Kicking sleepers out of bed: Macrophages promote reactivation of dormant Cryptococcus neoformans by extracellular vesicle release and non-lytic exocytosis.

Macrophages play a key role in disseminated cryptococcosis, a deadly fungal disease caused by Cryptococcus neoformans. This opportunistic infection can arise following the reactivation of a poorly characterized latent infection attributed to dormant C. neoformans. Here, we investigated the mechanisms underlying reactivation of dormant C. neoformans using an in vitro co-culture model of viable but non-culturable (VBNC; equivalent of dormant) yeast cells with bone marrow-derived murine macrophages (BMDMs). Comparative transcriptome analysis of BMDMs incubated with log, stationary phase or VBNC cells of C. neoformans showed that VBNC cells elicited a reduced transcriptional modification of the macrophage but retaining the ability to regulate genes important for immune response, such as NLRP3 inflammasome-related genes. We further confirmed the maintenance of the low immunostimulatory capacity of VBNC cells using multiplex cytokine profiling, and analysis of cell wall composition and dectin-1 ligands exposure. In addition, we evaluated the effects of classic (M1) or alternative (M2) macrophage polarization on VBNC cells. We observed that intracellular residence sustained dormancy, regardless of the polarization state of macrophages and despite indirect detection of pantothenic acid (or its derivatives), a known reactivator for VBNC cells, in the C. neoformans-containing phagolysosome. Notably, M0 and M2, but not M1 macrophages, induced extracellular reactivation of VBNC cells by the secretion of extracellular vesicles and non-lytic exocytosis. Our results indicate that VBNC cells retain the low immunostimulatory profile required for persistence of C. neoformans in the host. We also describe a pro-pathogen role of macrophage-derived extracellular vesicles in C. neoformans infection and reinforce the impact of non-lytic exocytosis and the macrophage profile on the pathophysiology of cryptococcosis.

Tryptophan-metabolizing gut microbes regulate adult neurogenesis via the aryl hydrocarbon receptor.

While modulatory effects of gut microbes on neurological phenotypes have been reported, the mechanisms remain largely unknown. Here, we demonstrate that indole, a tryptophan metabolite produced by tryptophanase-expressing gut microbes, elicits neurogenic effects in the adult mouse hippocampus. Neurogenesis is reduced in germ-free (GF) mice and in GF mice monocolonized with a single-gene tnaA knockout (KO) mutant Escherichia coli unable to produce indole. External administration of systemic indole increases adult neurogenesis in the dentate gyrus in these mouse models and in specific pathogen-free (SPF) control mice. Indole-treated mice display elevated synaptic markers postsynaptic density protein 95 and synaptophysin, suggesting synaptic maturation effects in vivo. By contrast, neurogenesis is not induced by indole in aryl hydrocarbon receptor KO (AhR-/-) mice or in ex vivo neurospheres derived from them. Neural progenitor cells exposed to indole exit the cell cycle, terminally differentiate, and mature into neurons that display longer and more branched neurites. These effects are not observed with kynurenine, another AhR ligand. The indole-AhR-mediated signaling pathway elevated the expression of ß-catenin, Neurog2, and VEGF-α genes, thus identifying a molecular pathway connecting gut microbiota composition and their metabolic function to neurogenesis in the adult hippocampus. Our data have implications for the understanding of mechanisms of brain aging and for potential next-generation therapeutic opportunities.

Urban mining from biomass, brine, sewage sludge, phosphogypsum and e-waste for reducing the environmental pollution: Current status of availability, potential, and technologies with a focus on LCA and TEA.

Rapid industrialization, improved standards of living, growing economies and ever-increasing population has led to the unprecedented exploitation of the finite and non-renewable resources of minerals in past years. It was observed that out of 100 BMT of raw materials processed annually only 10% is recycled back. This has resulted in a strenuous burden on natural or primary resources of minerals (such as ores) having limited availability. Moreover, severe environmental concerns have been raised by the huge piles of waste generated at landfill sites. To resolve these issues, 'Urban Mining' from waste or secondary resources in a Circular Economy' concept is the only sustainable solution. The objective of this review is to critically examine the availability, elemental composition, and the market potential of the selected secondary resources such as lignocellulosic/algal biomass, desalination water, sewage sludge, phosphogypsum, and e-waste for minerals sequestration. This review showed that, secondary resources have potential to partially replace the minerals required in different sectors such as macro and microelements in agriculture, rare earth elements (REEs) in electrical and electronics industry, metals in manufacturing sector and precious elements such as gold and platinum in ornamental industry. Further, inputs from the selected life cycle analysis (LCA) & techno economic analysis (TEA) were discussed which showed that although, urban mining has a potential to reduce the greenhouse gaseous (GHG) emissions in a sustainable manner however, process improvements through innovative, novel and cost-effective pathways are essentially required for its large-scale deployment at industrial scale in future.

