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Hardened steels, which are resistant to corrosive chemical environments, are quite popular in the medical fields for various tooling applications exposed to low temperature. Using deep cryogenic treatments hardness and corrosion resistance of steel can be significantly improve for better compatibility of steels in biomedical applications. The present investigation examines the microstructure and evaluates hardness values of deep cryogenically heat treated AISI M2 steel. Studies on cryogenically treated tool steel show microstructural changes in the material can influence the life of the tools significantly. Through pertinent analyses of the experimental results this investigation infers that deep cryogenic treatment of AISI M2 steel increases the hardness of specimens marginally with the increase in the number of tempering cycle. The treatment further provides opportunity to exploit the possible use to hardened steels in robotic arms operative at low temperature research labs.

Purpose>This study aims to identify and gauge the sustainability indicators (SUSIs) for sustainable Hydroelectric Power (HEP) project development. It examines major SUSIs under the social, economic and environmental (SEE) fronts and categorizes them under push and pull impacts which helps to identify challenges and opportunities associated with projects. Additionally, the study calculates an empirical sustainability index (SI) to assess the sustainability level of HEP. Finally, the study suggests mitigation measures across stakeholders, which will optimize government/developer/investor investments.Design/methodology/approach>This paper examines the interaction of sustainable HEP development with SUSIs using Uttarakhand as a study area. Additionally, SI has been developed quantitatively. For the indicator classification, the authors conducted a literature review and secondary survey of all affected parties, including investors, developers, NGOs and villagers. The fuzzy logic theory (FLT) is used to determine the SI of the study area and classify projects in their level of sustainability. On the basis of expert opinion and literature review, mitigation measures are proposed across stakeholders.Findings>The authors found that there is a mixed effect of SUSIs on HEP development across various projects in Uttarakhand. Furthermore, the authors suggest that index-based assessment and planned collaboration play a significant role in sustainable HEP development. Mitigation measures should be suggested to all affected stakeholders based on specific project issues, i.e. collaborations, training, public awareness campaigns, and initiatives by the government that would improve sustainability conditions.Research limitations/implications>In addition to supporting the ongoing and upcoming initiatives launched by the Government of India, including the Green Energy Corridor, independent power producers (IPPs); and the India-Renewable Resources Development Project with IDA and participates in Net zero target.Practical implications>The structured, sustainable HEP planning suggested in the study will help to conserve society, economy, save resources and in parallel reduce the cost and time of developers and policymakers. This will also help to improve the socioeconomic status of the villagers and prolong the life of the project.Originality/value>The innovative SI-based push-pull approach identifies a sustainable HEP project planning.

Purpose The study aims to identify the severe socioeconomic, environmental, and ecological impacts caused by the construction of mega and large hydro-power plants in Uttarakhand, India. In addition to identifying the attributes, the study creates an integrated index that will assist in the development of sustainable hydro-power. Design/methodology/approach The methodology used for this impact identification was based on extensive literature review, focused expert discussions and further validation through a primary survey among the stakeholders in the hydropower sector. The sustainability index (SI) was estimated using the fuzzy logic theory. Findings The study area SI shows that few projects are in extreme zones, and through suggestive measures, few project sites can be made viable for long-term sustainable project site. A Hydropower Sustainability Assessment Protocol–based conceptual model is also proposed for mitigation of impacts. Originality/value Hydropower plays an essential role in access to cleaner and cheaper sources of energy; it defines the usage of water resources toward inflation-free green energy and holds spectacular operational flexibility. Despite the significant advantages associated with hydroelectric power projects, there are adverse side effects as well. The water-based power sector industry contributes to any nation through both economic and environmental ways. Although one-third of the power business in India is carried out through water-based hydropower projects, recent trends in water-based hydropower projects show significant socioeconomic and environmental impacts that create a debate about the sustainability of these projects.

