Spatial prioritization of dugong habitats in India can contribute towards achieving the 30 × 30 global biodiversity target.

Indian coastal waters are critical for dugong populations in the western Indian Ocean. Systematic spatial planning of dugong habitats can help to achieve biodiversity conservation and area-based protection targets in the region. In this study, we employed environmental niche modelling to predict suitable dugong habitats and identify influencing factors along its entire distribution range in Indian waters. We examined data on fishing pressures collected through systematic interview surveys, citizen-science data, and field surveys to demarcate dugong habitats with varying risks. Seagrass presence was the primary factor in determining dugong habitat suitability across the study sites. Other variables such as depth, bathymetric slope, and Euclidean distance from the shore were significant factors, particularly in predicting seasonal suitability. Predicted suitable habitats showed a remarkable shift from pre-monsoon in Palk Bay to post-monsoon in the Gulf of Mannar, indicating the potential of seasonal dugong movement. The entire coastline along the Palk Bay-Gulf of Mannar region was observed to be at high to moderate risk, including the Gulf of Mannar Marine National Park, a high-risk area. The Andaman Islands exhibited high suitability during pre- and post-monsoon season, whereas the Nicobar Islands were highly suitable for monsoon season. Risk assessment of modelled suitable areas revealed that < 15% of high-risk areas across Andaman and Nicobar Islands and Palk Bay and Gulf of Mannar, Tamil Nadu, fall within the existing protected areas. A few offshore reef islands are identified under high-risk zones in the Gulf of Kutch, Gujarat. We highlight the utility of citizen science and secondary data in performing large-scale spatial ecological analysis. Overall, identifying synoptic scale 'Critical Dugong Habitats' has positive implications for the country's progress towards achieving the global 30 × 30 target through systematic conservation planning.

A novel inhibitory BAK antibody enables assessment of non-activated BAK in cancer cells.

BAX and BAK are pro-apoptotic members of the BCL2 family that are required to permeabilize the mitochondrial outer membrane. The proteins can adopt a non-activated monomeric conformation, or an activated conformation in which the exposed BH3 domain facilitates binding either to a prosurvival protein or to another activated BAK or BAX protein to promote pore formation. Certain cancer cells are proposed to have high levels of activated BAK sequestered by MCL1 or BCLXL, thus priming these cells to undergo apoptosis in response to BH3 mimetic compounds that target MCL1 or BCLXL. Here we report the first antibody, 14G6, that is specific for the non-activated BAK conformer. A crystal structure of 14G6 Fab bound to BAK revealed a binding site encompassing both the α1 helix and α5-α6 hinge regions of BAK, two sites involved in the unfolding of BAK during its activation. In mitochondrial experiments, 14G6 inhibited BAK unfolding triggered by three diverse BAK activators, supporting crucial roles for both α1 dissociation and separation of the core (α2-α5) and latch (α6-α9) regions in BAK activation. 14G6 bound the majority of BAK in several leukaemia cell lines, and binding decreased following treatment with BH3 mimetics, indicating only minor levels of constitutively activated BAK in those cells. In summary, 14G6 provides a new means of assessing BAK status in response to anti-cancer treatments.

Structure of the BAK-activating antibody 7D10 bound to BAK reveals an unexpected role for the α1-α2 loop in BAK activation.

Pro-apoptotic BAK and BAX are activated by BH3-only proteins to permeabilise the outer mitochondrial membrane. The antibody 7D10 also activates BAK on mitochondria and its epitope has previously been mapped to BAK residues in the loop connecting helices α1 and α2 of BAK. A crystal structure of the complex between the Fv fragment of 7D10 and the BAK mutant L100A suggests a possible mechanism of activation involving the α1-α2 loop residue M60. M60 mutants of BAK have reduced stability and elevated sensitivity to activation by BID, illustrating that M60, through its contacts with residues in helices α1, α5 and α6, is a linchpin stabilising the inert, monomeric structure of BAK. Our data demonstrate that BAK's α1-α2 loop is not a passive covalent connector between secondary structure elements, but a direct restraint on BAK's activation.

Structure of detergent-activated BAK dimers derived from the inert monomer.

