DEFECTIVE EMBRYO AND MERISTEMS genes are required for cell division and gamete viability in Arabidopsis

The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1’s primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.

Hinge Influences in Murine IgG Binding to Cryptococcus neoformans Capsule

Decades of studies on antibody structure led to the tenet that the V region binds antigens while the C region interacts with immune effectors. In some antibodies, however, the C region affects affinity and/or specificity for the antigen. One such case is that of the 3E5 antibodies, a family of monoclonal murine IgGs in which the mIgG3 isotype has different fine specificity to the Cryptococcus neoformans capsule polysaccharide than the other mIgG isotypes. Our group serendipitously found another pair of mIgG1/mIgG3 antibodies based on the 2H1 hybridoma to the C. neoformans capsule that recapitulated the differences observed with 3E5. In this work, we report the molecular basis of the constant domain effects on antigen binding using recombinant antibodies. As with 3E5, immunofluorescence experiments show a punctate pattern for 2H1-mIgG3 and an annular pattern for 2H1-mIgG1. Also as observed with 3E5, 2H1-mIgG3 bound on ELISA to both acetylated and non-acetylated capsular polysaccharide, whereas 2H1-mIgG1 only bound well to the acetylated form, consistent with differences in fine specificity. In engineering hybrid mIgG1/mIgG3 antibodies, we found that switching the 2H1-mIgG3 hinge for its mIgG1 counterpart changed the immunofluorescence pattern to annular, but a 2H1-mIgG1 antibody with a mIgG3 hinge still had an annular pattern. The hinge is thus necessary but not sufficient for these changes in binding to the antigen. This important role for the constant region in binding of antibodies to the antigen could affect the design of therapeutic antibodies and our understanding of their function in immunity.Key pointsKey point 1- 2H1 antibodies recapitulate differences between mIgG isotypes observed with 3E5.Key point 2 – The hinge region is necessary but not sufficient for these differences.Key point 3 - The antibody constant region can also play a role in mIgG binding to antigen.

Punctate pattern and Milky Way appearance caused by PML in a patient with AIDS

Introduction: Progressive multifocal leukoencephalopathy (PML) is an opportunistic infection caused by reactivation of JC virus in the central nervous system and is an important differential diagnosis in patients with subacute focal neurologic deficits. Magnetic resonance imaging (MRI) is the most sensitive tool for detection of early manifestations of the disease1 . Case Report: A 42-year-old woman with a previous diagnosis of HIV infection, without treatment for the last two years, presented with progressive headache, left hemiparesis, hypoesthesia and homonymous hemianopia in the last two months. CD4 cell count was 16 cel/mm3 and viral load of 99.364 copies/mL. Brain MRI revealed multifocal, subcortical and confluent hyperintense T2/FLAIR lesions in the right parietooccipital lobe, crossing the midline by the corpus callosum, with hyperintense peripheral rim on DWI-image and a central hypointense core, without mass effect. On T2-weighted image, typical multiple punctiform hyperintensities formed the Punctate Pattern, which is known as the Milky Way appearance when nearby a larger PML lesion. The final diagnosis was confirmed by the detection of JC virus on cerebrospinal fluid by PCR. Discussion: The punctate pattern is characterized by at least three punctiform (<5mm) hyperintense lesions on T2/FLAIR images, with or without contrast enhancement. It has already been described in PML, neurosarcoidosis, hematologic diseases, CLIPPERS and CNS vasculitis. Recently it was described as a highly specific feature of PML related to natalizumab, even in pre-symptomatic stages. Further studies are required stablish its incidence in patients with PML from other causes2.

Neuroimaging Insights Into Early Stages of HIV-Progressive Multifocal Leukoencephalopathy: A Case Report

This report aims to enhance the understanding of early longitudinal neuroimaging features of progressive multifocal leukoencephalopathy (PML) in human immunodeficiency virus (HIV). Neuroimaging has become crucial in the diagnosis and early recognition of PML. Recognition of magnetic resonance imaging (MRI) features in the early stages of PML is paramount to avoid misdiagnosis and facilitate the delivery of treatments aimed at reducing disease progression. A 49-year-old white man with HIV presented with 4-month progressive left-sided weakness. Neurological examination revealed mild cognitive impairment, left-sided hemiparesis, and somatosense impairment to all modalities. Brain MRI revealed a punctate pattern with innumerable T2-FLAIR (fluid attenuated inversion recovery) hyperintensities in the cortex, brainstem, cerebellum, subcortical, and periventricular areas. Susceptibility-weighted imaging (SWI) revealed hypointensities involving subcortical U-fibers and cortical architecture. A comprehensive diagnostic evaluation was inconclusive. John Cunningham virus (JCV) PCR in cerebrospinal fluid (CSF) was indeterminate. He was started on antiretroviral therapy. Repeat brain MRI performed 1.5 months later, in the setting of further neurological decline, demonstrated progression of the T2-hyperintensities into a large confluent white matter lesion in the right frontoparietal lobe. Despite an indeterminate JCV PCR, the appearance and characteristic progression of the lesions in successive imaging in the setting of severe immunosuppression, with extensive negative infectious workup, was indicative of PML. This clinical experience illustrates unique neuroimaging features of HIV-PML in early stages and its progression over time. It especially highlights the relevance of the SWI sequence in the diagnosis and features observed with disease evolution. Short-term imaging follow-up may assist with the recognition of MRI features consistent with the biology of the infection.