FRET reveals multiple interaction states between two component signalling system proteins of M. tuberculosis.

BACKGROUND: Two component signalling involves interaction between sensor kinase (SK) and response regulator (RR) proteins which depends on their phosphorylation status. METHODS: In this study we report the development of an in vitro FRET assay for studying interaction between fluorescently tagged SK and RR proteins. RESULTS: Using TCS proteins of Mycobacterium tuberculosis, we demonstrate that phosphorylation status of SK affects the SK-RR interaction, which varies from one TCS to another. The observation was strengthened by recordings from mutant SK and RR proteins. The assay retained the specificity/crosstalk potential of the participating proteins and reflected the inherent phosphotransfer potentials. CONCLUSIONS: SK and RR proteins interact with each other in unphosphorylated state and the phosphorylation affects the interaction between SK and RR, which was reflected as reduction in FRET ratio. GENERAL SIGNIFICANCE: A non-radioactive, in vitro FRET based assay is reported, which can be utilized for studying genome-wide partner screening, identifying crosstalk or specificity in TCSs.

Mycobacterium tuberculosis response regulators, DevR and NarL, interact in vivo and co-regulate gene expression during aerobic nitrate metabolism.

Mycobacterium tuberculosis genes Rv0844c/Rv0845 encoding the NarL response regulator and NarS histidine kinase are hypothesized to constitute a two-component system involved in the regulation of nitrate metabolism. However, there is no experimental evidence to support this. In this study, we established M. tuberculosis NarL/NarS as a functional two-component system and identified His(241) and Asp(61) as conserved phosphorylation sites in NarS and NarL, respectively. Transcriptional profiling between M. tuberculosis H37Rv and a ΔnarL mutant strain during exponential growth in broth cultures with or without nitrate defined an ∼30-gene NarL regulon that exhibited significant overlap with DevR-regulated genes, thereby implicating a role for the DevR response regulator in the regulation of nitrate metabolism. Notably, expression analysis of a subset of genes common to NarL and DevR regulons in M. tuberculosis ΔdevR, ΔdevSΔdosT, and ΔnarL mutant strains revealed that in response to nitrite produced during aerobic nitrate metabolism, the DevRS/DosT regulatory system plays a primary role that is augmented by NarL. Specifically, NarL itself was unable to bind to the narK2, acg, and Rv3130c promoters in phosphorylated or unphosphorylated form; however, its interaction with DevR∼P resulted in cooperative binding, thereby enabling co-regulation of these genes. These findings support the role of physiologically derived nitrite as a metabolic signal in mycobacteria. We propose NarL-DevR binding, possibly as a heterodimer, as a novel mechanism for co-regulation of gene expression by the DevRS/DosT and NarL/NarS regulatory systems.

The two-component signalling networks of Mycobacterium tuberculosis display extensive cross-talk in vitro.

Two-component systems (TCSs), which contain paired sensor kinase and response regulator proteins, form the primary apparatus for sensing and responding to environmental cues in bacteria. TCSs are thought to be highly specific, displaying minimal cross-talk, primarily due to the co-evolution of the participating proteins. To assess the level of cross-talk between the TCSs of Mycobacterium tuberculosis, we mapped the complete interactome of the M. tuberculosis TCSs using phosphotransfer profiling. Surprisingly, we found extensive cross-talk among the M. tuberculosis TCSs, significantly more than that in the TCSs in Escherichia coli or Caulobacter crescentus, thereby offering an alternate to specificity paradigm in TCS signalling. Nearly half of the interactions we detected were significant novel cross-interactions, unravelling a potentially complex signalling landscape. We classified the TCSs into specific 'one-to-one' and promiscuous 'one-to-many' and 'many-to-one' circuits. Using mathematical modelling, we deduced that the promiscuous signalling observed can explain several currently confounding observations about M. tuberculosis TCSs. Our findings suggest an alternative paradigm of bacterial signalling with significant cross-talk between TCSs yielding potentially complex signalling landscapes.

