Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast.

Although many prokaryotes have glycolysis alternatives, it's considered as the only energy-generating glucose catabolic pathway in eukaryotes. Here, we managed to create a hybrid-glycolysis yeast. Subsequently, we identified an inositol pyrophosphatase encoded by OCA5 that could regulate glycolysis and respiration by adjusting 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) levels. 5-InsP7 levels could regulate the expression of genes involved in glycolysis and respiration, representing a global mechanism that could sense ATP levels and regulate central carbon metabolism. The hybrid-glycolysis yeast did not produce ethanol during growth under excess glucose and could produce 2.68 g/L free fatty acids, which is the highest reported production in shake flask of Saccharomyces cerevisiae. This study demonstrated the significance of hybrid-glycolysis yeast and determined Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration, which may shed light on the role of inositol pyrophosphates in regulating eukaryotic metabolism.

Matched analysis of circulating selenium with the breast cancer selenotranscriptome: a multicentre prospective study.

INTRODUCTION: Low serum selenium and altered tumour RNA expression of certain selenoproteins are associated with a poor breast cancer prognosis. Selenoprotein expression stringently depends on selenium availability, hence circulating selenium may interact with tumour selenoprotein expression. However, there is no matched analysis to date. METHODS: This study included 1453 patients with newly diagnosed breast cancer from the multicentric prospective Sweden Cancerome Analysis Network - Breast study. Total serum selenium, selenoprotein P and glutathione peroxidase 3 were analysed at time of diagnosis. Bulk RNA-sequencing was conducted in matched tumour tissues. Fully adjusted Cox regression models with an interaction term were employed to detect dose-dependent interactions of circulating selenium with the associations of tumour selenoprotein mRNA expression and mortality. RESULTS: 237 deaths were recorded within ~ 9 years follow-up. All three serum selenium biomarkers correlated positively (p < 0.001). All selenoproteins except for GPX6 were expressed in tumour tissues. Single cell RNA-sequencing revealed a heterogeneous expression pattern in the tumour microenvironment. Circulating selenium correlated positively with tumour SELENOW and SELENON expression (p < 0.001). In fully adjusted models, the associations of DIO1, DIO3 and SELENOM with mortality were dose-dependently modified by serum selenium (p < 0.001, p = 0.020, p = 0.038, respectively). With increasing selenium, DIO1 and SELENOM associated with lower, whereas DIO3 expression associated with higher mortality. Association of DIO1 with lower mortality was only apparent in patients with high selenium [above median (70.36 µg/L)], and the HR (95%CI) for one-unit increase in log(FPKM + 1) was 0.70 (0.50-0.98). CONCLUSIONS: This first unbiased analysis of serum selenium with the breast cancer selenotranscriptome identified an effect-modification of selenium on the associations of DIO1, SELENOM, and DIO3 with prognosis. Selenium substitution in patients with DIO1-expressing tumours merits consideration to improve survival.

Induction of Breast Cancer Cell Apoptosis by TRAIL and Smac Mimetics: Involvement of RIP1 and cFLIP.

Smac mimetics are a group of compounds able to facilitate cell death in cancer cells. TNF-related apoptosis-inducing ligand (TRAIL) is a death receptor ligand currently explored in combination with Smac mimetics. The molecular mechanisms determining if the combination treatment results in apoptosis are however not fully understood. In this study, we aimed to shed light on these mechanisms in breast cancer cells. Three breast cancer cell lines, MDA-MB-468, CAMA-1 and MCF-7, were used to evaluate the effects of Smac mimetic LCL-161 and TRAIL using cell death assays and Western blot. The combination treatment induces apoptosis and caspase-8 cleavage in MDA-MB-468 and CAMA-1 but not in MCF-7 cells and downregulation of caspase-8 blocked apoptosis. Downregulation, but not kinase inhibition, of receptor-interacting protein 1 (RIP1) suppressed apoptosis in CAMA-1. Apoptosis is preceded by association of RIP1 with caspase-8. Downregulating cellular FLICE-like inhibitory protein (c-FLIP) resulted in increased caspase cleavage and some induction of apoptosis by TRAIL and LCL-161 in MCF-7. In CAMA-1, c-FLIP depletion potentiated TRAIL-induced caspase cleavage and LCL-161 did not increase it further. Our results lend further support to a model where LCL-161 enables the formation of a complex including RIP1 and caspase-8 and circumvents c-FLIP-mediated inhibition of caspase activation.