A body of data supports the existence of core (α2-α5) dimers of BAK and BAX in the oligomeric, membrane-perturbing conformation of these essential apoptotic effector molecules. Molecular structures for these dimers have only been captured for truncated constructs encompassing the core domain alone. Here, we report a crystal structure of BAK α2-α8 dimers (i.e., minus its flexible N-terminal helix and membrane-anchoring C-terminal segment) that has been obtained through the activation of monomeric BAK with the detergent C12E8. Core dimers are evident, linked through the crystal by contacts via latch (α6-α8) domains. This crystal structure shows activated BAK dimers with the extended latch domain present. Our data provide direct evidence for the conformational change converting BAK from inert monomer to the functional dimer that destroys mitochondrial integrity. This dimer is the smallest functional unit for recombinant BAK or BAX described so far.

Glow-in-the-Dark Patterned PET Nonwoven Using Air-Atmospheric Plasma Treatment and Vitamin B2-Derivative (FMN).

Flavin mononucleotide (FMN) derived from Vitamin B2, a bio-based fluorescent water-soluble molecule with visible yellow-green fluorescence, has been used in the scope of producing photoluminescent and glow-in-the-dark patterned polyester (PET) nonwoven panels. Since the FMN molecule cannot diffuse inside the PET fiber, screen printing, coating, and padding methods were used in an attempt to immobilize FMN molecules at the PET fiber surface of a nonwoven, using various biopolymers such as gelatin and sodium alginate as well as a water-based commercial polyacrylate. In parallel, air atmospheric plasma activation of PET nonwoven was carried for improved spreading and adhesion of FMN bearing biopolymer/polymer mixture. Effectively, the plasma treatment yielded a more hydrophilic PET nonwoven, reduction in wettability, and surface roughness of the plasma treated fiber with reduced water contact angle and increased capillary uptake were observed. The standard techniques of morphological properties were explored by a scanning electron microscope (SEM) and atomic force microscopy (AFM). Films combining each biopolymer and FMN were formed on PS (polystyrene) Petri-dishes. However, only the gelatin and polyacrylate allowed the yellow-green fluorescence of FMN molecule to be maintained on the film and PET fabric (seen under ultraviolet (UV) light). No yellow-green fluorescence of FMN was observed with sodium alginate. Thus, when the plasma-activated PET was coated with the gelatin mixture or polyacrylate bearing FMN, the intense photoluminescent yellow-green glowing polyester nonwoven panel was obtained in the presence of UV light (370 nm). Screen printing of FMN using a gelatin mixture was possible. The biopolymer exhibited appropriate viscosity and rheological behavior, thus creating a glow-in-the-dark pattern on the polyester nonwoven, with the possibility of one expression in daylight and another in darkness (in presence of UV light). A bio-based natural product such as FMN is potentially an interesting photoluminescent molecule with which textile surface pattern designers may create light-emitting textiles and interesting aesthetic expressions.

Robust autoactivation for apoptosis by BAK but not BAX highlights BAK as an important therapeutic target.

BAK and BAX, which drive commitment to apoptosis, are activated principally by certain BH3-only proteins that bind them and trigger major rearrangements. One crucial conformation change is exposure of their BH3 domain which allows BAK or BAX to form homodimers, and potentially to autoactivate other BAK and BAX molecules to ensure robust pore formation and cell death. Here, we test whether full-length BAK or mitochondrial BAX that are specifically activated by antibodies can then activate other BAK or BAX molecules. We found that antibody-activated BAK efficiently activated BAK as well as mitochondrial or cytosolic BAX, but antibody-activated BAX unexpectedly proved a poor activator. Notably, autoactivation by BAK involved transient interactions, as BAK and BAX molecules it activated could dissociate and homodimerize. The results suggest that BAK-driven autoactivation may play a substantial role in apoptosis, including recruitment of BAX to the mitochondria. Hence, directly targeting BAK rather than BAX may prove particularly effective in inhibiting unwanted apoptosis, or alternatively, inducing apoptosis in cancer cells.

Toward Bioluminescent Materials by Plasma Treatment of Microfibrous Nonwovens, Followed by Immobilization of One or Both Enzyme(s) (Luciferase and FMN Reductase) Involved in Luminescent Bacteria.

