Use of VESsel GENeration with Optical Coherence Tomography Angiography and Fluorescein Angiography for Detection and Quantification of Vascular Changes in Mild and Moderate Diabetic Retinopathy.

(1) Background: Previously, VESsel GENeration (VESGEN) software was used to map and quantify vascular changes observed on fluorescein angiography (FA) in subjects (n = 15 eyes) with retinal pathology ranging from mild non-proliferative diabetic retinopathy (NPDR) to proliferative diabetic retinopathy (PDR). In the current study, we used VESGEN for the assessment of individuals with early-stage NPDR imaged by FA (Cohort 1) and by optical coherence tomography angiography (OCTA; Cohort 2). (2) Methods: Cohort 1 included type 2 diabetics (T2D), represented 21 eyes (ranging from no DR to moderate DR), and also included nondiabetic controls (NDC; n = 15 eyes). Cohort 2 consisted of 23 eyes from T2D subjects (including no DR subjects and moderate DR subjects) and NDC (n = 18 eyes). (3) Results: In the FA-VESGEN study, total tortuosity (Tv) of microvessels (G ≥ 6) increased in T2D with mild DR compared to the controls. In contrast, the VESGEN analysis of OCTA images showed that vessel length (characterized as density) was lower in T2D subjects before the diagnosis of DR and following the diagnosis of DR when compared to the controls. Additionally, T2D showed a significant decrease in vessel area (density). (4) Conclusions: FA elucidated the vessel morphology of small-generation microvessels to a greater degree than OCTA; however, OCTA identified changes in vessel density better than FA. VESGEN analysis can be used with both standard FA and OCTA to facilitate our understanding of early events in DR, including before the clinical diagnosis of DR.

A Rare Case of Dapsone Hypersensitivity Syndrome and Leukemoid Reaction With Coexisting Hepatitis E in a Pediatric Patient.

Dapsone is extensively used for a variety of infectious, immunological, and hypersensitivity disorders. Dapsone can cause several adverse effects, the most serious being dapsone hypersensitivity syndrome (DHS), which is potentially fatal. DHS is characterized by triad of eruptions, fever, and organ involvement (including liver, kidney, hematological system, etc.). DHS can develop several weeks to as late as 6 months after treatment initiation with dapsone. Here, we report a case of DHS and leukemoid reaction with coexisting hepatitis E in a 10-year-old girl. Three weeks prior to the current admission, she was treated with dapsone (1 mg/kg/day in two divided doses) for 8 days by a local doctor for lichen nitidus. She was managed successfully for DHS with intravenous (IV) steroids followed by the oral steroid. This case is being reported to highlight the importance to ensure timely diagnosis of DHS and its appropriate management. Patients started on dapsone for various clinical conditions need to be observed carefully for the development of the DHS. If this occurs, DHS can be mistaken for the progression of the primary disease. If dapsone is not withdrawn, it could have deleterious and potentially fatal effects due to major organ dysfunction.

Acute Dengue Hemorrhagic Encephalitis in a Child: A Case Report.

Expanded dengue syndrome includes unusual or atypical manifestations of dengue fever by involving various organ systems. There have been increasing reports of dengue fever with unusual manifestations. Even though dengue virus is considered as a non-neurotropic virus, central nervous system complications have been reported. We are reporting a 4-year-old child who presented with acute dengue hemorrhagic encephalitis along with classical features of dengue infection and magnetic resonance imaging findings, suggestive of hemorrhage in the thalamus and cerebellum.

Risk tolerance to MS therapies: Survey results from the NARCOMS registry.

