BAP1 Malignant Pleural Mesothelioma Mutations in Caenorhabditis elegans Reveal Synthetic Lethality between ubh-4/BAP1 and the Proteasome Subunit rpn-9/PSMD13.

The deubiquitinase BAP1 (BRCA1-associated protein 1) is associated with BAP1 tumor predisposition syndrome (TPDS). BAP1 is a tumor suppressor gene whose alterations in cancer are commonly caused by gene mutations leading to protein loss of function. By CRISPR-Cas, we have generated mutations in ubh-4, the BAP1 ortholog in Caenorhabditis elegans, to model the functional impact of BAP1 mutations. We have found that a mimicked BAP1 cancer missense mutation (UBH-4 A87D; BAP1 A95D) resembles the phenotypes of ubh-4 deletion mutants. Despite ubh-4 being ubiquitously expressed, the gene is not essential for viability and its deletion causes only mild phenotypes without affecting 20S proteasome levels. Such viability facilitated an RNAi screen for ubh-4 genetic interactors that identified rpn-9, the ortholog of human PSMD13, a gene encoding subunit of the regulatory particle of the 26S proteasome. ubh-4[A87D], similarly to ubh-4 deletion, cause a synthetic genetic interaction with rpn-9 inactivation affecting body size, lifespan, and the development of germ cells. Finally, we show how ubh-4 inactivation sensitizes animals to the chemotherapeutic agent Bortezomib, which is a proteasome inhibitor. Thus, we have established a model to study BAP1 cancer-related mutations in C. elegans, and our data points toward vulnerabilities that should be studied to explore therapeutic opportunities within the complexity of BAP1 tumors.

Subcutaneous entomophthoramycosis in a child presenting as panniculitis: a case report from Bihar, India.

A 5-year-boy from Bihar, India was admitted to a tertiary care hospital with painful swelling over both lower limbs and buttocks, which had been increasing progressively for the past 1 year. The condition was initially undiagnosed and was later misdiagnosed as non-infective panniculitis, delaying treatment. Subsequently, the patient was diagnosed with subcutaneous entomophthoramycosis caused by Basidiobolus spp. A preliminary diagnosis was made by considering the history, clinical features, radiological findings and histopathological examination of the biopsied tissue. The confirmatory diagnosis was made using conventional techniques on aspirated pus, which included KOH wet mount and fungal culture on Sabouraud dextrose agar tubes incubated at 28°C and 37°C, respectively. Lactophenol cotton blue mount and slide culture were performed for identification of the fungal isolate. The patient responded well to oral itraconazole and oral potassium iodide. Delayed diagnosis and extensive involvement in a rare case of subcutaneous entomophthoramycosis causing panniculitis emphasizes the importance of correct diagnosis and appropriate, effective treatment.

Blastocystis spp. infection in cases of diarrhea: A pilot study from a tertiary care teaching hospital in Rishikesh, Uttarakhand, with a brief review of literature.

CONTEXT: Intestinal parasitic infections (IPI) are among the most common infections throughout the world. Blastocystis spp. is a mysterious parasite which is commonly encountered in tropical countries. Its pathogenic status is unknown and there is a paucity of literature about this organism from the state of Uttarakhand, India. AIMS: The aim was to estimate the prevalence of Blastocystis spp. in diarrheal stools. SETTINGS AND DESIGN: This was a cross-sectional study conducted from January 2018 to July 2019. SUBJECTS AND METHODS: Nonrepetitive stool samples of 187 consecutive patients of diarrhea attending the inpatient department and outpatient department of a tertiary care teaching hospital located in Rishikesh, Uttarakhand, were collected after obtaining informed written consent. These samples were subjected to wet mount microscopy and permanent staining. STATISTICAL ANALYSIS USED: Fisher's exact test and Kappa coefficient were used in this study. RESULTS: The mean age ± standard deviation of the patients was 36.04 ± 11.31 years with a male-to-female ratio of 1.49:1. The prevalence of IPI was 36.09%. Giardia intestinalis was the most common parasite. Blastocystis spp. was observed in 6.42% of the stool samples, majority of which were obtained from cases of chronic diarrhea. Moderate agreement (0.48) was observed between wet mount microscopy and permanent staining in the identification of Blastocystis spp. CONCLUSIONS: This is the first study to assess the burden and role of different epidemiological and clinical profiles of Blastocystis spp. in Uttarakhand. More studies are required to know its pathogenesis and its role as opportunistic pathogen.