Bioluminescent living organisms emit light through a specific biocatalyzed reaction involving a luciferin substrate and a luciferase enzyme. The present work investigated the possibility of creating optimal luminescence by immobilization of one or both the enzymes Luciferase (Luc) and FMN reductase (Red) involved in a bioluminescent bacterial system onto a plasma-activated microfibrous PET nonwoven. Parameters affecting the catalytic activity and efficiency of the bacterial system in aqueous medium were determined by luminescence intensity measurements using a luminometer. Two types of plasma, air atmospheric plasma (ATMP) and cold remote plasma (CRPNO) treatment, were used to activate the PET nonwoven. Further, one or both enzyme(s) were immobilized using a physical adsorption technique, without or with the use of natural biopolymers (gelatin and starch) and bovine serum albumin-BSA protein, to improve enzyme stability and activity. Coimmobilization of both Red and Luc enzymes on the CRPNO plasma-activated nonwoven in the presence of BSA led to the maximum luminescence. As high as 60,000 RLU equivalent to that of an LED light used for calibration was observed and showed stable intensity up to 6 min. Fiber surface analysis was tested using wettability tests (water contact angle and capillary uptake), while scanning electron microscopy, atomic force microscopy, and electron spectroscopy for chemical analysis showed changes in fiber surface morphology and chemical functional groups. A considerable increase in "N" atom content after coimmobilization of enzymes in the presence of BSA was detected. This study is the first successful attempt to use a biomimetic strategy for immobilization of enzymes involved in bacterial luminescence on a plasma-activated microfibrous nonwoven in an attempt to attain bioluminescent materials.

Color-changing intensified light-emitting multifunctional textiles via digital printing of biobased flavin.

Flavin mononucleotide (biobased flavin), widely known as FMN, possesses intrinsic fluorescence characteristics. This study presents a sustainable approach for fabricating color-changing intensified light-emitting textiles using the natural compound FMN via digital printing technologies such as inkjet and chromojet. The FMN based ink formulation was prepared at 5 different concentrations using water and glycerol-based systems and printed on cotton duck white (CD), mercerized cotton (MC), and polyester (PET) textile woven samples. After characterizing the printing inks (viscosity and surface tension), the photophysical and physicochemical properties of the printed textiles were investigated using FTIR, UV/visible spectrophotometry, and fluorimetry. Furthermore, photodegradation properties were studied after irradiation under UV (370 nm) and visible (white) light. Two prominent absorption peaks were observed at around 370 nm and 450 nm on K/S spectral curves because of the functionalization of FMN on the textiles via digital printing along with the highest fluorescence intensities obtained for cotton textiles. Before light irradiation, the printed textiles exhibited greenish-yellow fluorescence at 535 nm for excitation at 370 nm. The fluorescence intensity varied as a function of the FMN concentration and the solvent system (water/glycerol). With 0.8 and 1% of FMN, the fluorescence of the printed textiles persisted even after prolonged light irradiation; however, the fluorescence color shifted from greenish-yellow color to turquoise blue then to white, with the fluorescence quantum efficiency values (φ) increasing from 0.1 to a value as high as 1. Photodegradation products of the FMN with varying fluorescence wavelengths and intensities would explain the results. Thus, a color-changing light-emitting fluorescent textile was obtained after prolonged light irradiation of textile samples printed using biobased flavin. Furthermore, multifunctional properties such as antibacterial properties against E. coli were observed only for the printed cotton textile while increased ultraviolet protection was observed for both cotton and polyester printed fabrics for the high concentration of FMN water-based and glycerol-based formulations. The evaluation of fluorescence properties using digital printing techniques aimed to provide more sustainable solutions, both in terms of minimum use of biobased dye and obtaining the maximum yield.

Study of photoluminescence property on cellulosic fabric using multifunctional biomaterials riboflavin and its derivative Flavin mononucleotide.