The combination of TRAIL and the Smac mimetic LCL-161 induces an irreversible phenotypic change of MCF-7 breast cancer cells.

INTRODUCTION: Breast cancer is the most common malignancy affecting women. Although the prognosis generally is good, a substantial number of patients still suffer from relapse, emphasizing the need for novel treatments. Smac mimetics were developed to facilitate cell death by blocking inhibitor of apoptosis proteins (IAPs). It has been suggested that TNF-related apoptosis inducing ligand (TRAIL) can be used together with Smac mimetics to induce cancer cell death. METHODS: Cell viability was studied with Trypan blue staining and Annexin V assay, siRNA was used to downregulate specific proteins, protein levels were estimated with Western blot, and mRNA levels were analyzed with qPCR, microarray and RNA-seq. For global expression, groups were compared with principal component analysis and the limma package in R. Gene enrichment was analyzed with Fisher's test. For other experiments, significance of difference was tested by one-way ANOVA, followed by Tukey's HSD test. RESULTS: The combination of Smac mimetic LCL-161 and TRAIL induces an irreversible change in phenotype, but not cell death, of luminal MCF-7 breast cancer cells. The cells become small and circular and dissociate from each other and the effect could not be reversed by returning the cells to regular growth medium. The morphology change could be prevented by caspase inhibition using z-VAD-FMK and downregulation of caspase-8. Caspase-7 is also indicated to be of importance since downregulation of this caspase resulted in fewer morphologically changed cells. Enrichment analyses of changes in global gene expression demonstrated that genes associated with estrogen receptor (ER) signaling are downregulated, whereas nuclear factor kappa B- (NF-κB) and interferon- (IFN) driven genes are upregulated in altered cells. However, inhibition of these pathways did not influence the change in morphology. Induction of IFN-induced genes were potentiated but NF-ĸB-driven genes were slightly suppressed by caspase inhibition. CONCLUSIONS: The results demonstrate that LCL-161 and TRAIL can irreversibly alter the MCF-7 breast cancer cell phenotype. However, the changes in morphology and global gene expression are mediated via separate pathways.

Chemical disarming of isoniazid resistance in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) killed more people in 2017 than any other single infectious agent. This dangerous pathogen is able to withstand stresses imposed by the immune system and tolerate exposure to antibiotics, resulting in persistent infection. The global tuberculosis (TB) epidemic has been exacerbated by the emergence of mutant strains of Mtb that are resistant to frontline antibiotics. Thus, both phenotypic drug tolerance and genetic drug resistance are major obstacles to successful TB therapy. Using a chemical approach to identify compounds that block stress and drug tolerance, as opposed to traditional screens for compounds that kill Mtb, we identified a small molecule, C10, that blocks tolerance to oxidative stress, acid stress, and the frontline antibiotic isoniazid (INH). In addition, we found that C10 prevents the selection for INH-resistant mutants and restores INH sensitivity in otherwise INH-resistant Mtb strains harboring mutations in the katG gene, which encodes the enzyme that converts the prodrug INH to its active form. Through mechanistic studies, we discovered that C10 inhibits Mtb respiration, revealing a link between respiration homeostasis and INH sensitivity. Therefore, by using C10 to dissect Mtb persistence, we discovered that INH resistance is not absolute and can be reversed.

Combinatorial molecule screening identified a novel diterpene and the BET inhibitor CPI-203 as differentiation inducers of primary acute myeloid leukemia cells.

Combination treatment has proven effective for patients with acute promyelocytic leukemia, exemplifying the importance of therapy targeting multiple components of oncogenic regulation for a successful outcome. However, recent studies have shown that the mutational complexity of acute myeloid leukemia (AML) precludes the translation of molecular targeting into clinical success. Here, as a complement to genetic profiling, we used unbiased, combinatorial in vitro drug screening to identify pathways that drive AML and to develop personalized combinatorial treatments. First, we screened 513 natural compounds on primary AML cells and identified a novel diterpene (H4) that preferentially induced differentiation of FLT3 wild-type AML, while FLT3-ITD/mutations conferred resistance. The samples responding to H4, displayed increased expression of myeloid markers, a clear decrease in the nuclear-cytoplasmic ratio and the potential of re-activation of the monocytic transcriptional program reducing leukemia propagation in vivo. By combinatorial screening using H4 and molecules with defined targets, we demonstrated that H4 induces differentiation by the activation of the protein kinase C (PKC) signaling pathway, and in line with this, activates PKC phosphorylation and translocation of PKC to the cell membrane. Furthermore, the combinatorial screening identified a bromo- and extra-terminal domain (BET) inhibitor that could further improve H4-dependent leukemic differentiation in FLT3 wild-type monocytic AML. These findings illustrate the value of an unbiased, multiplex screening platform for developing combinatorial therapeutic approaches for AML.

