Indirect Immunofluorescence Microscopy Research Articles

State-of-the-art diagnosis of autoimmune blistering diseases.

Autoimmune blistering disorders (AIBDs) are a heterogeneous group of approximately a dozen entities comprising pemphigus and pemphigoid disorders and dermatitis herpetiformis. The exact diagnosis of AIBDs is critical for both prognosis and treatment and is based on the clinical appearance combined with the detection of tissue-bound and circulating autoantibodies. While blisters and erosions on the skin and/or inspectable mucosal surfaces are typical, lesions may be highly variable with erythematous, urticarial, prurigo-like, or eczematous manifestations. While direct immunofluorescence microscopy (IFM) of a perilesional biopsy is still the diagnostic gold standard, the molecular identification of the major target antigens opened novel therapeutic avenues. At present, most AIBDs can be diagnosed by the detection of autoantigen-specific serum antibodies by enzyme-linked immunosorbent assay (ELISA) or indirect IFM when the clinical picture is known. This is achieved by easily available and highly specific and sensitive assays employing recombinant immunodominant fragments of the major target antigens, i.e., desmoglein 1 (for pemphigus foliaceus), desmoglein 3 (for pemphigus vulgaris), envoplakin (for paraneoplastic pemphigus), BP180/type XVII collagen (for bullous pemphigoid, pemphigoid gestationis, and mucous membrane pemphigoid), laminin 332 (for mucous membrane pemphigoid), laminin β4 (for anti-p200 pemphigoid), type VII collagen (for epidermolysis bullosa acquisita and mucous membrane pemphigoid), and transglutaminase 3 (for dermatitis herpetiformis). Indirect IFM on tissue substrates and in-house ELISA and immunoblot tests are required to detect autoantibodies in some AIBD patients including those with linear IgA disease. Here, a straightforward modern approach to diagnosing AIBDs is presented including diagnostic criteria according to national and international guidelines supplemented by long-term in-house expertise.

Display of porcine epidemic diarrhea virus spike protein B-cell linear epitope on Lactobacillus mucosae G01 S-layer surface induce a robust immunogenicity in mice

The Porcine epidemic diarrhea virus (PEDV) presents a substantial risk to the domestic pig industry, resulting in extensive and fatal viral diarrhea among piglets. Recognizing the mucosal stimulation triggered by PEDV and harnessing the regulatory impact of lactobacilli on intestinal function, we have developed a lactobacillus-based vaccine that is carefully designed to elicit a strong mucosal immune response. Through bioinformatics analysis, we examined PEDV S proteins to identify B-cell linear epitopes that meet the criteria of being non-toxic, soluble, antigenic, and capable of neutralizing the virus. In this study, a genetically modified strain of Lactobacillus mucosae G01 (L.mucosae G01) was created by utilizing the S layer protein (SLP) as a scaffold for surface presentation. Chimeric immunodominant epitopes with neutralizing activity were incorporated at various sites on SLP. The successful expression of SLP chimeric immunodominant epitope 1 on the surface of L.mucosae G01 was confirmed through indirect immunofluorescence and transmission electron microscopy, revealing the formation of a transparent membrane. The findings demonstrate that the oral administration of L.mucosae G01, which expresses the SLP chimeric immunodominant gene epitope1, induces the production of secreted IgA in the intestine and feces of mice. Additionally, there is an elevation in IgG levels in the serum. Moreover, the levels of cytokines IL-2, IL-4, IFN-γ, and IL-17 are significantly increased compared to the negative control group. These results suggest that L. mucosae G01 has the ability to deliver exogenous antigens and elicit a specific mucosal immune response against PEDV. This investigation presents new possibilities for immunoprophylaxis against PEDV-induced diarrhea.

GCRV-II invades monocytes/macrophages and induces macrophage polarization and apoptosis in tissues to facilitate viral replication and dissemination.