Comparative study of papanicolaou smear and colposcopy in the evaluation of cervical lesions

Background: Cervical cancer is the third most common type of cancer among females. Study aims to critically evaluate the sensitivity and specificity of colposcopy versus papanicolaou (Pap) smear in the early detection of dysplasias. Its secondary objective to correlate the findings in the evaluation of unhealthy cervix by cytology, colposcopy and colposcopy guided biopsy.Methods: This was a tertiary care teaching hospital based, prospective, cross sectional study done in Department of Obstetrics and Gynaecology, at Vydehi Institute of Medical Sciences and Research Centre, Bangalore, conducted on 200 women attending Gynaecology OPD.Results: PAP smear was taken for all 200 patients. 73% of smear was found to be normal, 11% showed inflammatory atypia, 9% showed low grade squamous intraepithelial lesion (LSIL), 3.5% showed atypical squamous cells of undetermined significance (ASCUS) and 3.5% showed High Grade Squamous Intraepithelial Lesion (HSIL). Among the 200 cases studied, 38% (76/200) were diagnosed as colposcopically abnormal. Among the abnormal cases, AW areas were diagnosed in 4%. Punctate pattern of vessels was seen in 5% of women. Normal findings was present in 62%, Erosion cervix in 6%, inflammatory changes were seen in 6% and polyps were diagnosed in 7.5%, leucoplakia was found in 2% and unsatisfactory colposcopy finding was seen in 4% and underwent endocervical curettage. 32 cases out of 200 women were positive on Pap smear. 66 out of 200 women were positive on Biopsy. Pap smear was positive in 22 out of 66 biopsy proven positive cases.Conclusions: The commonest presenting complaint was vaginal discharge (182/200; 91% of the patients. the PAP smear is found to have sensitivity of 33.33% and specificity of 92.54%. colposcopy is found to have sensitivity of 81.82% and specificity of 82.84%.

Protein arginine methyltransferases interact with intraflagellar transport particles and change location during flagellar growth and resorption

Changes in protein by posttranslational modifications comprise an important mechanism for the control of many cellular processes. Several flagellar proteins are methylated on arginine residues during flagellar resorption; however, the function is not understood. To learn more about the role of protein methylation during flagellar dynamics, we focused on protein arginine methyltransferases (PRMTs) 1, 3, 5, and 10. These PRMTs localize to the tip of flagella and in a punctate pattern along the length, very similar, but not identical, to that of intraflagellar transport (IFT) components. In addition, we found that PRMT 1 and 3 are also highly enriched at the base of the flagella, and the basal localization of these PRMTs changes during flagellar regeneration and resorption. Proteins with methyl arginine residues are also enriched at the tip and base of flagella, and their localization also changes during flagellar assembly and disassembly. PRMTs are lost from the flagella of fla10-1 cells, which carry a temperature-sensitive mutation in the anterograde motor for IFT. The data define the distribution of specific PRMTs and their target proteins in flagella and demonstrate that PRMTs are cargo for translocation within flagella by the process of IFT.

The odd one out: Arabidopsis reticulon20 has a role in lipid biosynthesis

The family of reticulon proteins has been shown to be involved in a variety of functions in eukaryotic cells including tubulation of the endoplasmic reticulum (ER), formation of cell plates and primary plasmodesmata. Reticulons are integral ER membrane proteins characterised by a reticulon homology domain comprising four transmembrane domains which results in the reticulons sitting in the membrane in a W-topology. Here we report on a subgroup of reticulons with an extended N-terminal domain and in particular on arabidopsis reticulon 20. We show that reticulon 20 is located in a unique punctate pattern on the ER membrane. Its closest homologue reticulon 19 labels the whole ER. We show that mutants in RTN20 or RTN19, respectively, display a significant change in sterol composition in the roots indicating a role in lipid biosynthesis or regulation. A third homologue in this family - 3BETAHSD/D1- is localised to ER exit sites resulting in an intriguing location difference for the three proteins.

N-cadherin relocalization during cardiac trabeculation

During cardiac trabeculation, cardiomyocytes delaminate from the outermost (compact) layer to form complex muscular structures known as trabeculae. As these cardiomyocytes delaminate, the remodeling of adhesion junctions must be tightly coordinated so cells can extrude from the compact layer while remaining in tight contact with their neighbors. In this study, we examined the distribution of N-cadherin (Cdh2) during cardiac trabeculation in zebrafish. By analyzing the localization of a Cdh2-EGFP fusion protein expressed under the control of the zebrafish cdh2 promoter, we initially observed Cdh2-EGFP expression along the lateral sides of embryonic cardiomyocytes, in an evenly distributed pattern, and with the occasional appearance of punctae. Within a few hours, Cdh2-EGFP distribution on the lateral sides of cardiomyocytes evolves into a clear punctate pattern as Cdh2-EGFP molecules outside the punctae cluster to increase the size of these aggregates. In addition, Cdh2-EGFP molecules also appear on the basal side of cardiomyocytes that remain in the compact layer. Delaminating cardiomyocytes accumulate Cdh2-EGFP on the surface facing the basal side of compact layer cardiomyocytes, thereby allowing tight adhesion between these layers. Importantly, we find that blood flow/cardiac contractility is required for the transition from an even distribution of Cdh2-EGFP to the formation of punctae. Furthermore, using time-lapse imaging of beating hearts in conjunction with a Cdh2 tandem fluorescent protein timer transgenic line, we observed that Cdh2-EGFP molecules appear to move from the lateral to the basal side of cardiomyocytes along the cell membrane, and that Erb-b2 receptor tyrosine kinase 2 (Erbb2) function is required for this relocalization.