Inflammatory macrophage derived TNFα downregulates estrogen receptor α via FOXO3a inactivation in human breast cancer cells.

Patients with estrogen receptor α positive (ERα+) breast cancer can respond to endocrine therapy, but treatment resistance is common and associated with downregulation of ERα expression in the dormant residual cells. Here we show, using long-term NSG xenograft models of human breast cancer and primary human monocytes, in vitro primary cell cultures and tumors from breast cancer patients, that macrophage derived tumor necrosis factor alpha (TNFα) downregulates ERα in breast cancer cells via inactivation of the transcription factor Forkhead box O transcription factor 3a (FOXO3a). Moreover, presence of tumor associated macrophages in the primary tumor of breast cancer patients, was associated with ERα negativity, and with worse prognosis in patients with ERα+ tumors. We propose that pro-inflammatory macrophages, despite being tumoricidal, may have direct effects on tumor progression and endocrine resistance in breast cancer patients. Our findings suggest that TNFα antagonists should be evaluated for treatment of ERα+ breast cancer.

Refinement of breast cancer molecular classification by miRNA expression profiles.

BACKGROUND: Accurate classification of breast cancer using gene expression profiles has contributed to a better understanding of the biological mechanisms behind the disease and has paved the way for better prognostication and treatment prediction. RESULTS: We found that miRNA profiles largely recapitulate intrinsic subtypes. In the case of HER2-enriched tumors a small set of miRNAs including the HER2-encoded mir-4728 identifies the group with very high specificity. We also identified differential expression of the miR-99a/let-7c/miR-125b miRNA cluster as a marker for separation of the Luminal A and B subtypes. High expression of this miRNA cluster is linked to better overall survival among patients with Luminal A tumors. Correlation between the miRNA cluster and their precursor LINC00478 is highly significant suggesting that its expression could help improve the accuracy of present day's signatures. CONCLUSIONS: We show here that miRNA expression can be translated into mRNA profiles and that the inclusion of miRNA information facilitates the molecular diagnosis of specific subtypes, in particular the clinically relevant sub-classification of luminal tumors.

Corticosteroids protect infected cells against mycobacterial killing in vitro.

The effect of corticosteroids on human physiology is complex and their use in tuberculosis patients remains controversial. In a high-throughput screening approach designed to discover virulence inhibitors, several corticosteroids were found to prevent cytolysis of fibroblasts infected with mycobacteria. Further experiments with Mycobacterium tuberculosis showed anti-cytolytic activity in the 10 nM range, but no effect on bacterial growth or survival in the absence of host cells at 20 µM. The results from a panel of corticosteroids with various affinities to the glucocorticoid- and mineralocorticoid receptors indicate that the inhibition of cytolysis most likely is mediated through the glucocorticoid receptor. Using live-imaging of M. tuberculosis-infected human monocyte-derived macrophages, we also show that corticosteroids to some extent control intracellular bacteria. In vitro systems with reduced complexity are to further study and understand the interactions between bacterial infection, immune defense and cell signaling.

Agreement between molecular subtyping and surrogate subtype classification: a contemporary population-based study of ER-positive/HER2-negative primary breast cancer.