Grass carp reovirus (GCRV), particularly the highly prevalent type II GCRV (GCRV-II), causes huge losses in the aquaculture industry. However, little is known about the mechanisms by which GCRV-II invades grass carp and further disseminates among tissues. In the present study, monocytes/macrophages (Mo/Mφs) were isolated from the peripheral blood of grass carp and infected with GCRV-II. The results of indirect immunofluorescent microscopy, transmission electron microscopy, real-time quantitative RT-PCR (qRT-PCR), western blot (WB), and flow cytometry analysis collectively demonstrated that GCRV-II invaded Mo/Mφs and replicated in them. Additionally, we observed that GCRV-II induced different types (M1 and M2) of polarization of Mo/Mφs in multiple tissues, especially in the brain, head kidney, and intestine. To assess the impact of different types of polarization on GCRV-II replication, we recombinantly expressed and purified the intact cytokines CiIFN-γ2, CiIL-4/13A, and CiIL-4/13B and successfully induced M1 and M2 type polarization of macrophages using these cytokines through in vitro experiments. qRT-PCR, WB, and flow cytometry analyses showed that M2 macrophages had higher susceptibility to GCRV-II infection than other types of Mo/Mφs. In addition, we found GCRV-II induced apoptosis of Mo/Mφs to facilitate virus replication and dissemination and also detected the presence of GCRV-II virus in plasma. Collectively, our findings indicated that GCRV-II could invade immune cells Mo/Mφs and induce apoptosis and polarization of Mo/Mφs for efficient infection and dissemination, emphasizing the crucial role of Mo/Mφs as a vector for GCRV-II infection.IMPORTANCEType II grass carp reovirus (GCRV) is a prevalent viral strain and causes huge losses in aquaculture. However, the related dissemination pathway and mechanism remain largely unclear. Here, our study focused on phagocytic immune cells, monocytes/macrophages (Mo/Mφs) in blood and tissues, and explored whether GCRV-II can invade Mo/Mφs and replicate and disseminate via Mo/Mφs with their differentiated type M1 and M2 macrophages. Our findings demonstrated that GCRV-II infected Mo/Mφs and replicated in them. Furthermore, GCRV-II infection induces an increased number of M1 and M2 macrophages in grass carp tissues and a higher viral load in M2 macrophages. Furthermore, GCRV-II induced Mo/Mφs apoptosis to release viruses, eventually infecting more cells. Our study identified Mo/Mφs as crucial components in the pathway of GCRV-II dissemination and provides a solid foundation for the development of treatment strategies for GCRV-II infection.

Isolation and Genomic Characterization of the G6P[1]‐Type Sheep Rotavirus in China

Rotavirus A (RVA) is a prevalent cause of enteric diarrhea in infants, bovine, pigs, and sheep globally. Currently, the G6P[1]‐type rotaviruses are prevalent in sheep or goat in Bangladesh, Turkey, and Uganda. However, this genotype has not been reported in Chinese sheep or goat. Therefore, 12 anal swabs were collected from diarrheal sheep in Gansu Province, China, in 2023 and tested for rotavirus using reverse transcription‐polymerase chain reaction (RT‐PCR). Pathological sections and immunohistochemistry were used to observe pathological changes and rotavirus antigens in the duodenum, respectively. The sheep rotavirus was isolated in MA‐104 cells and characterized through indirect immunofluorescence and transmission electron microscopy. The genes of the strain were obtained using the next‐generation sequencing technology and analyzed phylogenetically. One sheep was positive for rotavirus by RT‐PCR, and immunohistochemistry revealed numerous rotavirus antigens in the apical portion of the duodenal villi. Transmission electron microscopy revealed that the strain was characterized by virus particles that were “wheel‐shaped” and measured 70–80 nm in size. The gene constellations of this strain is G6‐P[1]‐I2‐R2‐C2‐M2‐A11‐N2‐T6‐E2‐H3. BLASTn and phylogenetic tree analyses suggest that this strain is likely a recombinant of human rotavirus, goat rotavirus, and bovine rotavirus. The comparison of amino acid similarities revealed three differences in the key antigenic epitopes of the VP7 and VP4 proteins between the GO34 strain and this study strain despite the identical gene constellations of the two strains. To date, this is the first report of this constellation of RVA being found in sheep.