Tissue-Specific Impact of Autophagy Genes on the Ubiquitin-Proteasome System in C. elegans.

The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the two main eukaryotic intracellular proteolytic systems involved in maintaining proteostasis. Several studies have reported on the interplay between the UPS and ALP, however it remains largely unknown how compromised autophagy affects UPS function in vivo. Here, we have studied the crosstalk between the UPS and ALP by investigating the tissue-specific effect of autophagy genes on the UPS at an organismal level. Using transgenic Caenorhabditis elegans expressing fluorescent UPS reporters, we show that the downregulation of the autophagy genes lgg-1 and lgg-2 (ATG8/LC3/GABARAP), bec-1 (BECLIN1), atg-7 (ATG7) and epg-5 (mEPG5) by RNAi decreases proteasomal degradation, concomitant with the accumulation of polyubiquitinated proteasomal substrates in a tissue-specific manner. For some of these genes, the changes in proteasomal degradation occur without a detectable alteration in proteasome tissue expression levels. In addition, the lgg-1 RNAi-induced reduction in proteasome activity in intestinal cells is not dependent on sqst-1/p62 accumulation. Our results illustrate that compromised autophagy can affect UPS in a tissue-specific manner, and demonstrate that UPS does not function as a direct compensatory mechanism in an animal. Further, a more profound understanding of the multilayered crosstalk between UPS and ALP can facilitate the development of therapeutic options for various disorders linked to dysfunction in proteostasis.

Curvularia lunata causing orbital cellulitis in a diabetic patient: An old fungus in a new territory.

BACKGROUND AND PURPOSE: Rhinocerebral mycosis is a rapidly invasive infection in diabetic patients with an unfavorable course. Herein, we report a rare case of orbital cellulitis caused by Curvularia lunata following fungal rhinosinusitis in a diabetic male patient. CASE REPORT: A 35-year-old male with uncontrolled diabetes presented to the emergency department of our center with high-grade fever accompanied by chills and rigors, severe diffuse headache, and projectile vomiting with swelling and loss of vision in the right eye. The tissue sample from surgical debridement showed pigmented hyphae; in addition, Curvularia lunata was isolated in culture. Imaging was indicative of orbital extension. Therefore, the patient was diagnosed with fungal rhinosinusitis with orbital cellulitis. The patient was subjected to extensive surgical debridement, along with antifungals. Rhinosinusitis resolved; however, the loss of vision was irreversible. CONCLUSION: Orbital cellulitis is a very rare but life-threatening complication of fungal rhinosinusitis. Very few cases of orbital cellulitis following fungal rhinosinusitis have been reported in the literature. Early and prompt diagnosis can save the life of a patient.

Clinico-epidemiological spectrum of strongyloidiasis in India: Review of 166 cases.

Strongyloidiasis is frequently asymptomatic but can cause disseminated disease and variable presentations. Diagnosis is often delayed or misdirected either due to poor degree of clinical suspicion or clinical imitation of other gastrointestinal conditions. This infection is not infrequent and several cases from all over India have been reported barring few states from central India. We reviewed 166 cases published in English literature from India; from 2001 till 2018 including 2 recent cases from our institute. The mean age of presentation was 35 years with male female ratio of 2.8:1. The duration of disease at the time of presentation varied from 15 days to 10 years. Most important predisposing factor identified in the study was HIV (13.3%) and steroid therapy (6.6%). Most common modality of diagnosis was by stool microscopy (69.3%). Radiological investigations were ordered in 33.7% patients before stool microscopy. Ivermectin was the most common treatment regimen with cure rate of 97.6%. Better awareness and early clinical suspicion of the disease with stool microscopy and adequate therapy are necessary to improve the outcome. Strongyloidiasis is rather widely prevalent infection with variable symptomatology and calls for a close coordination from family physicians and microbiologists.

