the force change value during gait was also effective in understanding the characteristics of the gait in the initial contact-loading response in patients with trochanteric fractures. Additionally, gliding is related not only to impairments such as pain but also to disabilities such as those affecting gait

Alteration of <em>POLDIP3</em> Splicing Associated with Loss of Function of TDP-43 in Tissues Affected with ALS

<div><p>Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease caused by selective loss of motor neurons. In the ALS motor neurons, TAR DNA-binding protein of 43 kDa (TDP-43) is dislocated from the nucleus to cytoplasm and forms inclusions, suggesting that loss of a nuclear function of TDP-43 may underlie the pathogenesis of ALS. TDP-43 functions in RNA metabolism include regulation of transcription, mRNA stability, and alternative splicing of pre-mRNA. However, a function of TDP-43 in tissue affected with ALS has not been elucidated. We sought to identify the molecular indicators reflecting on a TDP-43 function. Using exon array analysis, we observed a remarkable alteration of splicing in the polymerase delta interacting protein 3 (<em>POLDIP3</em>) as a result of the depletion of TDP-43 expression in two types of cultured cells. In the cells treated with TDP-43 siRNA, wild-type <em>POLDIP3</em> (variant-1) decreased and <em>POLDIP3</em> lacking exon 3 (variant-2) increased. The RNA binding ability of TDP-43 was necessary for inclusion of <em>POLDIP3</em> exon 3. Moreover, we found an increment of <em>POLDIP3</em> variant-2 mRNA in motor cortex, spinal cord and spinal motor neurons collected by laser capture microdissection with ALS. Our results suggest a loss of TDP-43 function in tissues affected with ALS, supporting the hypothesis that a loss of function of TDP-43 underlies the pathogenesis of ALS.</p> </div

Reduction of TDP-43 expression makes cells smaller.

<p>(<b>A</b>) A flow cytometer was used to obtain forward scatter (FSC) histograms of SH-SY5Y cells transfected with siRNA targeting POLDIP3 (blue) or TDP-43 (red) or non-targeting control (black). The FSC histogram is one representative transfection from which 10,000 cells were counted. (<b>B</b>) Data represent the mean with standard error of forward scatter of the siRNA-transfected cells from three independent experiments. Note that down-regulation of TDP-43 leads to a reduction in cell size as well as POLDIP3 depression. Asterisk indicates significant difference (*<i>P</i><0.01, Tukey multiple comparison test). (<b>C and D</b>) Cell lysates from the SHSY5Y cells stably expressed GFP-tagged POLDIP3 variants were subjected to immunoblotting for POLDIP3 (<b>C</b>) or TDP-43 (<b>D</b>). Anti-actin immunoblotting served as a loading control. (<b>E</b>) Recovery rate of the cell size in the TDP-43-depleted SH-SY5Y with or without expression of GFP-tagged POLDIP3 variant. Data represent the mean with standard error from three independent experiments. Note that the cell size recovery rate is significantly increased in the cells expressing POLDIP3 variant-1 compared to in the cells expressing variant-2. Asterisk indicates significant difference (*<i>P</i><0.01, Student <i>t</i> test).</p

TDP-43 RRM1 domain is necessary for including <i>POLDIP3</i> exon 3.

<p>(<b>A</b>) TDP-43 binds to <i>POLDIP3</i> mRNA. Whole cell lysate from HeLa cells was immunoprecipitated with anti-TDP-43 or control rabbit IgG, subjected to the isolation of RNA and RT-PCR analysis using primers located in exon 5 of <i>POLDIP3</i>. (<b>B–F</b>) Analysis of <i>POLDIP3</i> splicing by TDP-43 with mutated in RRM domains. (<b>B</b>) Schematic diagram of the constructs for wild-type (WT) and mutated TDP-43 expression plasmid (mRRM1, mRRM2, and mRRM1/2). Phe (F) to Leu (L) alterations were introduced in RRM1, RRM2, or both motifs of the TDP-43. Boxes indicate RRM motif. Numbers indicate the location of substituted amino acids. (<b>C</b>) TDP-43 depletion caused by siRNA targeting 3′-UTR of <i>TDP-43</i> (TDP-43 3′-UTR siRNA) was rescued by ectopic expression of WT and mutated TDP-43 in HEK293T cells. Mutated TDP-43s were similarly expressed. HEK293T cells were transfected with control (lane 1) or TDP-43 siRNA targeting for 3′-UTR region (lanes 2–6), followed by transfection with empty vector (EV) or various TDP-43 expression plasmids. TDP-43 expression was assessed by probing with anti-TDP-43 antibody. Anti-actin immunoblotting served as a loading control. (<b>D</b>–<b>F</b>) <i>POLDIP3</i> mRNA levels in culture cells transfected with <i>TDP-43</i> 3′-UTR siRNA as a fold change relative to those of cells transfected with control siRNA. The amounts of variant-1 (<b>D</b>), variant-2 (<b>E</b>) or total <i>POLDIP3</i> mRNA (<b>F</b>) were quantified by real-time qRT-PCR using the primers indicated with arrows in Fig. 2A. In this experiment, <i>RPLP1</i> and <i>RPS18</i> were used as reference genes. Data represent the mean with standard error from three independent experiments. Asterisk indicates significant difference among <i>TDP-43</i> 3′-UTR siRNA-transfected cells (*<i>P</i><0.01, Tukey multiple comparison test).</p

Genes with altered splicing by depletion of TDP-43 in culture cells.

<p>(<b>A</b>) Western blot analysis of TDP-43 in HeLa and SH-SY5Y cells with or without TDP-43 siRNA. TDP-43 and actin were shown by immunoblotting with anti-TDP-43 (top) and anti-actin (bottom) antibody, respectively. (<b>B</b>) Microarray analyses were performed in control (transfected with siControl non-targeting pool) and TDP-43 knockdown HeLa and SH-SY5Y cells in triplicate. Venn diagram shows overlapping of altered splicing genes between HeLa and SH-SY5Y cells. Only 15 genes overlapped between them. From the array data, we calculated the <i>P</i> value and the splicing index (|SI|). Both <i>P</i><0.05 and |SI|>0.5 were used as thresholds to identify the genes with altered splicing. (<b>C</b>) Exon structure diagram of the main isoform (top) and candidate variant (bottom) for <i>POLDIP3</i> and <i>HP1BP3.</i> From the exon array result, we selected the candidate splicing variant that was induced by the depletion of TDP-43. The name of each transcript is labeled on the side of the exon structure. The red box indicates the exons that are expected to be altered by TDP-43 depletion. The black bars indicate the position of the primers we used in this experiment. The gene views show the expression of exons as determined by analyzing the results of exon array in HeLa cells using Genespring GX. The expression levels are shown on a log2 scale; the error bars show standard errors of means. TDP-43 siRNA, red circle; control siRNA, blue square. (<b>D</b>) qRT-PCR analysis revealed that the splicing alteration of <i>POLDIP3</i> and <i>HP1BP3</i> gene were validated in both HeLa and SH-SY5Y cells. <i>RPLP1</i> and <i>RPS18</i> were used as reference genes. Data represent the mean with standard error from three independent experiments. Asterisk indicates significant difference (*<i>P</i><0.01, Student <i>t</i> test).</p