Phosphorylation impacts GLE1 nuclear localization and association with DDX1

Gle1 regulates gene expression at multiple steps from transcription to mRNA export to translation under stressed and non-stressed conditions. To better understand Gle1 function in stressed human cells, specific antibodies were generated that recognized the phosphorylation of threonine residue 102 (T102) in Gle1. A series of in vitro kinase assays indicated that T102 phosphorylation serves as a priming event for further phosphorylation in Gle1's N-terminal low complexity cluster. Indirect immunofluorescence microscopy with the anti-Gle1-pT102 antibodies revealed that basally phosphorylated Gle1 was pre-dominantly nuclear with punctate distribution; however, under sodium arsenite-induced stress, more cytoplasmic localization was detected. Immunoprecipitation with the anti-Gle1-pT102 antibody resulted in co-isolation of Gle1-pT102 with the DEAD-box protein DDX1 in a phosphatase sensitive manner. This suggested Gle1 phosphorylation might be linked to its role in regulating DDX1 during transcription termination. Notably, whereas the total Gle1-DDX1 association was decreased when Gle1 nucleocytoplasmic shuttling was disrupted, co-isolation of Gle1-pT102 and DDX1 increased under the same conditions. Taken together, these studies demonstrated that Gle1 phosphorylation impacts its cellular distribution and potentially drives nuclear Gle1 functions in transcription termination. We propose a model wherein phosphorylation of Gle1 either reduces its nucleocytoplasmic shuttling capacity or increases its binding affinity with nuclear interaction partners.

Retrospective Assessment of Antinuclear Antibody by Indirect Immunofluorescence Microscopy and Immunoblot in Patients with Polyarthritis

Retrospective Assessment of Antinuclear Antibody by Indirect Immunofluorescence Microscopy and Immunoblot in Patients with Polyarthritis

Calcineurin associates with centrosomes and regulates cilia length maintenance.

Calcineurin, or protein phosphatase 2B (PP2B), the Ca2+ and calmodulin-activated phosphatase and target of immunosuppressants, has many substrates and functions that remain uncharacterized. By combining rapid proximity-dependent labeling with cell cycle synchronization, we mapped the spatial distribution of calcineurin in different cell cycle stages. While calcineurin-proximal proteins did not vary significantly between interphase and mitosis, calcineurin consistently associated with multiple centrosomal and/or ciliary proteins. These include POC5, which binds centrins in a Ca2+-dependent manner and is a component of the luminal scaffold that stabilizes centrioles. We show that POC5 contains a calcineurin substrate motif (PxIxIT type) that mediates calcineurin binding in vivo and in vitro. Using indirect immunofluorescence and ultrastructure expansion microscopy, we demonstrate that calcineurin colocalizes with POC5 at the centriole, and further show that calcineurin inhibitors alter POC5 distribution within the centriole lumen. Our discovery that calcineurin directly associates with centriolar proteins highlights a role for Ca2+ and calcineurin signaling at these organelles. Calcineurin inhibition promotes elongation of primary cilia without affecting ciliogenesis. Thus, Ca2+ signaling within cilia includes previously unknown functions for calcineurin in maintenance of cilia length, a process that is frequently disrupted in ciliopathies.

Methanolic extract of Teucrium persicum up-regulates and induces the membrane restoration of E-cadherin protein in PC-3 cells

ABSTRACT Teucrium persicum Boiss. an Iranian endemic plant is used in Iranian traditional medicine. E-cadherin transmembrane protein participates in adherens junctions and is the main partner for β-catenin protein. The GC-MS analysis was used to detect the chemical constituents of the methanolic extract. Its effects on the transcription of the E-cadherin encoding gene, cellular levels, and localization of E-cadherin protein in PC-3 cells were investigated. About 70 chemical constituents were identified. Indirect immunofluorescence microscopy and western blotting results revealed the restoration of E-cadherin protein at cell adhesion contact sites in cells treated with T. persicum extract. Gene expression studies revealed that the extract increased the transcription of the E-cadherin encoding gene in PC-3 cells. These results suggest that T. persicum extract may contain potent compounds that provide further support for the anticancer properties of T. persicum. Surely, detailed molecular investigations are needed to find the mechanism(s) behind these effects.