PURPOSE: Oestrogen receptor-positive (ER+) and human epidermal receptor 2-negative (HER2-) breast cancers are classified as Luminal A or B based on gene expression, but immunohistochemical markers are used for surrogate subtyping. The aims of this study were to examine the agreement between molecular subtyping (MS) and surrogate subtyping and to identify subgroups consisting mainly of Luminal A or B tumours. METHODS: The cohort consisted of 2063 patients diagnosed between 2013-2017, with primary ER+/HER2- breast cancer, analysed by RNA sequencing. Surrogate subtyping was performed according to three algorithms (St. Gallen 2013, Maisonneuve and our proposed Grade-based classification). Agreement (%) and kappa statistics (κ) were used as concordance measures and ROC analysis for luminal distinction. Ki67, progesterone receptor (PR) and histological grade (HG) were further investigated as surrogate markers. RESULTS: The agreement rates between the MS and St. Gallen 2013, Maisonneuve and Grade-based classifications were 62% (κ = 0.30), 66% (κ = 0.35) and 70% (κ = 0.41), respectively. PR did not contribute to distinguishing Luminal A from B tumours (auROC = 0.56). By classifying HG1-2 tumours as Luminal A-like and HG3 as Luminal B-like, agreement with MS was 80% (κ = 0.46). Moreover, by combining HG and Ki67 status, a large subgroup of patients (51% of the cohort) having > 90% Luminal A tumours could be identified. CONCLUSIONS: Agreement between MS and surrogate classifications was generally poor. However, a post hoc analysis showed that a combination of HG and Ki67 could identify patients very likely to have Luminal A tumours according to MS.

Yeast mitochondria: an overview of mitochondrial biology and the potential of mitochondrial systems biology.

Mitochondria are dynamic organelles of endosymbiotic origin that are essential components of eukaryal cells. They contain their own genetic machinery, have multicopy genomes and like their bacterial ancestors they consist of two membranes. However, the majority of the ancestral genome has been lost or transferred to the nuclear genome of the host, preserving only a core set of genes involved in oxidative phosphorylation. Mitochondria perform numerous biological tasks ranging from bioenergetics to production of protein co-factors, including heme and iron-sulfur clusters. Due to the importance of mitochondria in many cellular processes, mitochondrial dysfunction is implicated in a wide variety of human disorders. Much of our current knowledge on mitochondrial function and dysfunction comes from studies using Saccharomyces cerevisiae. This yeast has good fermenting capacity, rendering tolerance to mutations that inactivate oxidative phosphorylation and complete loss of mitochondrial DNA. Here, we review yeast mitochondrial metabolism and function with focus on S. cerevisiae and its contribution in understanding mitochondrial biology. We further review how systems biology studies, including mathematical modeling, has allowed gaining new insight into mitochondrial function, and argue that this approach may enable us to gain a holistic view on how mitochondrial function interacts with different cellular processes.

Cathepsin K Contributes to Cavitation and Collagen Turnover in Pulmonary Tuberculosis.

Cavitation in tuberculosis enables highly efficient person-to-person aerosol transmission. We performed transcriptomics in the rabbit cavitary tuberculosis model. Among 17 318 transcripts, we identified 22 upregulated proteases. Five type I collagenases were overrepresented: cathepsin K (CTSK), mast cell chymase-1 (CMA1), matrix metalloproteinase 1 (MMP-1), MMP-13, and MMP-14. Studies of collagen turnover markers, specifically, collagen type I C-terminal propeptide (CICP), urinary deoxypyridinoline (DPD), and urinary helical peptide, revealed that cavitation in tuberculosis leads to both type I collagen destruction and synthesis and that proteases other than MMP-1, MMP-13, and MMP-14 are involved, suggesting a key role for CTSK. We confirmed the importance of CTSK upregulation in human lung specimens, using immunohistochemical analysis, which revealed perigranulomatous staining for CTSK, and we showed that CTSK levels were increased in the serum of patients with tuberculosis, compared with those in controls (3.3 vs 0.3 ng/mL; P = .005).

The expression pattern of matrix-producing tumor stroma is of prognostic importance in breast cancer.

BACKGROUND: There are several indications that the composition of the tumor stroma can contribute to the malignancy of a tumor. Here we utilized expression data sets to identify metagenes that may serve as surrogate marker for the extent of matrix production and vascularization of a tumor and to characterize prognostic molecular components of the stroma. METHODS: TCGA data sets from six cancer forms, two breast cancer microarray sets and one mRNA data set of xenografted tumors were downloaded. Using the mean correlation as distance measure compact clusters with genes representing extracellular matrix production (ECM metagene) and vascularization (endothelial metagene) were defined. Explorative Cox modeling was used to identify prognostic stromal gene sets. RESULTS: Clustering of stromal genes in six cancer data sets resulted in metagenes, each containing three genes, representing matrix production and vascularization. The ECM metagene was associated with poor prognosis in renal clear cell carcinoma and in lung adenocarcinoma but not in other cancers investigated. Explorative Cox modeling using gene pairs identified gene sets that in multivariate models were prognostic in breast cancer. This was validated in two microarray sets. Two notable genes are TCF4 and P4HA3 which were included in the sets associated with positive and negative prognosis, respectively. Data from laser-microdissected tumors, a xenografted tumor data set and from correlation analyses demonstrate the stroma specificity of the genes. CONCLUSIONS: It is possible to construct ECM and endothelial metagenes common for several cancer forms. The molecular composition of matrix-producing cells, rather than the extent of matrix production seem to be important for breast cancer prognosis.

