Deciphering the BH3 code for the neutralization of pro-survival Bcl-2 proteins in membranes

The proteins of Bcl-2 family, the pro-survival and the pro-apoptotic tightly regulates the process of apoptosis. The pro-survival proteins show a specific interaction pattern with BH3 domain of BH3 only proteins, determining the cellular fate during apoptotic stress. This interaction specificity is pivotal in designing BH3 mimetics, a class of anticancer drug molecules based on the BH3 domain of BH3 only proteins showing promising results in clinical trials. The role of the mitochondrial outer membrane in exhibiting Bcl2 complex interactome is extensively studied recently. Overall most studies addressed so far on the interactions of BH3 peptides and the truncated Bcl-2 proteins are reported in the solution / cytosolic environment while the quantitative interactions in membranes are still missing. To tackle this, we systematically quantified the library of BH3 peptides using two-color fluorescence correlation spectroscopy in solution and in the model membrane. We further extended our investigations to isolated yeast mitochondria using ensemble FRET and in mammalian cancer cell lines using a high throughput screening called BH3 profiling. We show that BH3 peptides derived from Hrk and Bim are the most effective in disrupting cBid/Bcl-xL complexes, which correlates with their response in mitochondria and in cells. Moreover, to understand the activation process of pro-apoptotic effector protein Bax on membranes, we designed an ​in-vitro system to investigate its autoactivation by the recruitment of inactive cytosolic Bax molecules by active membrane-bound Bax. Furthermore, ​in vitro studies also showed active membrane-bound Bax recruits Bcl-xL to the membrane, which retrotranslocates active Bax back into the cytosol, thereby maintaining membrane integrity. Quantitative analysis showed that Bax retrotranslocation activity potentiates Bcl-xL antiapoptotic activity by at least 10 fold. Overall, these findings highlights the importance of the membrane in Bcl-2 family interactions and thereby screening peptides that can disrupt specific interactions of these proteins in the membrane and can improve cancer therapies.Der mitochondriale Apoptosesignalweg wird durch die anti- und pro-apoptotischen Vertreter der Bcl-2 Proteinfamilie kontrolliert. Die anti-apoptotischen Proteine spielen eine zentrale Rolle, in dem sie durch die selektive Interaktion mit der BH3 Domäne der BH3-only Proteinen den apoptotischen Prozess regulieren. Basierend auf dieser Interaktion und der BH3 Domäne werden BH3-Mimetika, eine Gruppe von anti-Krebs Medikamenten, entworfen. BH3-Mimetika zeigen in klinischen Studien eine signifikante Aktivierung von Apoptose, indem sie diese Interaktion stören. Der Einfluss der äußeren Mitochondrienmembran auf die Interaktion der Bcl-2 Proteinen wird zur Zeit detailliert untersucht. Bisher wurde ausschließlich die Interaktion der BH3 Peptiden mit den verkürzten Formen der Bcl-2 Proteinen nur in Lösung und im Zytoplasma untersucht, während quantitative Interaktionen in Membranen komplett außer Acht gelassen wurden. Um diese Fragestellung anzugehen, haben wir systematisch die Interaktion bestehender BH3 Peptiden mit Hilfe der zwei Farben Fluoreszenz-Korrelations- Spektroskopie, sowohl in Lösung als auch in Modell Membranen, quantifiziert. Wir erweiterten unsere Untersuchungen auf isolierte Hefe Mitochondrien mit Hilfe der FRET Analyse und auf Säugetier Krebszelllinien mit Hilfe des Hochdurchsatz-Screenings BH3 Profiling. Wir zeigen, dass die BH3 Peptide, die von Hrk und Bim abgeleitet wurden, die effektivesten in der Störung des cBid / Bcl-xL Komplexes seien. Diese Daten spiegeln die Antwort in Mitochondrien und in Zellen wieder. Um den Aktivierungsprozess des pro-apoptotischen Effektorproteins Bax an Membranen zu verstehen, haben wir ein in-vitro Assay entwickelt, das erlaubt, die Autoaktivierung von Bax zu untersuchen. Das aktive, membrangebundene Bax rekrutiert dabei das inaktive, zytoplasmatische Bax zu der Membran. Des Weiteren haben in-vitro Analysen gezeigt, dass membrangebundenes Bax, Bcl-xL zu der Membran rekrutiert. Bcl-xL retrotranloziert aktives, nicht oligomerisiertes Bax zurück in das Zytoplasma, um die Membranintegrität aufrecht zu erhalten. Quantitative Analysen zeigen, dass die Bax-retrotranslokationsaktivität die anti-apoptotische Bcl-xL-Aktivität um mindestens das 10fache verstärkt. Unsere Ergebnisse zeigen die Wichtigkeit der Membran für die Interaktion der Bcl-2 Proteinen auf, deshalb ist das Screening von Peptiden, die die Interaktion dieser Proteine an der Membran stört von großer Bedeutung für die Krebstherapie