Computer-aided classification of indirect immunofluorescence patterns on esophagus and split skin for the detection of autoimmune dermatoses.

Autoimmune bullous dermatoses (AIBD) are rare diseases that affect human skin and mucous membranes. Clinically, they are characterized by blister formation and/or erosions. Depending on the structures involved and the depth of blister formation, they are grouped into pemphigus diseases, pemphigoid diseases, and dermatitis herpetiformis. Classification of AIBD into their sub-entities is crucial to guide treatment decisions. One of the most sensitive screening methods for initial differentiation of AIBD is the indirect immunofluorescence (IIF) microscopy on tissue sections of monkey esophagus and primate salt-split skin, which are used to detect disease-specific autoantibodies. Interpretation of IIF patterns requires a detailed examination of the image by trained professionals automating this process is a challenging task with these highly complex tissue substrates, but offers the great advantage of an objective result. Here, we present computer-aided classification of esophagus and salt-split skin IIF images. We show how deep networks can be adapted to the specifics and challenges of IIF image analysis by incorporating segmentation of relevant regions into the prediction process, and demonstrate their high accuracy. Using this semi-automatic extension can reduce the workload of professionals when reading tissue sections in IIF testing. Furthermore, these results on highly complex tissue sections show that further integration of semi-automated workflows into the daily workflow of diagnostic laboratories is promising.

Role of host ciliate Paramecium bursaria mitochondria and trichocysts for symbiotic Chlorella variabilis attachment beneath the host cell cortex.

Symbiotic Chlorella variabilis is encased in the perialgal vacuole (PV) membrane of ciliate Paramecium bursaria. The PV membrane is stably anchored below the host cell cortex by adhesion to host mitochondria. Host trichocysts, which are defensive organelles against predators, are present in the mitochondria and PV membrane vicinity. The mechanism by which PV attaches beneath the host cell cortex remains unknown. When P. bursaria is centrifuged at high speed, the symbiotic algae are displaced from the host cell cortex and concentrate at the posterior end. When centrifugation is stopped, the dislocated algae reattach beneath the host cell cortex with fast cytoplasmic streaming. The densities of mitochondria and trichocysts before and after centrifugation were compared using indirect immunofluorescence microscopy with monoclonal antibodies. Almost all trichocysts were shed by high-speed centrifugation, but dislocated algae could reattach even in the absence of trichocysts. In contrast, host mitochondria were unaffected in localization and number, and the dislocated algae also reattached. These findings suggest trichocysts are unnecessary for algal relocalization and that mitochondria are colocalized with the algae. However, many mitochondria were also present in the cell's anterior region without symbiotic algae. Therefore, not all areas with mitochondria contained algae, but there was an algal localization bias within the host cell.

Analysis of glycans in a Burnt-on/Baked-on (BoBo) model food soil using Microarray Polymer Profiling (MAPP) and immunofluorescence microscopy

Burning of food materials during cooking can increase the difficulty in removal from solid surfaces, forming residual food soils. Using molecular probe-based technologies, the aim of this work was to elucidate the composition and relative abundance of glycans within a Burnt-On/Baked-On (BoBo) model food soil and investigate enzyme systems that may facilitate soil breakdown. Microarray Polymer Profiling identified xylan, arabinoxylan, mixed-linkage glucan and mannan as target substrates for the enzymatic cleaning of BoBo residues from surfaces. Indirect immunofluorescence microscopy revealed that burning resulted in extensive structural modifications and degradation of the three-dimensional architecture of constituent polysaccharide matrices. Results from high-throughput enzyme screening indicate that inclusion of xylan depolymerising enzymes in automatic dishwashing detergents may improve cleaning of recalcitrant, plant glycan-rich BoBo soils. Collectively, this study provides new insight into the composition and removal chemistry of complex, multi-component food soils.