Mycobacterium tuberculosis is the cause of one of the most important infectious diseases in humans, which leads to 1.4 million deaths every year 1 . Specialized protein transport systems—known as type VII secretion systems (T7SSs)—are central to the virulence of this pathogen, and are also crucial for nutrient and metabolite transport across the mycobacterial cell envelope 2 , 3 . Here we present the structure of an intact T7SS inner-membrane complex of M. tuberculosis . We show how the 2.32-MDa ESX-5 assembly, which contains 165 transmembrane helices, is restructured and stabilized as a trimer of dimers by the MycP 5 protease. A trimer of MycP 5 caps a central periplasmic dome-like chamber that is formed by three EccB 5 dimers, with the proteolytic sites of MycP 5 facing towards the cavity. This chamber suggests a central secretion and processing conduit. Complexes without MycP 5 show disruption of the EccB 5 periplasmic assembly and increased flexibility, which highlights the importance of MycP 5 for complex integrity. Beneath the EccB 5 –MycP 5 chamber, dimers of the EccC 5 ATPase assemble into three bundles of four transmembrane helices each, which together seal the potential central secretion channel. Individual cytoplasmic EccC 5 domains adopt two distinctive conformations that probably reflect different secretion states. Our work suggests a previously undescribed mechanism of protein transport and provides a structural scaffold to aid in the development of drugs against this major human pathogen. A cryo-electron microscopy structure of the inner membrane complex of the ESX-5 type VII secretion system of Mycobacterium tuberculosis reveals an important role of interactions with MycP 5 protease for complex integrity.

Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow-growing mycobacteria.

Type VII secretion (T7S) systems are specialized machineries used by mycobacterial pathogens to transport important virulence factors across their highly hydrophobic cell envelope. There are up to five mycobacterial T7S systems, named ESX-1 to ESX-5, at least three of which specifically secrete a different subset of substrates. The T7S substrates or substrate complexes are defined by the general secretion motif YxxxD/E. However this motif does not determine system specificity. Here, we show that the substrate domain recognized by the EspG chaperone is the determinant factor for this specificity. We first show that the introduction of point mutations into the EspG 1 -binding domain of the ESX-1 substrate pair PE35/PPE68_1 affects their secretion. Subsequently, we demonstrate that replacing this domain by the EspG 5 -binding domain of the ESX-5 substrate PPE18 resulted in EspG 5 dependence and exclusive rerouting to the ESX-5 system. This rerouting of PE35/PPE68_1 to the ESX-5 system had a negative effect on the secretion of endogenous ESX-5 substrates.

The pathogen Mycobacterium tuberculosis employs a range of ESX-1 substrates to manipulate the host and build a successful infection. Although the importance of ESX-1 secretion in virulence is well established, the characterization of its individual components and the role of individual substrates is far from complete. Here, we describe the functional characterization of the Mycobacterium marinum accessory ESX-1 proteins EccA1, EspG1 and EspH, i.e. proteins that are neither substrates nor structural components. Proteomic analysis revealed that EspG1 is crucial for ESX-1 secretion, since all detectable ESX-1 substrates were absent from the cell surface and culture supernatant in an espG1 mutant. Deletion of eccA1 resulted in minor secretion defects, but interestingly, the severity of these secretion defects was dependent on the culture conditions. Finally, espH deletion showed a partial secretion defect; whereas several ESX-1 substrates were secreted in normal amounts, secretion of EsxA and EsxB was diminished and secretion of EspE and EspF was fully blocked. Interaction studies showed that EspH binds EspE and therefore could function as a specific chaperone for this substrate. Despite the observed differences in secretion, hemolytic activity was lost in all M. marinum mutants, implying that hemolytic activity is not strictly correlated with EsxA secretion. Surprisingly, while EspH is essential for successful infection of phagocytic host cells, deletion of espH resulted in a significantly increased virulence phenotype in zebrafish larvae, linked to poor granuloma formation and extracellular outgrowth. Together, these data show that different sets of ESX-1 substrates play different roles at various steps of the infection cycle of M. marinum.

