Differentiating molecular etiologies of Angelman syndrome through facial phenotyping using deep learning

Angelman syndrome (AS) is caused by several genetic mechanisms that impair the expression of maternally‐inherited UBE3A through deletions, paternal uniparental disomy (UPD), UBE3A pathogenic variants, or imprinting defects. Current methods of differentiating the etiology require molecular testing, which is sometimes difficult to obtain. Recently, computer‐based facial analysis systems have been used to assist in identifying genetic conditions based on facial phenotypes. We sought to understand if the facial‐recognition system DeepGestalt could find differences in phenotype between molecular subtypes of AS. Images and molecular data on 261 individuals with AS ranging from 10 months through 32 years were analyzed by DeepGestalt in a cross‐validation model with receiver operating characteristic (ROC) curves generated. The area under the curve (AUC) of the ROC for each molecular subtype was compared and ranked from least to greatest differentiable phenotype. We determined that DeepGestalt demonstrated a high degree of discrimination between the deletion subtype and UPD or imprinting defects, and a lower degree of discrimination with the UBE3A pathogenic variants subtype. Our findings suggest that DeepGestalt can recognize subclinical differences in phenotype based on etiology and may provide decision support for testing.     

Expression of interleukin-6 in polymorphic reticulosis--immunohistochemical study of 5 cases.

Peripheral T cell lymphoma encompasses lymphomas with a variety of histologic appearances and clinical patterns. Recently, it has been suggested that almost all of the histologic features described under the name of polymorphic reticulosis(PR), lethal midline granuloma, and midline malignant reticulosis can be included in those generally described for malignant lymphomas of peripheral T cell origin(PTCL). There have been few studies of pathogenesis or tissue damage mechanism in PR patients. The need for a precise mechanism for tissue damage has important therapeutic implications. Using immunohistochemical methods with polyclonal anti IL-6 antibody, the authors describe 5 cases of PR with clinically and pathologically typical PR demonstrating a high expression of IL-6. According to classification, 2 cases of grade 1 PR showed the highest expressions, and 2 cases of grade 2 PR with atypical lymphoid cells showed moderate activity, but one case progressed into frank lymphoma(grade 3) and lost IL-6 expression. This strongly implies that some cases of PR have a different mechanism of tissue damage from frank PTCL, despite the one disease spectrum. Further studies on more cases may help clarify the pathogenesis.     

Molecular cloning and characterization of the 78-kilodalton glucose-regulated protein of Trypanosoma cruzi.

The protozoan Trypanosoma cruzi is the etiologic agent of Chagas' disease, an illness responsible for morbidity and death among millions of Latin Americans. Mice also develop this disease when infected with T. cruzi and are a useful model organism for the study of parasite-specific immune responses. To identify immunogenic T. cruzi antigens, serum from an infected mouse was used to isolate clones from a T. cruzi epimastigote cDNA expression library. One of these clones was found to encode the 78-kDa glucose-regulated protein (grp78), the endoplasmic reticular member of the 70-kDa heat shock protein (hsp70) family. Like the mammalian and yeast grp78s, the T. cruzi protein contains an endoplasmic reticular leader peptide and a carboxyl-terminal endoplasmic reticular retention sequence. T. cruzi grp78 is encoded by a tandemly arranged family of three genes located on a chromosome of 1.6 Mb. The effects on grp78 expression of heat shock and tunicamycin treatment, the latter of which specifically stimulates mammalian grp78, were investigated. While the level of the grp78 protein remained constant under all circumstances, grp78 mRNA was unaffected by heat shock but induced fivefold by tunicamycin. Finally, we found that grp78 is the most immunogenic of the T. cruzi heat shock proteins we have characterized, reacting strongly in immunoblots with sera from infected mice.     

Immunohistochemical analysis of p53 protein expression in benign and malignant skin tumors using a panel of anti-p53 antibodies.