Immobilization of ß-glucosidase onto silicon oxide nanoparticles and augment of phenolics in sugarcane juice.

Purified ß-glucosidase was immobilized on SiO2 nanoparticles with 52 % efficiency and 14.1 % yield. It had a temperature optima at 60 °C and pH optima of 5.0. Immobilized enzyme was fairly stable at 60-70 °C. After immobilization, the Km value of ß-glucosidase for p-nitrophenyl-ß-d-glucopyranoside (pNPG) increased from 0.9 to 1.074 mM and Vmax decreased from 3.5 to 1.513 U/mg. The immobilized enzyme showed improved storage stability at temperature 4 and 25 °C and was reusable for up to ten cycles with 70 % residual activity in pNPG and 60 % residual activity in sugarcane juice treatment. Sugarcane juice density, viscosity; surface tension etc. changed after treatment with immobilized ß-glucosidase. ß-Glucosidase treated sugarcane juice showed higher phenolics than untreated sugarcane juice. Caffeic acid which was absent in juice, was detected in ß-glucosidase treated juice at a concentration of about 1 mg/L.

Rv1027c-Rv1028c encode functional KdpDE two--component system in Mycobacterium tuberculosis.

In Mycobacteriumtuberculosis Rv1027c-Rv1028c genes are predicted to encode KdpDE two component system, which is highly conserved across all bacterial species. Here, we show that the system is functionally active and KdpD sensor kinase undergoes autophosphorylation and transfers phosphoryl group to KdpE, response regulator protein. We identified His(642) and Asp(52) as conserved phosphorylation sites in KdpD and KdpE respectively and by SPR analysis confirmed the physical interaction between them. KdpD was purified with prebound divalent ions and their importance in phosphorylation was established using protein refolding and ion chelation approaches. Genetically a single transcript encoded both KdpD and KdpE proteins. Overall, we report that M. tuberculosis KdpDE system operates like a canonical two component system.

Induction of Dormancy in Cryptococcus neoformans In Vitro: The HypNOS Protocol.

Cryptococcus neoformans is the second major cause of death in patients with HIV. During a latent infection, this pathogenic fungus survives in the host for years without causing symptoms of active disease. Upon favorable conditions, such as immunosuppression due to HIV infection, or other conditions (steroid use or organ transplantation), the yeast may reactivate and cause active cryptococcosis. Hence, dormancy is an important phase in the pathogenesis of C. neoformans. Additionally, C. neoformans also persists during antifungal treatment and causes disease recurrence, which is a major medical problem, especially in low- and middle-income countries. To survive in the host, yeast cells must react to the stresses they are exposed to and generate a cellular response that is favorable for yeast survival. A prominent strategy used by C. neoformans to combat challenging surroundings is dormancy, which may translate into a viable, but nonculturable phenotype (VBNC). This chapter describes an in vitro protocol to generate and characterize dormant Cryptococci.

Unveiling the potential of cellulose nanofibre based nitrogen fertilizer and its transformative effect on Vigna radiata (Mung Bean): nanofibre for sustainable agriculture.

Introduction: Fertilizer management is crucial to maintaining a balance between environmental health, plant health, and total crop yield. Farmers are overutilizing fertilizers with a mind set to enhance the productive capacity of the field, which adversely impacts soil fertility and causes serious environmental hazards. To mitigate the issues of over-utilization of fertilizers, controlled-release fertilizers were developed using nitrogen fertilizer (ammonium chloride) loaded on cellulose nanofibres (named CNF*N). Methodology: In this study, the effects of CNF*N were compared with commercial nitrogen fertilizer (ammonium chloride) on Vigna radiata (Mung) under greenhouse conditions. The pot experiment was conducted using six treatments: first treatment was control, where the plant was cultivated (T1); second treatment was T2, where the plant was cultivated with CNF to determine the impact of CNF on the plant; third was T3 where commercial ammonium chloride (24 mg/ 2 kg soil) was added to the plant; fourth was T4, where the plant was loaded with CNF, viz. CNF*N contains 4.8 mg of nitrogen; fifth was T5 CNF*N pellet contains 12 mg of nitrogen, and the last sixth treatment (T6) where CNF*N pellet containing 24 mg of nitrogen. Results: It indicated that the growth parameters were best achieved in T6 treatment. Plant height was at its maximum in the T6 treatment (44.4 ±0.1cm) after the second harvest, whereas the minimum plant height was observed in T1, which was 39.1 ±0.1 cm. Root-to-shoot weight ratio was also maximum in T6 (0.183± 0.02) and minimum in T1 (0.07± 0.01) after second harvesting. The significant difference among the treatments was determined with Tukey's honestly significant difference (HSD). The nitrogen content (available and total) was significantly higher in the T4, T5, and T6 treatments (0.22, 0.25, and 0.28%) as compared to the control treatments (T1 (0.12%), T2 (0.13%), and T3 (0.14%) during the second harvesting stage (90 days), as nitrogen plays a crucial role in the development of vegetative growth in Vigna radiata. The rate of controlled-release nitrogen-fertilizer was found to be optimal in terms of plant growth and soil nutrients; hence, it could potentially play a crucial role in improving soil health and the yield of the crop.