Flavins are ubiquitous in nature and participate in various biochemical reactions mainly in the form of coenzyme Flavin mononucleotide (FMN) or as precursor such as Riboflavin (RF). Both flavins, RF and FMN are multifunctional bio-based molecules yielding yellow coloration and exhibit photoluminescence, UV protection, and redox properties. The aim of the present research study was to investigate the diffusion method as a technique to obtain photoluminescent cellulosic fabric using multifunctional RF and FMN. The photoluminescent moiety RF and FMN exhibited three maximum absorbance peaks at about 270 nm, 370 nm and 446 nm in aqueous solution at pH 7. The solutions of RF and FMN with concentration 4% and 20% (owf) at pH 7 were prepared and used in diffusion method for cellulosic fabric dyeing. The study involved the determination of color performance and evaluation of luminescence property of the dyed fabric using UV-visible spectrophotometer and photoluminescence spectroscopy, respectively. Under monochromatic UV lamp exposure emitting at 370 nm, the dyed fabric showed an intense emission of greenish yellow color, which was later confirmed by the intense photoluminescence observed at a wavelength of about 570 nm. The study demonstrates the theoretical evaluation of quantum efficiency (φ) obtaining maximum φ value of 0.28. Higher color strength value and improved wash fastness were obtained by treatment with different biobased mordants such as tannic acid and citric acid as well as calcium chloride for both RF and FMN. Additionally, ultraviolet (UV) protection ability for both RF and FMN dyed fabric were determined and showed UPF factor of 50+ and 35 respectively. The work allowed us to explore the photoluminescence property of riboflavin and Flavin mononucleotide for its application in the field of textiles as a new scope of producing photoluminescent textile along with multifunctional properties such as coloration and UV protection.

BAX Activation: Mutations Near Its Proposed Non-canonical BH3 Binding Site Reveal Allosteric Changes Controlling Mitochondrial Association.

To elicit apoptosis, BAX metamorphoses from an inert cytosolic monomer into homo-oligomers that permeabilize the mitochondrial outer membrane (MOM). A long-standing puzzle is that BH3 domains apparently activate BAX by not only its canonical groove but also a proposed site involving helices α1 and α6. Our mutagenesis studies reveal that late steps like oligomerization require activation through the groove but probably not earlier steps like MOM association. Conversely, α1 or α6 obstruction and alanine mutagenesis scanning implicate these helices early in BAX activation. The α1 and α6 mutations lowered BH3 binding, altered the BAX conformation, and reduced its MOM translocation and integration; their exposure of the BAX α1-α2 loop allosterically sequestered its α9 membrane anchor in the groove. The crystal structure of an α6 mutant revealed additional allosteric effects. The results suggest that the α1 and α6 region drives MOM association and integration, whereas groove binding favors subsequent steps toward oligomerization.

Probing BAK and BAX Activation and Pore Assembly with Cytochrome c Release, Limited Proteolysis, and Oxidant-Induced Linkage.

Mitochondrial permeabilization is a key event in the intrinsic pathway of apoptosis, and is mediated by either of the BCL-2 family members BAK or BAX. These two proteins generate pores in the mitochondrial outer membrane that release factors such as cytochrome c into the cytosol to trigger caspase activation and apoptotic cell death. To generate pores, BAK and BAX undergo major changes including BAX translocation to the outer membrane, and partial unfolding, dimerization, and oligomerization. Here we describe biochemical protocols that can be used on most cell types to gain a population overview of BAK and BAX status.

Ensemble Properties of Bax Determine Its Function.

BAX and BAK are essential mediators of intrinsic apoptosis that permeabilize the mitochondrial outer membrane. BAX activation requires its translocation from cytosol to mitochondria where conformational changes cause its oligomerization. To better understand the critical step of translocation, we examined its blockade by mutation near the C terminus (P168G) or by antibody binding near the N terminus. Similarities in the crystal structures of wild-type and BAX P168G but significant other differences suggest that cytosolic BAX exists as an ensemble of conformers, and that the distribution of conformers within the ensemble determines the different functions of wild-type and mutant proteins. We also describe the structure of BAX in complex with an antibody, 3C10, that inhibits cytosolic BAX by limiting exposure of the membrane-associating helix α9, as does the P168G mutation. Our data for both means of BAX inhibition argue for an allosteric model of BAX regulation that derives from properties of the ensemble of conformers.