The expression of p53 in a variety of benign and malignant skin lesions has been first assessed in frozen sections and then compared with the results obtained in corresponding paraffin-embedded sections using various immunohistochemical staining methods with a panel of anti-p53 antibodies. Of the 48 benign and malignant skin lesions studied, 46(96%) had corresponding paraffin sections and immunohistochemical results obtained with DO7 on frozen and paraffin sections were concordant in 97%, qualitatively. Using streptavidin-biotin complex method, p53 was identified in 33% of dysplastic squamous lesions, 50% of squamous cell carcinomas (SCCs) and 36% of basal cell carcinomas (BCCs) on frozen section, whereas 25% of dysplastic squamous lesions, 40% of SCCs, and 32% of BCCs showed p53 positivity on paraffin-embedded sections. In frozen sections, the same regions of each specimen exhibited similar topographic patterns of positive immunoreactivity with both monoclonal antibodies, PAb 1801 and DO7. In contrast, immunohistochemical staining with polyclonal antibody, CM-1, gave poor morphologic resolution, although effective in paraffin-embedded sections.     

Regulation of IgE receptor expression on human peripheral blood lymphocytes by lymphocytosis promoting factor (LPF), lectins and dexamethasone.

Using a monoclonal anti-human Fc epsilon R antibody (H107), we found that lymphocytosis promoting factor (LPF), phytohaemagglutinin (PHA-P) and Concanavalin A (Con A) could induce Fc epsilon R, detected by immunofluorescence study, on normal human peripheral blood lymphocytes without IgE. The number of Fc epsilon R bearing lymphocytes was increased by stimulation with 3, 10 and 10 micrograms/ml of LPF, PHA-P and Con A, respectively, from 6.0 +/- 3.0/1000 cells to 26.0 +/- 7.9, 54.0 +/- 6.7 and 24.8 +/- 7.1/1000 cells, respectively. Although the induction of Fc epsilon R occurred neither in the separated T-enriched fraction (TEF) nor the T-depleted fraction (TDF), it recovered when the two fractions were mixed. The cell free supernatants from TEF stimulated with LPF or PHA-P could increase Fc epsilon R(+) cells in TDF, whereas those from TDF failed to increase them in TEF. The results suggest that the induction of Fc epsilon R occurs mainly on B lymphocytes by the soluble factor(s) formed by T cells stimulated with LPF or PHA-P. The induction of Fc epsilon R by stimulants was completely inhibited by 10(-6) M dexamethasone. It was demonstrated that the effects of dexamethasone on lymphocytes were dual: one was on B cells to inhibit responsive increases of Fc epsilon R, and the other was on T cells to suppress the formation of the soluble factor(s) which induced Fc epsilon R on B cells.     

Identification of Bacillus anthracis by rpoB sequence analysis and multiplex PCR

Comparative sequence analysis was performed upon Bacillus anthracis and its closest relatives, B. cereus and B. thuringiensis. Portions of rpoB DNA from 10 strains of B. anthracis, 16 of B. cereus, 10 of B. thuringiensis, 1 of B. mycoides, and 1 of B. megaterium were amplified and sequenced. The determined rpoB sequences (318 bp) of the 10 B. anthracis strains, including five Korean isolates, were identical to those of Ames, Florida, Kruger B, and Western NA strains. Strains of the "B. cereus group" were separated into two subgroups, in which the B. anthracis strains formed a separate clade in the phylogenetic tree. However, B. cereus and B. thuringiensis could not be differentiated. Sequence analysis confirmed the five Korean isolates as B. anthracis. Based on the rpoB sequences determined in the present study, multiplex PCR generating either B. anthracis-specific amplicons (359 and 208 bp) or cap DNA (291 bp) in a virulence plasmid could be used for the rapid differential detection and identification of virulent B. anthracis.     

