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.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.Peer reviewe

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

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

Clinical presentation and bacteriological profile of diabetic foot in Eastern Bihar, India

Background: Diabetes is a worldwide problem. A majority of diabetic patients develop foot ulcers in one point of time or other during the course of their illness. Chronic wound, especially non-healing types are the most common surgical conditions. The etiopathogenesis of diabetic foot lesions are multi-factorial like diabetic neuropathies, vasculopathy, poor control of diabetes and bacterial infection. The aim of the present study was to study various modes of presentation and microbiological profile in management of diabetic foot.Methods: 100 diagnosed cases of diabetic foot were studied over a period of three years in the department of General Surgery at Katihar Medical College with emphasis on clinical features and microbiological picture of diabetic foot and its complications.Results: Males are more prone to surgical complications three times than females because of more outdoor activities. It is more prevalent in age group 51-60 years (36%). In the present study, cases presenting with ulcer were maximum (52%) followed by cellulitis (20%), cases presenting with gangrene of toe or foot was minimum (12%). Staphylococcus Aureus was found in majority of cases of septic lesions on culture of pus (41%). Other organisms isolated were Pseudomonas, Klebsiella, E.Coli, Proteus etc.Conclusions: Diabetic foot has varied presentation. The prevalence of diabetes mellitus and its surgical complications can be attributed to poor patient knowledge, education and awareness of the disease. Patient education for care of feet such as pairing of nails, wearing proper footwear and prompt reporting to doctor in case of early lesions is essential.

Comparative Study of AFB Detection in Concentrated and Unconcentrated Sputum Sample by Ziehl-Neelsen Staining and Auramine-O Staining of Patients Attending the Microscopic Centre of RNTCP at Darbhanga Medical College & Hospital, Laheriasarai

Ziehl-Neelsen is a common bacteriological staining method used from a long time to stain acid-fast bacilli, especially Mycobacterium tuberculosis which causes mainly pulmonary tuberculosis. In recent technologies, fluorescent-staining is considered to be a more reliable method due to more intensive binding of mycolic acids of the bacilli to phenol auramine-O, so the tubercle bacilli is seen more clearly against black background.Objective: This study was done to compare the efficacy of conventional Ziehl-Neelsen (ZN) and Auramine-O (AO) fluorescent microscopy in detecting acid-fast bacilli in direct and concentrated sputum samples of patients attending the microscopic centre of RNTCP at Darbhanga Medical College & Hospital, Laheriasarai.Method: One thousand and fifty patients suspected of having pulmonary tuberculosis referred to the RNTCP centre of Darbhanga Medical College and Hospital was included in this study. Spot sputum sample was collected as the clinical sample. Direct smears were prepared from the mucopurulent part of the sputum with a sterile loop. Samples were then concentrated using modified Petroff’s method and smear prepared from the concentrated sediment. Both smears were then stained by ZN and AO staining method respectively.Result: Out of 1050 samples, 165 samples were positive by AO method in direct method and 166 samples were positive by AO method in concentrated method, 147 were positive by ZN staining in direct method and 156 samples were positive by ZN in concentrated method.Conclusion: FM definitely improves the diagnostic value of the sputum smear especially in patients with low density of bacilli that are likely to be missed on ZN-stained smears, concentrated method on both AO and ZN stain were more sensitive than direct method

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

18 alpha-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 18 alpha-glycyrrhetinic acid (18 alpha-GA; a previously shown proteasome activator in cell culture) results in enhanced levels of proteasome activities that lead to a skinhead-1- and proteasomeactivation-dependent life span extension. The elevated proteasome function confers lower paralysis rates in various AD nematode models accompanied by decreased A beta 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 18 alpha-GA treatment. Innovation: This is the first report of the use of 18 alpha-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.Peer reviewe

In-vitro Anti-Ulcer Activities of Mallotus Japonicus

Objectives: This study\u27s objective was to investigate whether or not a methanolic extract of Mallotus japonicas could decrease H+-K+ ATPase activity and neutralise acid. Materials and Methods: We assessed the total phenolic and flavonoid contents of the sample while it was exposed to varying amounts of standard esmoprazole and methanol extract. Results: The proton pump inhibitory activity of the extract from stomach mucosal homogenate was found to be significant (*P<0.05) and on par with the standard. Conclusions: Based on these findings, it may be concluded that the proton pump can be effectively blocked by the methanolic extract

XHX - A Framework for Optimally Secure Tweakable Block Ciphers from Classical Block Ciphers and Universal Hashing

Tweakable block ciphers are important primitives for designing cryptographic schemes with high security. In the absence of a standardized tweakable block cipher, constructions built from classical block ciphers remain an interesting research topic in both theory and practice. Motivated by Mennink\u27s F[2] publication from 2015, Wang et al. proposed 32 optimally secure constructions at ASIACRYPT\u2716, all of which employ two calls to a classical block cipher each. Yet, those constructions were still limited to n-bit keys and n-bit tweaks. Thus, applications with more general key or tweak lengths still lack support. This work proposes the XHX family of tweakable block ciphers from a classical block cipher and a family of universal hash functions, which generalizes the constructions by Wang et al. First, we detail the generic XHX construction with three independently keyed calls to the hash function. Second, we show that we can derive the hash keys in efficient manner from the block cipher, where we generalize the constructions by Wang et al.; finally, we propose efficient instantiations for the used hash functions

PIM-related kinases selectively regulate olfactory sensations in C. elegans

The mammalian PIM family of serine/threonine kinases regulate several cellular functions, such as cell survival and motility. Since PIM expression is observed in sensory organs, such as olfactory epithelium, we now wanted to explore the physiological roles of PIM kinascs 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 analysed 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 AWC(ON) 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 NM-related kinases, our data may have implications in regulation of also mammalian olfaction.Peer reviewe