Rare Antinuclear Antibody Patterns: Relevance in Routine Laboratory Reporting

Introduction: Uncommon patterns on Human Epithelial 2 (Hep 2) substrate during Antinuclear Antibody (ANA) screening by Indirect Immunofluorescence (IIF) microscopy are not routinely reported by many laboratories since their clinical relevance is not well established. Aim: To elucidate rare ANA patterns on Hep 2 and their possible association with clinical presentation. Materials and Methods: A retrospective study was conducted on ANA reports in the duration January 2021 to March 2022 at the Department of Laboratory Medicine, Dr. Sulaiman Al Habib Medical Group, Olaya Medical Complex, Riyadh, Saudi Arabia, to recognise rare ANA patterns. Gold standard method of IIF on Hep 2 was used for screening. Statistical evaluation was done to obtain frequencies of various ANA patterns. Those with frequency of less than 1% were classified as rare patterns. Results: Overall, 4207 consecutive ANA reports were evaluated out of which 1388 were positive and 210 (4.99%) demonstrated rare ANA patterns including nuclear, cytoplasmic and mitotic subtypes. Most commonly encountered among the rare ANA patterns was intercellular bridge (AC 27) with frequency of 0.78% (n=33). Systemic Lupus Erythematosus (SLE) (10/210) was the most often observed clinical association with rare cytoplasmic and mitotic patterns at titer ≥1:160. Conclusion: Uncommon ANA patterns may be useful in initial work-up of autoimmune illness hence, should be routinely reported. Further studies to enlighten the significance of these patterns, analogous antibodies could be of diagnostic relevance in autoimmune and other diseases.

An Optimized Stress Granule Detection Method: Investigation of UBQLN2 Effect on Stress Granule Formation.

Stress granules (SGs) are cytosolic ribonucleoprotein granules that form via a liquid-liquid phase separation in response to environmental stresses such as heat, oxidative, and osmotic changes. Due to the condensation of low complexity, hydrophobic regions in core SG components in these highly dynamic granules, defects in SG maintenance and formation have been linked to toxic aggregate formation in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia. However, efforts to dissect mechanisms regulating SG formation and maintenance have been limited by methods of tracking protein-SG localization. Here we describe a method for detecting and quantifying recruitment of cytosolically enriched proteins to SGs by indirect immunofluorescence microscopy. Using this method, we tracked the transient recruitment of the cytosolically enriched ubiquitin-like protein, ubiquilin 2 (UBQLN2), and a number of other factors into SGs, demonstrating its utility (Alexander et al., Proc Natl Acad Sci U S A 115:E11485-E11494, 2018).

Survival and prognostic factors in bullous pemphigoid: A retrospective cohort study.

Background Bullous pemphigoid is the most common subepidermal autoimmune blistering disease. Till now, the reported prognostic factors in bullous pemphigoid vary considerably. Aims The purpose of this study was to determine the overall survival rate and prognostic factors in bullous pemphigoid. Methods We conducted a retrospective cohort study on newly diagnosed bullous pemphigoid patients between July 2001 and November 2019 in a referral unit for autoimmune blistering skin diseases in Romania. Results One hundred forty-eight patients were included in the study. The Kaplan-Meier overall survival rates at 1, 3, 5 and 10 years were respectively 74.2% (95% confidence interval, 67.5-81.6%), 53.4% (45.7-62.2%), 43.6% (35.9-53%) and 31.3% (23.5-41.7%). The median follow-up among survivors was 48 months (interquartile range: 11-150). Ninety (60.8%) patients died during the follow-up period; of them, 38 (42.2%) had active disease at the time of death. Advanced age, neurological diseases, valvular heart disease, malignancies, use of statins, skin infections and extensive cutaneous involvement were linked to poorer outcomes, while the use of topical corticosteroids was associated with increased overall survival. Limitations This study lacks a control cohort to validate the obtained results. It was conducted in a retrospective manner in a single centre. In addition, indirect immunofluorescence microscopy was not performed in all patients. Conclusion Beyond ageing and neurological comorbidities, the prognosis of bullous pemphigoid patients was significantly influenced by the presence of skin infections, valvular heart disease, use of statins and extensive cutaneous involvement. Topical corticosteroid treatment was associated with increased survival in these patients.