BACKGROUND: There is little information about risk acceptance of multiple sclerosis (MS) patients to various MS therapies. OBJECTIVE: To determine MS patients׳ tolerance to risky therapies and identify associated characteristics. METHODS: MS patients from the North American Research Committee on Multiple Sclerosis (NARCOMS) Registry׳s online cohort were invited to complete questionnaires on decision making and risk tolerance (RT) to two therapeutic scenarios: a theoretical cure for MS [CureMS], with permanent reversal of all MS symptoms but a risk of immediate painless death; and natalizumab [NAT], a real-life scenario with benefits and risks as defined by Phase III trial results. RESULTS: The median RT for both scenarios was 1:10,000; 15-23% of respondents were not willing to take any risk for their MS therapy. Participants with greater disability or not taking any MS therapy showed a greater RT, while females and those caring for dependents had a lower RT. Females and older age were predictors of lower RT, while increasing disability and greater blunting attitude with respect to information seeking behavior were predictors of higher RT. CONCLUSION: MS patients displayed a wide range of RT for MS therapies. Our study identified gender, age, disability and information seeking behavior to be associated with RT.

Transforming growth factor beta (TGFbeta)-induced apoptosis: the rise & fall of Bim.

Transforming growth factor beta (TGFbeta) regulates essential cellular functions such as cellular proliferation, differentiation and apoptosis. Multiple apoptotic mediators and signaling pathways have been implicated in TGFbeta-induced apoptosis. Bim, a BH3-only protein, is critical for apoptosis in a variety of cell types. In resting cells, BimEL expression levels, the major and most abundant isoform, are controlled by Erk1/2-mediated phosphorylation, which targets BimEL for ubiquitination and degradation. We previously reported that TGFbeta induces the expression of the pro-apoptotic protein Bim through a Smad3-dependent mechanism to induce cell death in B-lymphocytes. A number of studies have shown TGFbeta to cause transcriptional induction of Bim in many cell types. Recently, we demonstrated that, in addition to its transcriptional effects on Bim, TGFbeta induces a MAPK phosphatase (MKP), MKP2/DUSP4, to rapidly increase BimEL levels by inactivation of Erk1/2, resulting in dephosphorylation and escape of BimEL from ubiquitin-mediated degradation. Our findings are of importance not only in the context that we implicate TGFbeta to increase BimEL levels through both an immediate post-translational regulatory mechanism and a long-term effect through transcriptional induction, but also in the context of implicating MKPs as regulatory players in apoptosis. Here we summarize these recent findings and their significance to our understanding of how TGFbeta mediates apoptosis, and we explore the possible regulatory mechanisms controlling Bim expression levels.

TGF beta-mediated BIM expression and apoptosis are regulated through SMAD3-dependent expression of the MAPK phosphatase MKP2.

Transforming growth factor-beta (TGFbeta) induces the expression of the pro-apoptotic protein BIM, and mediates apoptosis in hepatocytes and B lymphocytes. BIM is regulated through a post-translational mechanism involving ERK-dependent phosphorylation and ubiquitin-mediated proteasomal degradation. Here, we show that TGFbeta induces BIM through its rapid inhibition of ERK, thereby preventing the phosphorylation and degradation of BIM. TGFbeta, through a SMAD3-dependent mechanism, transcriptionally induces the mitogen-activated protein kinase (MAPK) phosphatase MKP2, encoded by an immediate early gene, to attenuate ERK and promote the accumulation of BIM protein. Overexpression of MKP2 in hepatocytes modulates ERK-mediated phosphorylation of BIM and apoptosis in the absence of TGFbeta, whereas its ablation in pro-B cells, derived from MKP2-deficient mice, protects cells from TGFbeta-mediated apoptosis, and blocks TGFbeta-induced ERK inhibition and BIM induction. Furthermore, in pro-B cells derived from SMAD3-deficient mice, induction of MKP2 by TGFbeta, inhibition of ERK, induction of BIM and apoptosis do not occur. Our results indicate that MKP2 mediates TGFbeta-dependent apoptosis by linking SMAD3 to the modulation of ERK activity and mitochondrial-mediated pro-apoptotic events.

A K52Q substitution in the globular domain of histone H1t modulates its nucleosome binding properties.