Novel OCTN2 mutations: No genotype–phenotype correlations: Early carnitine therapy prevents cardiomyopathy

Primary systemic carnitine deficiency or carnitine uptake defect (OMIM 212140) is a potentially lethal, autosomal recessive disorder characterized by progressive infantile‐onset cardiomyopathy, weakness, and recurrent hypoglycemic hypoketotic encephalopathy, which is highly responsive to L ‐carnitine therapy. Molecular analysis of the SLC22A5 (OCTN2) gene, encoding the high‐affinity carnitine transporter, was done in 11 affected individuals by direct nucleotide sequencing of polymerase chain reaction products from all 10 exons. Carnitine uptake (at Km of 5 μM) in cultured skin fibroblasts ranged from 1% to 20% of normal controls. Eleven mutations (delF23, N32S, and one 11‐bp duplication in exon 1; R169W in exon 3; a donor splice mutation [IVS3+1 G > A] in intron 3; frameshift mutations in exons 5 and 6; Y401X in exon 7; T440M, T468R and S470F in exon 8) are described. There was no correlation between residual uptake and severity of clinical presentation, suggesting that the wide phenotypic variability is likely related to exogenous stressors exacerbating carnitine deficiency. Most importantly, strict compliance with carnitine from birth appears to prevent the phenotype. © 2002 Wiley‐Liss, Inc.     

Microtubule- and dynein-mediated movement of Orientia tsutsugamushi to the microtubule organizing center

The host cell microfilaments and microtubules (MTs) are known to play a critical role in the life cycles of several pathogenic intracellular microbes by providing for successful invasion and promoting movement of the pathogen once inside the host cell cytoplasm. Orientia tsutsugamushi, an obligate intracellular bacterium, enters host cells by induced phagocytosis, escapes to the cytosol, and then replicates in the cytosol. ECV304 cells infected with O. tsutsugamushi revealed the colocalization of the MT organizing center (MTOC) and cytosolic orientiae by indirect immunofluorescence assay. Using immunofluorescence microscopy in the presence and absence of MT-depolymerizing agents (colchicine and nocodazole), it was shown that the cytosolic oriential movement was mediated by MTs. By transfection study (overexpression of dynamitin [also called p50], which is known to associate with dynein-dependent movement), the movement of O. tsutsugamushi to the MTOC was also mediated by dynein, the minus-end-directed MT-related motor. Although the significance of this movement in the life cycle of O. tsutsugamushi was not proven, we propose that the cytosolic O. tsutsugamushi bacteria use MTs and dyneins to propel themselves from the cell periphery to the MTOC.     

Differentiation of mycobacterial species by PCR-restriction analysis of DNA (342 base pairs) of the RNA polymerase gene (rpoB)

PCR amplification-restriction analysis (PRA) of rpoB DNA (342 bp), which comprises the Rif(r) region, was used for the differential identification of 49 mycobacteria. The DNA had been used previously for the identification of mycobacterial species by comparative sequence analysis (B. J. Kim et al., J. Clin. Microbiol. 37:1714-1720, 1999). Digestion with four restriction enzymes (HaeIII, HindII, MvaI, and AccII), which were selected on the basis of rpoB DNA sequences, generated distinctive PRA patterns that allowed not only the reference strains but also the clinical isolates of mycobacteria to be distinguished. Both rapidly and slowly growing mycobacteria were distinctly differentiated by HaeIII digestion of the amplified rpoB DNA. By HindII digestion the Mycobacterium tuberculosis complex was distinguished from the other mycobacteria. Furthermore, six subspecies of Mycobacterium kansasii (subspecies I to VI) as well as the closely related Mycobacterium gastri, and other closely related species, were distinguished by simultaneous digestion of MvaI and AccII. According to the rpoB PRA scheme, 240 strains of clinical isolates could be identified. It was also possible to detect and identify M. tuberculosis directly from sputa and bronchoalveolar lavage specimens. These results suggest that PRA of rpoB DNA is a simple and feasible method not only for the differentiation of culture isolates but also for the rapid detection and identification of pathogenic mycobacteria in primary clinical specimens.