Construction of Recombinant Rabies Virus Vectors Expressing H or F Protein of Peste des Petits Ruminants Virus.

Simple SummaryPeste des petits ruminants (PPR) is one of the most contagious and fatal diseases of small ruminants. In this study, two recombinant viruses rSRV9-H and rSRV9-F, which express the envelope glycoprotein H (hemagglutinin protein) or F (fusion protein) protein, respectively, were successfully generated with a rabies virus as vector. The constructed viruses had good proliferative activity and stability and provided potential bivalent inactivated vaccine candidate strains for the prevention of PPR and livestock rabies.Peste des petits ruminants (PPR) is one of the most contagious and fatal diseases of small ruminants in the world and is classified as a category A epidemic disease. It is the target of a global eradication campaign led by the Office International des Epizooties (OIE) and Food and Agriculture Organization of the United Nations (FAO). The PPR live attenuated vaccine is currently the most widely used and approved vaccine, but the use of this vaccine interferes with the serological testing of the PPR elimination program, and there is a potential safety risk. Viral vector vaccines are one of the most promising methods to solve this dilemma. In this study, the full-length infectious clone plasmid of rabies virus (RABV), pD-SRV9-PM-LASV, was used as the backbone, and the envelope glycoprotein H (hemagglutinin protein) or F (fusion protein) gene of PPRV was inserted into the backbone plasmid to construct the infectious clones pD-SRV9-PM-PPRV-H and pD-SRV9-PM-PPRV-F, which express the PPRV H and PPRV F genes, respectively. The correct construction of these infectious clones was verified after sequencing and double digestion. The infectious clones were transfected with a helper plasmid into BSR/T7 cells, and recombinant viruses were successfully rescued by direct immunofluorescence, indirect immunofluorescence, Western blotting, and transmission electron microscopy and named rSRV9-H and rSRV9-F. The results of growth kinetics studies indicated that the inserted gene did not affect virus proliferation. Stability studies revealed that the inserted target gene was stably expressed in recombinant RABV for at least 15 generations. In this study, the recombinant viruses rSRV9-H and rSRV9-F were successfully rescued. The constructed viruses had good proliferative activity and stability and provided potential bivalent inactivated vaccine candidate strains for the prevention of PPR and livestock rabies.

The effect of the BNT162b2 vaccine on antinuclear antibody and antiphospholipid antibody levels.

The Food and Drug Administration (FDA) approved the first SARS-CoV-2 mRNA vaccine (Pfizer-BioNTech) in December 2020. New adverse events have emerged since these vaccines have reached market. Although no clear association between messenger ribonucleic acid (mRNA) vaccines and autoimmunity has emerged, the significance of such an association warrants further exploration. After obtaining consent, a standardized survey on baseline characteristics and other relevant variables was conducted on unvaccinated individuals who were scheduled for vaccination and had not previously contracted COVID-19. Blood samples were collected from participants prior to the first dose, prior to the second dose, and 1 month after the second dose. All collected samples were tested for antinuclear antibody (ANA) titers using indirect immunofluorescence microscopy kits, and antiphospholipid (APS) immunoglobulin M (IgM) and immunoglobulin G (IgG) levels using an enzyme-linked immunoassay (ELISA) technique. ANA titers were positive for 9 participants out of 101 (8.9%) in the first pre-vaccination draw. For the second draw, the number of participants testing positive for ANA decreased to 5 (5%). For the last draw, 6 (5.9%) participants tested positive for ANA titers. One participant tested positive for APS IgM at the first pre-vaccination draw, 2 tested positive at the second draw, and 2 at the third draw. As for APS IgG titers, all participants tested negative in the three draws. McNemar’s test for two dependent categorical outcomes was conducted on all variables and did not show a statistical significance. The McNemar test of these two composite variables (i.e., ANA/APS, first draw vs. ANA/APS, second and third draws) did not show statistical significance. The 2-sided exact significance of the McNemar test was 1.0. The Friedman test also showed no significance (p = 0.459). No association was found between BNT162b2 vaccine administration and changes in APS and ANA titers. The benefits of the BNT162b2 vaccine significantly outweigh any possible risk of autoimmune dysregulation considering the current evidence.