PIM-Related Kinases Selectively Regulate Olfactory Sensations in Caenorhabditis elegans.

The mammalian PIM family of serine/threonine kinases regulate several cellular functions, such as cell survival and motility. Because PIM expression is observed in sensory organs, such as olfactory epithelium, we now wanted to explore the physiological roles of PIM kinases there. As our model organism, we used the Caenorhabditis elegans nematodes, which express two PIM-related kinases, PRK-1 and PRK-2. We demonstrated PRKs to be true PIM orthologs with similar substrate specificity as well as sensitivity to PIM-inhibitory compounds. When we analyzed the effects of pan-PIM inhibitors on C. elegans sensory functions, we observed that PRK activity is selectively required to support olfactory sensations to volatile repellents and attractants sensed by AWB and AWCON neurons, respectively, but is dispensable for gustatory sensations. Analyses of prk-deficient mutant strains confirmed these findings and suggested that PRK-1, but not PRK-2 is responsible for the observed effects on olfaction. This regulatory role of PRK-1 is further supported by its observed expression in the head and tail neurons, including AWB and AWC neurons. Based on the evolutionary conservation of PIM-related kinases, our data may have implications in regulation of also mammalian olfaction.

Fluorescent Tools for In Vivo Studies on the Ubiquitin-Proteasome System.

The ubiquitin-proteasome system (UPS) plays a key role in maintaining proteostasis by degrading most of the cellular proteins. Traditionally, UPS activity is studied in vitro, in yeast, or in mammalian cell cultures by using short-lived GFP-based UPS reporters. Here, we present protocols for two fluorescent tools facilitating real-time imaging of UPS activity in living animals. We have generated transgenic Caenorhabditis elegans (C. elegans) expressing a photoconvertible UbG76V-Dendra2 UPS reporter, which permits measurement of reporter degradation by the proteasome independently of reporter protein synthesis, and a fluorescent polyubiquitin-binding reporter for detection of the endogenous pool of Lys48-linked polyubiquitinated proteasomal substrates. These reporter systems facilitate cell- and tissue-specific analysis of UPS activity especially in young adult animals, but can also be used for studies during development, aging, and for example stress conditions.

18α-Glycyrrhetinic Acid Proteasome Activator Decelerates Aging and Alzheimer's Disease Progression in Caenorhabditis elegans and Neuronal Cultures.

AIMS: Proteasomes are constituents of the cellular proteolytic networks that maintain protein homeostasis through regulated proteolysis of normal and abnormal (in any way) proteins. Genetically mediated proteasome activation in multicellular organisms has been shown to promote longevity and to exert protein antiaggregation activity. In this study, we investigate whether compound-mediated proteasome activation is feasible in a multicellular organism and we dissect the effects of such approach in aging and Alzheimer's disease (AD) progression. RESULTS: Feeding of wild-type Caenorhabditis elegans with 18α-glycyrrhetinic acid (18α-GA; a previously shown proteasome activator in cell culture) results in enhanced levels of proteasome activities that lead to a skinhead-1- and proteasome activation-dependent life span extension. The elevated proteasome function confers lower paralysis rates in various AD nematode models accompanied by decreased Aß deposits, thus ultimately decelerating the progression of AD phenotype. More importantly, similar positive results are also delivered when human and murine cells of nervous origin are subjected to 18α-GA treatment. INNOVATION: This is the first report of the use of 18α-GA, a diet-derived compound as prolongevity and antiaggregation factor in the context of a multicellular organism. CONCLUSION: Our results suggest that proteasome activation with downstream positive outcomes on aging and AD, an aggregation-related disease, is feasible in a nongenetic manipulation manner in a multicellular organism. Moreover, they unveil the need for identification of antiaging and antiamyloidogenic compounds among the nutrients found in our normal diet. Antioxid. Redox Signal. 25, 855-869.