Pulmonary Spindle Cell Carcinoma: As Rare as a Hen's Teeth.

This case is about a 70-year-old man who presented with symptoms and laboratory reports that indicated differentials toward an infectious disease (pneumonia and tuberculosis). A lung mass was found in his chest X-ray and in the computerized tomography (CT) scan of his thorax. A biopsy was taken from the lung mass, and histopathological examination and immunohistochemical staining of the biopsy were done. The results revealed the presence of spindle cell carcinoma (SpCC) with vimentin and cytokeratin positivity. Spindle cell lung cancer is a rare type of non-small cell lung carcinoma, for which all available research indicates a poor prognosis. Due to the rarity of diagnosis, there is a dearth of information about the epidemiology and overall survival of affected patients.

Silk Industry Waste Protein-Derived Sericin Hybrid Nanoflowers for Antibiotics Remediation via Circular Economy.

Hybrid protein-copper nanoflowers have emerged as promising materials with diverse applications in biocatalysis, biosensing, and bioremediation. Sericin, a waste biopolymer from the textile industry, has shown potential for fabricating such nanoflowers. However, the influence of the molecular weight of sericin on nanoflower morphology and peroxidase-like activity remains unexplored. This work focused on the self-assembly of nanoflowers using high- and low-molecular-weight (HMW and LMW) silk sericin combined with copper(II) as an inorganic moiety. The peroxidase-like activity of the resulting nanoflowers was evaluated using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hydrogen peroxide (H2O2). The findings revealed that high-molecular-weight sericin hybrid nanoflowers (HMW-ShNFs) exhibited significantly higher peroxidase-like activity than low-molecular-weight sericin hybrid nanoflowers (LMW-ShNFs). Furthermore, HMW-ShNFs demonstrated superior reusability and storage stability, thereby enhancing their potential for practical use. This study also explored the application of HMW-ShNF for ciprofloxacin degradation to address the environmental and health hazards posed by this antibiotic in water. The results indicated that HMW-ShNFs facilitated the degradation of ciprofloxacin, achieving a maximum degradation of 33.2 ± 1% at pH 8 and 35 °C after 72 h. Overall, the enhanced peroxidase-like activity and successful application in ciprofloxacin degradation underscore the potential of HMW-ShNFs for a sustainable and ecofriendly remediation process. These findings open avenues for the further exploration and utilization of hybrid nanoflowers in various environmental applications.

Microbial Indoles: Key Regulators of Organ Growth and Metabolic Function.

Gut microbes supporting body growth are known but the mechanisms are less well documented. Using the microbial tryptophan metabolite indole, known to regulate prokaryotic cell division and metabolic stress conditions, we mono-colonized germ-free (GF) mice with indole-producing wild-type Escherichia coli (E. coli) or tryptophanase-encoding tnaA knockout mutant indole-non-producing E. coli. Indole mutant E. coli mice showed multiorgan growth retardation and lower levels of glycogen, cholesterol, triglycerides, and glucose, resulting in an energy deficiency despite increased food intake. Detailed analysis revealed a malfunctioning intestine, enlarged cecum, and reduced numbers of enterochromaffin cells, correlating with a metabolic phenotype consisting of impaired gut motility, diminished digestion, and lower energy harvest. Furthermore, indole mutant mice displayed reduction in serum levels of tricarboxylic acid (TCA) cycle intermediates and lipids. In stark contrast, a massive increase in serum melatonin was observed-frequently associated with accelerated oxidative stress and mitochondrial dysfunction. This observational report discloses functional roles of microbe-derived indoles regulating multiple organ functions and extends our previous report of indole-linked regulation of adult neurogenesis. Since indoles decline by age, these results imply a correlation with age-linked organ decline and levels of indoles. Interestingly, increased levels of indole-3-acetic acid, a known indole metabolite, have been shown to correlate with younger biological age, further supporting a link between biological age and levels of microbe-derived indole metabolites. The results presented in this resource paper will be useful for the future design of food intervention studies to reduce accelerated age-linked organ decline.