A comparison of the globular domain sequences of the somatic H1d and testis-specific H1t revealed a single substitution of lysine 52 in H1d to glutamine 54 in H1t, which is one of the three crucial residues within the second DNA binding site. The globular domains of both histones were modeled using the crystal structure of chicken GH5 as a template and was also docked onto the nucleosome structure. The glutamine residue in histone H1t forms a hydrogen bond with main chain carbonyl of methionine-52 (in H1t) and is spatially oriented away from the nucleosome dyad axis. A consequence of this change was a lower affinity of recombinant histone H1t towards Four-way junction DNA and reconstituted 5S mononucleosomes. When Gln-54 in Histone H1t was mutated to lysine, its binding affinity towards DNA substrates was comparable to that of histone H1d. The differential binding of histones H1d and H1t towards reconstituted mononucleosomes was also reflected in the chromatosome-stop assay.

Analysis of the initiator tRNA genes from a slow- and a fast-growing Mycobacterium.

Initiation of protein synthesis is a major post-transcriptional regulatory step in gene expression. The initiator tRNA gene from Mycobacterium smegmatis, a fast-growing mycobacterium, was characterized and compared with its counterpart from Mycobacterium tuberculosis, a slow-growing mycobacterium. In both mycobacteria, the functional initiator tRNA genes were found in a single copy. Unlike the M. tuberculosis initiator tRNA, the CCA end of the M. smegmatis initiator is not encoded in the gene, and it is most likely added post-transcriptionally. Transcription start site mapping allowed accurate assignment of the hexameric -10 and -35 promoter elements for both genes. These elements of the M. smegmatis initiator tRNA gene contain single nucleotide changes compared to their respective counterparts in the M. tuberculosis gene. Chloramphenicol acetyl transferase reporter assays suggested that the promoter of the initiator tRNA gene from M. smegmatis is twice as strong as that of M. tuberculosis, irrespective of whether the assays were performed in the fast-growing homologous host (M. smegmatis) or the slow-growing heterologous host (M. tuberculosis). Characterization of the M. smegmatis metU promoter, in this study, provides a valuable tool for the expression of genes in mycobacteria.

Identification of a 34 amino acid stretch within the C-terminus of histone H1 as the DNA-condensing domain by site-directed mutagenesis.

The C-terminus of histone H1 is necessary for the folding of polynucleosomal arrays into higher-order structure(s) and contains octapeptide repeats each having DNA binding S/TPKK motifs. These repeat motifs were earlier shown to mimic the DNA/chromatin-condensing properties of the C-terminus of histone H1 (Khadake, J. R., and Rao, M. R. S. (1995) Biochemistry 36, 1041-1051). In the present study, we have generated a series of C-terminal mutants of rat histone H1d and studied their DNA-condensation properties. The single proline to alanine mutation in the S/TPKK motifs either singly or in combination resulted in only a 20% decrease in the DNA-condensation property of histone H1. Deletion of all the three S/TPKK motifs resulted in a 45% decrease in DNA condensation. When the three octapeptide repeats encompassing the S/TPKK motifs were deleted, there was again a 45% decrease in DNA condensation. On the other hand, when the entire 34 amino acid stretch (residue 145-178) was deleted, there was nearly a 90% decrease in DNA condensation brought about by histone H1d. Interestingly, deletion of the 10 amino acid spacer between the octapeptide repeats (residues 161-170) also reduced the DNA condensation by 70%. Deletion of the region (residues 115-141) immediately before the 34 amino acid stretch and after the globular domain and the region (residues 184-218) immediately after the 34 amino acid stretch had only a marginal effect on DNA condensation. The importance of the 34 amino acid stretch, including the 10 amino acid spacer, was also demonstrated with the recombinant histone H1d C-terminus. We have also determined the induced alpha-helicity of histone H1 and its various mutants in the presence of 60% trifluoroethanol, and the experimentally determined induced helical contents agree with the theoretical predictions of secondary structural elements in the C-terminus of histone H1d. Thus, we have identified a 34 amino acid stretch in the C-terminus of histone H1d as the DNA-condensing domain.