Environmental study on water quality of Mayur River with reference to suitability for irrigation

ABSTRACT The farmers residing in the western fringe of Khulna city in Bangladesh use the sewage-fed water of the Mayur River for irrigation as good quality surface water is not available as well as higher cost in groundwater irrigation. The present study was undertaken to evaluate the suitability of this river water for irrigation during the hot summer months (March to May) since this period is more sensitive to crop agriculture in the study area. A total of 30 water samples, 10 in each month from 10 stations, were collected and analyzed for pH, EC, TDS, major cations (Na, K, Ca, Mg) and anions (Cl, HCO 3 , NO 3 , PO 4 , SO 4 ), Chemical data were used for calculation of SAR, Na%, RSC, PI, KI and MR for better understanding the suitability of river water for irrigation use. Wilcox diagram and USSL diagram were also adopted in the present study to verify the suitability of river water quality for irrigation. The results revealed that water of the Mayur River was alkaline in nature like major world rivers. Sodium was the most dominant cation throughout the sampling period, whereas sulfate was the most dominat anion in March and May, and chloride in April. The river water was found to be safe for irrigation with respect to pH and PI. However, some usual and calculated parameters like EC, TDS, hardness, alkalinity, chloride, sulfate, nitrate, sodium, Na%, SAR, KI, MR and Ca/Mg restricted the Mayr River water for use in irrigation. The RSC values indicated the water to be safe during April and May and permissible to severe in March. The USSL and Wilcox diagrams indicated high salinity in the water with high sodium being unsuitable for irrigation. The findings call for an immediate management plan to protect this invaluable resource

MERLIN: a novel BRET-based proximity biosensor for studying mitochondria–ER contact sites

The contacts between the ER and mitochondria play a key role in cellular functions such as the exchange of lipids and calcium between both organelles, as well as in apoptosis and autophagy signaling. The molecular architecture and spatiotemporal regulation of these distinct contact regions remain obscure and there is a need for new tools that enable tackling these questions. Here, we present a new bioluminescence resonance energy transfer-based biosensor for the quantitative analysis of distances between the ER and mitochondria that we call MERLIN (Mitochondria-ER Length Indicator Nanosensor). The main advantages of MERLIN compared with available alternatives are that it does not rely on the formation of artificial physical links between the two organelles, which could lead to artifacts, and that it allows to study contact site reversibility and dynamics. We show the applicability of MERLIN by characterizing the role of the mitochondrial dynamics machinery on the contacts of this organelle with the ER.We thank Peter McCormick for helpful advice and discussion and Carolin Stegmuller, Sabine Schafer, Iris Koch, Maria Zarani, Astrid Schauss, Christian Jungst, Felix Babatz, and Marina Nikolova for technical support. This work has been partially supported by the Deutsche Forschungsgemeinschaft (FOR2036 GA1641/2-1 and GA1641/2-2) and the European Research Council (StG 309966)

Studies on cooking of tussah silk cocoon

61-67<span style="font-size:11.0pt;line-height:115%; font-family:" calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-fareast-font-family:="" "times="" new="" roman";mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:="" minor-latin;mso-bidi-font-family:calibri;mso-bidi-theme-font:minor-latin;="" mso-ansi-language:en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="">An attempt bas been made to optimize the cooking conditions of tussah silk cocoon (Antheraea mylitta D.) with ethylenediamine using the Box and Behnken factorial design for three variables and three levels for each variable. It is observed that the duration and temperature of treatment as well as the concentration of ethylenediamine significantly influence the cocoon shell weight loss and silk filament recovery. Silk recovery improves with the shell weight loss of up to 11.5% after which no further improvement in silk recovery is observed. The mechanical properties of tussah silk filament have very little correlation with the process parameters. For better silk recovery, the following cooking conditions are recommended: ethylenediamine, 10% on the weight of cocoon; temperature, 80ᵒC; duration of treatment, 30 min; and meterial-to-liquor ratio, 1:30. Different races of tussah silk cocoon require adjustment in the duration of treatment. The hardness of water up to 1100 ppm has no significant effect on the recovery of silk in ethylenediamine cooking system.</span