Pathogenic mycobacteria contain up to five type VII secretion (T7S) systems, ESX-1 to ESX-5. One of the conserved T7S components is the serine protease mycosin (MycP). Strikingly, whereas MycP is essential for secretion, the protease activity of MycP 1 in Mycobacterium tuberculosis has been shown to be dispensable for secretion. The essential role of MycP therefore remains unclear. Here we show that MycP 1 and MycP 5 of M. marinum have similar phenotypes, confirming that MycP has a second unknown function that is essential for its T7S system. To investigate whether this role is related to proper functioning of the T7S membrane complex, we first analyzed the composition of the ESX-1 membrane complex and showed that this complex consists of EccBCDE 1 , similarly to what was previously shown for ESX-5. Surprisingly, while mycosins are not an integral part of these purified core complexes, we noticed that the stability of both the ESX-1 complex and the ESX-5 complex is compromised in the absence of their MycP subunit. Additional interaction studies showed that, although mycosins are not part of the central ESX membrane complex, they loosely associate with this complex. We hypothesize that this MycP association with the core membrane complex is crucial for the integrity and functioning of the T7S machinery. IMPORTANCE Among the major virulence factors of pathogenic mycobacteria are the type VII secretion (T7S) systems. Three of these systems, ESX-1, ESX-3, and ESX-5, have been shown to be crucial for virulence or viability. Here we describe the function of mycosin proteases, which are conserved components within these systems. We show that MycP 1 and MycP 5 have a second, proteolytic-independent function which is essential for the T7S system. We additionally found that this second essential role is related to the stabilization and proper functioning of their respective ESX membrane core complexes. Finally, we found that this is mediated by a loose association of MycP with the complex. Understanding the essential role of mycosins in type VII secretion systems, which play central roles in the virulence and viability of pathogenic mycobacteria, may provide new intervention strategies to treat tuberculosis. Among the major virulence factors of pathogenic mycobacteria are the type VII secretion (T7S) systems. Three of these systems, ESX-1, ESX-3, and ESX-5, have been shown to be crucial for virulence or viability. Here we describe the function of mycosin proteases, which are conserved components within these systems. We show that MycP 1 and MycP 5 have a second, proteolytic-independent function which is essential for the T7S system. We additionally found that this second essential role is related to the stabilization and proper functioning of their respective ESX membrane core complexes. Finally, we found that this is mediated by a loose association of MycP with the complex. Understanding the essential role of mycosins in type VII secretion systems, which play central roles in the virulence and viability of pathogenic mycobacteria, may provide new intervention strategies to treat tuberculosis.

Mycobacterium tuberculosis requires a large number of secreted and exported proteins for its virulence, immune modulation and nutrient uptake. Most of these proteins are transported by the different type VII secretion systems 1 , 2 . The most recently evolved type VII secretion system, ESX-5, secretes dozens of substrates belonging to the PE and PPE families, which are named for conserved proline and glutamic acid residues close to the amino terminus 3 , 4 . However, the role of these proteins remains largely elusive 1 . Here, we show that mutations of ppe38 completely block the secretion of two large subsets of ESX-5 substrates, that is, PPE-MPTR and PE_PGRS, together comprising >80 proteins. Importantly, hypervirulent clinical M. tuberculosis strains of the Beijing lineage have such a mutation and a concomitant loss of secretion 5 . Restoration of PPE38-dependent secretion partially reverted the hypervirulence phenotype of a Beijing strain, and deletion of ppe38 in moderately virulent M. tuberculosis increased virulence. This indicates that these ESX-5 substrates have an important role in virulence attenuation. Phylogenetic analysis revealed that deletion of ppe38 occurred at the branching point of the ‘modern’ Beijing sublineage and is shared by Beijing outbreak strains worldwide, suggesting that this deletion may have contributed to their success and global distribution 6 , 7 . A mutation in ppe38 , commonly seen in Beijing-lineage strains, alters protein secretion and increases virulence of Mycobacterium tuberculosis .

Mycobacteria are characterized by their impermeable outer membrane, which is rich in mycolic acids 1 . To transport substrates across this complex cell envelope, mycobacteria rely on type VII (also known as ESX) secretion systems 2 . In Mycobacterium tuberculosis , these ESX systems are essential for growth and full virulence and therefore represent an attractive target for anti-tuberculosis drugs 3 . However, the molecular details underlying type VII secretion are largely unknown, due to a lack of structural information. Here, we report the molecular architecture of the ESX-5 membrane complex from Mycobacterium xenopi determined at 13 Å resolution by electron microscopy. The four core proteins of the ESX-5 complex (EccB 5 , EccC 5 , EccD 5 and EccE 5 ) assemble with equimolar stoichiometry into an oligomeric assembly that displays six-fold symmetry. This membrane-associated complex seems to be embedded exclusively in the inner membrane, which indicates that additional components are required to translocate substrates across the mycobacterial outer membrane. Furthermore, the extended cytosolic domains of the EccC ATPase, which interact with secretion effectors, are highly flexible, suggesting an as yet unseen mode of substrate interaction. Comparison of our results with known structures of other bacterial secretion systems demonstrates that the architecture of type VII secretion system is fundamentally different, suggesting an alternative secretion mechanism. Single-particle analysis of electron microscopy structures elucidates the mycobacterial ESX-5 type VII secretion system membrane complex.