Impaired production of skin barrier lipid acylceramides and abnormal localization of PNPLA1 due to ichthyosis-causing mutations in PNPLA1

BackgroundPNPLA1 is a causative gene of autosomal recessive congenital ichthyosis. The transacylase PNPLA1 produces ω-O-acylceramides (acylceramides), lipids essential for the skin barrier function, by catalyzing the transfer of a linoleic acid from triglycerides to ω-hydroxyceramides. ObjectiveWe aimed to validate the involvement of PNPLA1 mutations found in ichthyosis patients in the pathogenesis and elucidate the correlation between the effects of these mutations on acylceramide-producing activity and ichthyosis pathology. MethodsAcylceramide-producing activity of PNPLA1 mutants was investigated using a cell-based assay system, in which wild-type PNPLA1 or each PNPLA1 mutant was co-overexpressed with the enzymes involved in acylceramide synthesis. The effect of each mutation on the ABHD5-dependent lipid droplet localization of PNPLA1 was examined through indirect immunofluorescence microscopy. ResultsOf 16 PNPLA1 missense mutations, 15 mutations, except the C216R mutation, resulted in a complete loss of acylceramide-producing activity, while the C216R mutation weakly affected this activity. Intracellular localization of mutants with no activity varied among mutants. Two mutants (S19L and D172N) localized in lipid droplets, and eight mutants (S53L, S53W, A59V, T125N, D129E, R166C, P234S, and P235L) partially localized there. Five mutants (A34P, A34T, S53P, K141E, and P163L) localized throughout the cytosol. ConclusionThe PNPLA1 missense mutations examined in this study are responsible for ichthyosis pathology. The weak effect of C216R mutation on acylceramide-producing activity correlates with the mild symptoms of the ichthyosis patient. Sixteen PNPLA1 mutants were classified into four groups based on their acylceramide-producing activity and localization.

Mapping Glucose Uptake, Transport and Metabolism in the Bovine Lens Cortex.

Purpose: To spatially correlate the pattern of glucose uptake to glucose transporter distributions in cultured lenses and map glucose metabolism in different lens regions. Methods: Ex vivo bovine lenses were incubated in artificial aqueous humour containing normoglycaemic stable isotopically-labelled (SIL) glucose (5 mM) for 5 min-20 h. Following incubations, lenses were frozen for subsequent matrix-assisted laser desorption/ionisation (MALDI) imaging mass spectrometry (IMS) analysis using high resolution mass spectrometry. Manually dissected, SIL-incubated lenses were subjected to gas chromatography-mass spectrometry (GC-MS) to verify the identity of metabolites detected by MALDI-IMS. Normal, unincubated lenses were manually dissected into epithelium flat mounts and fibre cell fractions and then subjected to either gel-based proteomic analysis (Gel-LC/MS) to detect facilitative glucose transporters (GLUTs) by liquid chromatography tandem mass spectrometry (LC-MS/MS). Indirect immunofluorescence and confocal microscopy of axial lens sections from unincubated fixed lenses labelled with primary antibodies specific for GLUT 1 or GLUT 3 were utilised for protein localisation. Results: SIL glucose uptake at 5 min was concentrated in the equatorial region of the lens. At later timepoints, glucose gradually distributed throughout the epithelium and the cortical lens fibres, and eventually the deeper lens nucleus. SIL glucose metabolites found in glycolysis, the sorbitol pathway, the pentose phosphate pathway, and UDP-glucose formation were mapped to specific lens regions, with distinct regional signal changes up to 20 h of incubation. Spatial proteomic analysis of the lens epithelium detected GLUT1 and GLUT3. GLUT3 was in higher abundance than GLUT1 throughout the epithelium, while GLUT1 was more abundant in lens fibre cells. Immunohistochemical mapping localised GLUT1 to epithelial and cortical fibre cell membranes. Conclusion: The major uptake site of glucose in the bovine lens has been mapped to the lens equator. SIL glucose is rapidly metabolised in epithelial and fibre cells to many metabolites, which are most abundant in the metabolically more active cortical fibre cells in comparison to central fibres, with low levels of metabolic activity observed in the nucleus.