Controlled release fertilizer delivery system derived from rice straw cellulose nanofibres: a circular economy based solution for sustainable development.

Recently, the development of sustainable and environmentally friendly biomaterials has gained the attention of researchers as potential alternatives to petroleum-based materials. Biomaterials are a promising candidate to mitigate sustainability issues due to their renewability, biodegradability, and cost-effectiveness. Thus, the purpose of this study is to explore a cost-effective biomaterial-based delivery system for delivering fertilizers to plants. To achieve this, rice straw (agro-waste) was selected as a raw material for the extraction of cellulose. The cellulose was extracted through alkali treatment (12% NaOH), followed by TEMPO-based oxidation. The cellulose nanofibers were characterized using Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy, and transmission electron microscopy. In scanning electron microscopy, a loosening of the fibrillar structure in cellulose nanofibers (CNFs) was observed with a diameter of 17 ± 4 nm. The CNFs were loaded with nitrogen-based fertilizer (ammonium chloride) in 1:1, 1:2, and 2:1 (w/w) proportions. The loading was estimated through surface charge variation; in the case of the 1:1 sample, maximum reductions in surface charge were seen from -42.0 mV to -12.8 mV due to the binding of positive ammonium ions. In the release kinetics study, a controlled release pattern was observed at 1:1, which showed a 58% cumulative release of ammonium ions within 8 days. Thus, the study paves the way for value-added uses of rice straw as an alternative to the current environmentally harmful practices.

Bio-based cellulose nanofibers (CNFs) from rice straw via circular economy approach.Controlled release fertilizers for sustainable agriculture.Nanotechnology for precision agriculture and decarbonization via agricultural waste management.

Rice straw-derived cellulose: a comparative study of various pre-treatment technologies and its conversion to nanofibres.

Rice straw is a waste product generated after the harvesting of rice crops and is commonly disposed of by burning it off in open fields. This study explored the potential for the extraction and conversion of cellulose to cellulose nanofibres (CNFs) to be used as smart delivery systems for fertilizers applications. In this study, alkali, steam explosion, and organosolv treatments were investigated for cellulose extraction efficiency. The morphological characterization of cellulose showed smooth fibrillar structures. Fourier transform infrared spectroscopy represented significant removal of non-cellulosic components in treatments. The crystallinity increased from 52.2 to 65% in CNFs after fibrillation. Cellulose nanofibres (CNFs) had an average diameter of 37.4 nm and - 25.2 mV surface charges as determined by SEM and zeta potential, respectively, which have desired properties for holding fertilizers. Therefore, this study paves the way for value-added uses of rice straw as alternatives to current environmentally harmful practices.

Survey of Indian Dental Professionals Regarding the Use of Computer-Aided Design/Computer-Aided Manufacture (CAD/CAM) Technology.

Objective In recent years, digital processes like computer-aided design/computer-aided manufacture (CAD/CAM) have been implemented in dentistry. On the use and reporting of this technology by dentists, there is no published information. The goal of this study was to determine whether CAD/CAM technology had infiltrated Indian dentistry practices and to look into the relationship between various demographic parameters and respondents' answers about using or not using this technology. Materials and methods A sample of Indian dentists, both users and non-users of CAD/CAM, were sent about 500 online surveys. It sought to shed light on the usage style, materials, advantages, and limitations of CAD/CAM dentistry, as well as their perceived advantages and access hurdles. Statistical analysis was conducted to determine the impact of numerous demographic factors, including country of employment, dentist experience, educational attainment, and the nature of the job. Results There were 132 total replies that were finished. The majority of respondents didn't use any aspect of a digital workflow, and the biggest obstacles to using CAD/CAM were the upfront expenses and a lack of perceived advantages over traditional techniques. The likelihood of using CAD/CAM technology was highest among dentists who primarily performed private practice (P<0.0001). A third of users thought that their training was insufficient, even though the majority of users were either self-taught or schooled by firms. The majority of respondents (60.6%) believed that CAD/CAM would have a significant future role. Conclusion Most respondents said they had never used any aspect of a digital process. Although most dentists who responded to the study thought CAD/CAM would play a significant role in the future, the majority were still interested in integrating it into their workflow. Dentists continue to have certain concerns regarding the chairside CAD/CAM restorations' quality.