Molecular characterization of protein kinase C delta (PKCδ)-Smac interactions.

BACKGROUND: Protein kinase C δ (PKCδ) is known to be an important regulator of apoptosis, having mainly pro- but also anti-apoptotic effects depending on context. In a previous study, we found that PKCδ interacts with the pro-apoptotic protein Smac. Smac facilitates apoptosis by suppressing inhibitor of apoptosis proteins (IAPs). We previously established that the PKCδ-Smac complex dissociates during induction of apoptosis indicating a functional importance. Because the knowledge on the molecular determinants of the interaction is limited, we aimed at characterizing the interactions between PKCδ and Smac. RESULTS: We found that PKCδ binds directly to Smac through its regulatory domain. The interaction is enhanced by the PKC activator TPA and seems to be independent of PKCδ catalytic activity since the PKC kinase inhibitor GF109203X did not inhibit the interaction. In addition, we found that C1 and C2 domains from several PKC isoforms have Smac-binding capacity. CONCLUSIONS: Our data demonstrate that the Smac-PKCδ interaction is direct and that it is facilitated by an open conformation of PKCδ. The binding is mediated via the PKCδ regulatory domain and both the C1 and C2 domains have Smac-binding capacity. With this study we thereby provide molecular information on an interaction between two apoptosis-regulating proteins.

Mycobacterium tuberculosis dysregulates MMP/TIMP balance to drive rapid cavitation and unrestrained bacterial proliferation.

Active tuberculosis (TB) often presents with advanced pulmonary disease, including irreversible lung damage and cavities. Cavitary pathology contributes to antibiotic failure, transmission, morbidity and mortality. Matrix metalloproteinases (MMPs), in particular MMP-1, are implicated in TB pathogenesis. We explored the mechanisms relating MMP/TIMP imbalance to cavity formation in a modified rabbit model of cavitary TB. Our model resulted in consistent progression of consolidation to human-like cavities (100% by day 28), with resultant bacillary burdens (>10(7) CFU/g) far greater than those found in matched granulomatous tissue (10(5) CFU/g). Using a novel, breath-hold computed tomography (CT) scanning and image analysis protocol, we showed that cavities developed rapidly from areas of densely consolidated tissue. Radiological change correlated with a decrease in functional lung tissue, as estimated by changes in lung density during controlled pulmonary expansion (R(2) = 0.6356, p < 0.0001). We demonstrated that the expression of interstitial collagenase (MMP-1) was specifically greater in cavitary compared to granulomatous lesions (p < 0.01), and that TIMP-3 significantly decreased at the cavity surface. Our findings demonstrated that an MMP-1/TIMP imbalance is associated with the progression of consolidated regions to cavities containing very high bacterial burdens. Our model provided mechanistic insight, correlating with human disease at the pathological, microbiological and molecular levels. It also provided a strategy to investigate therapeutics in the context of complex TB pathology. We used these findings to predict a MMP/TIMP balance in active TB and confirmed this in human plasma, revealing the potential of MMP/TIMP levels as key components of a diagnostic matrix aimed at distinguishing active from latent TB (PPV = 92.9%, 95% CI 66.1-99.8%, NPV = 85.6%; 95% CI 77.0-91.9%).

In vivo prediction of tuberculosis-associated cavity formation in rabbits.

The presence of cavitary lesions in patients with tuberculosis poses a significant clinical concern due to the risk of infectivity and the risk of antibiotic treatment failure. We describe 2 algorithms that use noninvasive positron emission tomography (PET) and computed tomography (CT) to predict the development of cavitary lesions in rabbits. Analysis of the PET region of interest predicted cavitary disease with 100% sensitivity and 76% specificity, and analysis of the CT region of interest predicted cavitary disease with 83.3% sensitivity and 76.9% specificity. Our results show that restricting our analysis to regions with high [(18)F]-fluorodeoxyglucose uptake provided the best combination of sensitivity and specificity.

Cord blood insulinoma-associated protein 2 autoantibodies are associated with increased risk of type 1 diabetes in the population-based Diabetes Prediction in Skåne study.

AIMS/HYPOTHESIS: The aim of this study was to examine the effect of cord blood autoantibodies on the risk for type 1 diabetes in children followed prospectively from birth. METHODS: The Diabetes Prediction in Skåne (DiPiS) study consists of 35,853 children from the general population born during 2000-2004. Samples were collected at birth and analysed for HLA genotypes and autoantibodies to glutamate decarboxylase 65 (GAD65), insulin and insulinoma-associated protein 2 (IA-2). After adjusting for HLA, sex, maternal age and parental type 1 diabetes, independent associations with risk of diabetes were assessed using multivariate Cox proportional hazards models. RESULTS: In total, 151 children (0.4%) had developed type 1 diabetes by the end of 2013 at a median age of 5.8 years (0.8-12.2 years). In the multivariate analysis, the presence of IA-2 autoantibodies (IA-2A) in cord blood (HR 6.88, 95% CI 1.46,32.4; p = 0.003), but not maternal diabetes (HR 1.38, 95% CI 0.24,7.84; p = 0.71), was associated with risk of developing type 1 diabetes. No increased risk could be seen for the presence of autoantibodies to GAD65 or insulin. CONCLUSIONS/INTERPRETATION: Our study indicates that the presence of cord blood IA-2A superimposes maternal diabetes and other cord blood islet autoantibodies as a predictor of type 1 diabetes development in the child. These findings may be of significance for future screening and study protocols on type 1 diabetes prediction.

Prognostic stromal gene signatures in breast cancer.

INTRODUCTION: Global gene expression analysis of tumor samples has been a valuable tool to subgroup tumors and has the potential to be of prognostic and predictive value. However, tumors are heterogeneous, and homogenates will consist of several different cell types. This study was designed to obtain more refined expression data representing different compartments of the tumor. METHODS: Formalin-fixed paraffin-embedded stroma-rich triple-negative breast cancer tumors were laser-microdissected, and RNA was extracted and processed to enable microarray hybridization. Genes enriched in stroma were identified and used to generate signatures by identifying correlating genes in publicly available data sets. The prognostic implications of the signature were analyzed. RESULTS: Comparison of the expression pattern from stromal and cancer cell compartments from three tumors revealed a number of genes that were essentially specifically expressed in the respective compartments. The stroma-specific genes indicated contribution from fibroblasts, endothelial cells, and immune/inflammatory cells. The gene set was expanded by identifying correlating mRNAs using breast cancer mRNA expression data from The Cancer Genome Atlas. By iterative analyses, 16 gene signatures of highly correlating genes were characterized. Based on the gene composition, they seem to represent different cell types. In multivariate Cox proportional hazard models, two immune/inflammatory signatures had opposing hazard ratios for breast cancer recurrence also after adjusting for clinicopathological variables and molecular subgroup. The signature associated with poor prognosis consisted mainly of C1Q genes and the one associated with good prognosis contained HLA genes. This association with prognosis was seen for other cancers as well as in other breast cancer data sets. CONCLUSIONS: Our data indicate that the molecular composition of the immune response in a tumor may be a powerful predictor of cancer prognosis.

Fermentation performance and physiology of two strains of Saccharomyces cerevisiae during growth in high gravity spruce hydrolysate and spent sulphite liquor.

BACKGROUND: Lignocellulosic materials are a diverse group of substrates that are generally scarce in nutrients, which compromises the tolerance and fermentation performance of the fermenting organism. The problem is exacerbated by harsh pre-treatment, which introduces sugars and substances inhibitory to yeast metabolism. This study compares the fermentation behaviours of two yeast strains using different types of lignocellulosic substrates; high gravity dilute acid spruce hydrolysate (SH) and spent sulphite liquor (SSL), in the absence and presence of yeast extract. To this end, the fermentation performance, energy status and fermentation capacity of the strains were measured under different growth conditions. RESULTS: Nutrient supplementation with yeast extract increased sugar uptake, cell growth and ethanol production in all tested fermentation conditions, but had little or no effect on the energy status, irrespective of media. Nutrient-supplemented medium enhanced the fermentation capacity of harvested cells, indicating that cell viability and reusability was increased by nutrient addition. CONCLUSIONS: Although both substrates belong to the lignocellulosic spruce hydrolysates, their differences offer specific challenges and the overall yields and productivities largely depend on choice of fermenting strain.