Molecular insights into the microtubules depolymerizing activity of the IL-8 receptor B antagonist SB225002.

We have previously reported the novel off-target microtubules destabilizing activity of SB225002, a compound that was originally designed as a selective and potent IL-8 receptor B antagonist. In the present study we investigated the reversibility of SB225002 antimitotic effect and provided additional mechanistic insights underlying cell death induction in SW480 human colorectal adenocarcinoma cells. Mitotically arrested cells by SB225002 treatment were isolated by shake-off, and their identity was verified by both flow cytometry and immunoblotting. The reversibility of SB225002 antimitotic effects was investigated by flow cytometry and immunoblotting. Prometaphase arrested cells were imaged via indirect immunofluorescence and confocal microscopy. Activation of CHK1 in mitotically arrested cells was assessed by immunoblotting, and the relationship between CHK1 and mitotic arrest was examined via siRNA-mediated knockdown of CHK1. JNK signaling was evaluated via immunoblotting as well as pharmacological inhibition, followed by flow cytometry. The role of reactive oxygen species (ROS) in cytotoxicity was evaluated by ROS scavenging and flow cytometry. Following SB225002 washout, the mitotic checkpoint was abrogated, and cell cycle perturbations were gradually restored with induction of cell death. Mechanistically, CHK1 checkpoint was activated by SB225002 and occurred downstream of the mitotic checkpoint. In addition, SB225002 activated JNK signaling which contributed to cell death and restrained polyploidy. Furthermore, SB225002 increased intracellular ROS which played a role in mediating SB225002 cytotoxicity. Findings of the present study warrants further development of SB225002 as a lead compound that uniquely targets microtubules dynamics and IL-8 signaling.

Determination and Prevalence of Antinuclear Antibody (ANA) Patterns in Autoimmune Disorders in a Tertiary Care Hospital, Jaipur

Introduction: Autoimmunity is the immune response of antibodies against normal cellular components. With the advent of serological testing for autoimmune disorders, detection of anti-nuclear antibodies (ANA) by indirect immunofluorescence method became the gold standard. ANA screening is non-specific, reliable and a quick way and identifies immunofluorescent patterns linked to autoimmune disorders. Aim: This study was undertaken to identify the prevalence of ANA patterns amongst the various demo graph presented in a tertiary care hospital in Jaipur, Rajasthan. Material and Method: 100 serum samples were screened by indirect immunofluorescence microscopy in the duration of July 2021 to October 2021. Results: 27% of the serum samples presented with positive ANA patterns out of which 62.96% were nuclear speckled, followed by 22.22% of nuclear homogenous pattern and 7.40% were observed to be cytoplasmic speckled. The age bracket with higher positivity were 20-40 years (28%) and 40-60 (28%). There was female predominance noted in the ANA positivity. Conclusion: Nuclear speckled was the most frequent pattern, few unusual patterns were also observed. An observational study is needed to understand not only the epidemiology of autoimmune disorders but also the predictive value of ANA IIF in clinical setup, besides considering the possibility of taking up IIF as a screening tool for autoimmune disorders. Keywords: Antinuclear antibody (ANA), indirect immunofluorescence (IIF), systemic lupus erythematosus (SLE), autoantibody.