Algal polysaccharides for 3D printing: A review.

Algae hold particular promise as a feedstock for biomaterials, as they are capable of producing a wide variety of polymers with the properties required for 3D printing. However, the use of algal polymers has been limited to alginate, agar, carrageenan, and ulvan extracted from seaweeds. Diverse algal taxa beyond seaweeds have yet to be explored. In this comprehensive review, we discuss available algal biomaterials, their properties, and emerging applications in 3D printing techniques. We also identify elite algal strains to be used in 3D printing and comment on both advantages and limitations of algal biomass as a printing material. Global 3D printing market trends and material demands are also critically analyzed. Finally, the future prospects, opportunities, and challenges for using algal polymers in 3D printing market for a sustainable economy are discussed. We hope this review will provide a foundation for exploring the 3D printable biomaterials from algae.

Understanding the excited state dynamics and redox behavior of highly luminescent and electrochemically active Eu(III)-DES complex.

Deep eutectic solvents (DES) are considered a novel class of environmentally benign molecular solvents that are considered as potential solvents for nuclear fuel reprocessing, material recycling, and many other technological applications in both research and industry. However, there is a complete dearth of understanding pertaining to the behavior of metal ions in DES. Herein, we have investigated the speciation, complexation behavior, photochemistry, and redox properties and tried to obtain insight into the chemical aspects of the europium ion in DES (synthesized from heptyltriphenylphosphonium bromide and decanoic acid). The same has been probed using time-resolved photoluminescence (TRPL), cyclic voltammetry (CV), synchrotron-based extended X-ray absorption fine structure (EXAFS) spectroscopy, and density functional theory (DFT) calculations. TRPL indicated the stabilization of europium in the +3 oxidation state, favoring the potential of the Eu(III)-DES complex to emit red light under near UV excitation and the existence of inefficient energy transfer between DES and Eu3+. EXAFS analysis revealed the presence of Eu-O and Eu-Br, which represent the local surroundings of Eu3+ in the Eu(III)-DES complex. TRPL measurement has also suggested two distinct local environments of europium ions in the complex. DFT calculations supported the EXAFS findings, confirming that the Eu(III)-DES structure involves not only the oxygen atom of decanoic acid but also the oxygen atoms from the nitrate ions, contributing to the local coordination of Eu(III). Electrochemical studies demonstrated that the redox reaction of Eu(III)/Eu(II) in DES displays quasi-reversible behavior. The reaction rate was observed to increase with higher temperatures. The findings of this study can contribute to the understanding of the fundamental properties and potential applications of this luminescent and electrochemically active complex and pave the way for further studies and the development of novel materials with enhanced luminescent and electrochemical properties.

The Ring Fencing Mechanism: a case study of innovative self-financing approach for energy efficiency upgrades.

This paper introduces an enhanced energy auditing process including a ring-fencing mechanism for the selection of energy efficiency measures considering a multi-stage planning of the energy retrofitting project. The proposed ring-fencing approach enables SMEs to overcome the barrier of lack of capital for the implementation of energy efficiency measures by implementing first no-cost measures and only after that energy savings are accumulated considering the installation of low-cost, medium-cost and finally high-cost measures. The advantages of the proposed methodology are illustrated by means of three case studies, where a variety of energy efficiency measures were first identified throughout the auditing process involving three different SMEs, then most effective measures were selected and scheduled to be implemented according to a multi-annual plan while considering budget and operational constraints. The results of the pilot studies show that the business owners have improved their decision-making with respect to energy efficiency upgrades by engaging in the auditing process and accepting the recommendations about the suggested interventions to maximize financial (and environmental) benefits.