Pore formation by dimeric Bak and Bax: an unusual pore?

Apoptotic cell death via the mitochondrial pathway occurs in all vertebrate cells and requires the formation of pores in the mitochondrial outer membrane. Two Bcl-2 protein family members, Bak and Bax, form these pores during apoptosis, and how they do so has been investigated for the last two decades. Many of the conformation changes that occur during their transition to pore-forming proteins have now been delineated. Notably, biochemical, biophysical and structural studies indicate that symmetric homodimers are the basic unit of pore formation. Each dimer contains an extended hydrophobic surface that lies on the outer membrane, and is anchored at either end by a transmembrane domain. Membrane-remodelling events such as positive membrane curvature have been reported to accompany apoptotic pore formation, suggesting Bak and Bax form lipidic pores rather than proteinaceous pores. However, it remains unclear how symmetric dimers assemble to porate the membrane. Here, we review how clusters of dimers and their lipid-mediated interactions provide a molecular explanation for the heterogeneous assemblies of Bak and Bax observed during apoptosis.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'.

Disordered clusters of Bak dimers rupture mitochondria during apoptosis.

During apoptosis, Bak and Bax undergo major conformational change and form symmetric dimers that coalesce to perforate the mitochondrial outer membrane via an unknown mechanism. We have employed cysteine labelling and linkage analysis to the full length of Bak in mitochondria. This comprehensive survey showed that in each Bak dimer the N-termini are fully solvent-exposed and mobile, the core is highly structured, and the C-termini are flexible but restrained by their contact with the membrane. Dimer-dimer interactions were more labile than the BH3:groove interaction within dimers, suggesting there is no extensive protein interface between dimers. In addition, linkage in the mobile Bak N-terminus (V61C) specifically quantified association between dimers, allowing mathematical simulations of dimer arrangement. Together, our data show that Bak dimers form disordered clusters to generate lipidic pores. These findings provide a molecular explanation for the observed structural heterogeneity of the apoptotic pore.

Epigenetic control of mitochondrial cell death through PACS1-mediated regulation of BAX/BAK oligomerization.

PCAF and ADA3 associate within the same macromolecular complexes to control the transcription of many genes, including some that regulate apoptosis. Here we show that PCAF and ADA3 regulate the expression of PACS1, whose protein product is a key component of the machinery that sorts proteins among the trans-Golgi network and the endosomal compartment. We describe a novel role for PACS1 as a regulator of the intrinsic pathway of apoptosis and mitochondrial outer membrane permeabilization. Cells with decreased PACS1 expression were refractory to cell death mediated by a variety of stimuli that operate through the mitochondrial pathway, including human granzyme B, staurosporine, ultraviolet radiation and etoposide, but remained sensitive to TRAIL receptor ligation. The mitochondria of protected cells failed to release cytochrome c as a result of perturbed oligomerization of BAX and BAK. We conclude that PCAF and ADA3 transcriptionally regulate PACS1 and that PACS1 is a key regulator of BAX/BAK oligomerization and the intrinsic (mitochondrial) pathway to apoptosis.

Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies.

During apoptosis, Bak and Bax are activated by BH3-only proteins binding to the α2-α5 hydrophobic groove; Bax is also activated via a rear pocket. Here we report that antibodies can directly activate Bak and mitochondrial Bax by binding to the α1-α2 loop. A monoclonal antibody (clone 7D10) binds close to α1 in non-activated Bak to induce conformational change, oligomerization, and cytochrome c release. Anti-FLAG antibodies also activate Bak containing a FLAG epitope close to α1. An antibody (clone 3C10) to the Bax α1-α2 loop activates mitochondrial Bax, but blocks translocation of cytosolic Bax. Tethers within Bak show that 7D10 binding directly extricates α1; a structural model of the 7D10 Fab bound to Bak reveals the formation of a cavity under α1. Our identification of the α1-α2 loop as an activation site in Bak paves the way to develop intrabodies or small molecules that directly and selectively regulate these proteins.

Apoptotic pore formation is associated with in-plane insertion of Bak or Bax central helices into the mitochondrial outer membrane.

The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores.