Structure-guided mutagenesis of Henipavirus receptor-binding proteins reveals molecular determinants of receptor usage and antibody-binding epitopes.

Nipah virus (NiV) is a highly lethal, zoonotic Henipavirus (HNV) that causes respiratory and neurological signs and symptoms in humans. Similar to other paramyxoviruses, HNVs mediate entry into host cells through the concerted actions of two surface glycoproteins: a receptor-binding protein (RBP) that mediates attachment and a fusion glycoprotein (F) that triggers fusion in an RBP-dependent manner. NiV uses ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as entry receptors. Ghana virus (GhV), a novel HNV identified in a Ghanaian bat, uses EFNB2 but not EFNB3. In this study, we employ a structure-informed approach to identify receptor-interfacing residues and systematically introduce GhV-RBP residues into a NiV-RBP backbone to uncover the molecular determinants of EFNB3 usage. We reveal two regions that severely impair EFNB3 binding by NiV-RBP and EFNB3-mediated entry by NiV pseudotyped viral particles. Further analyses uncovered two-point mutations (NiVN557SGhV and NiVY581TGhV) pivotal for this phenotype. Moreover, we identify NiV interaction with Y120 of EFNB3 as important for the usage of this receptor. Beyond these EFNB3-related findings, we reveal two domains that restrict GhV binding of EFNB2, confirm the HNV-head as an immunodominant target for polyclonal and monoclonal antibodies, and describe putative epitopes for GhV- and NiV-specific monoclonal antibodies. Cumulatively, the work presented here generates useful reagents and tools that shed insight to residues important for NiV usage of EFNB3, reveal regions critical for GhV binding of EFNB2, and describe putative HNV antibody-binding epitopes. IMPORTANCE: Hendra virus and Nipah virus (NiV) are lethal, zoonotic Henipaviruses (HNVs) that cause respiratory and neurological clinical features in humans. Since their initial outbreaks in the 1990s, several novel HNVs have been discovered worldwide, including Ghana virus. Additionally, there is serological evidence of zoonotic transmission, lending way to concerns about future outbreaks. HNV infection of cells is mediated by the receptor-binding protein (RBP) and the Fusion protein (F). The work presented here identifies NiV RBP amino acids important for the usage of ephrin-B3 (EFNB3), a receptor highly expressed in neurons and predicted to be important for neurological clinical features caused by NiV. This study also characterizes epitopes recognized by antibodies against divergent HNV RBPs. Together, this sheds insight to amino acids critical for HNV receptor usage and antibody binding, which is valuable for future studies investigating determinants of viral pathogenesis and developing antibody therapies.

An in vitro experimental pipeline to characterize the epitope of a SARS-CoV-2 neutralizing antibody.

IMPORTANCE: The COVID-19 pandemic remains a significant public health concern for the global population; the development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread so long as they recognize and interact with circulating variants. The epitope and binding specificity of a neutralizing anti-SARS-CoV-2 Spike receptor-binding domain antibody clone against many SARS-CoV-2 variants of concern were characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis and VSV-spike neutralization studies. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

PGM3 inhibition shows cooperative effects with erastin inducing pancreatic cancer cell death via activation of the unfolded protein response.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor patient prognosis. Remarkably, PDAC is one of the most aggressive and deadly tumor types and is notorious for its resistance to all types of treatment. PDAC resistance is frequently associated with a wide metabolic rewiring and in particular of the glycolytic branch named Hexosamine Biosynthetic Pathway (HBP). Methods: Transcriptional and bioinformatics analysis were performed to obtain information about the effect of the HBP inhibition in two cell models of PDAC. Cell count, western blot, HPLC and metabolomics analyses were used to determine the impact of the combined treatment between an HBP's Phosphoglucomutase 3 (PGM3) enzyme inhibitor, named FR054, and erastin (ERA), a recognized ferroptosis inducer, on PDAC cell growth and survival. Results: Here we show that the combined treatment applied to different PDAC cell lines induces a significant decrease in cell proliferation and a concurrent enhancement of cell death. Furthermore, we show that this combined treatment induces Unfolded Protein Response (UPR), NFE2 Like BZIP Transcription Factor 2 (NRF2) activation, a change in cellular redox state, a greater sensitivity to oxidative stress, a major dependence on glutamine metabolism, and finally ferroptosis cell death. Conclusion: Our study discloses that HBP inhibition enhances, via UPR activation, the ERA effect and therefore might be a novel anticancer mechanism to be exploited as PDAC therapy.

An in vitro experimental pipeline to characterize the binding specificity of SARS-CoV-2 neutralizing antibodies.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has led to over 760 million cases and >6.8 million deaths worldwide. We developed a panel of human neutralizing monoclonal antibodies (mAbs) targeting the SARS-CoV-2 Spike protein using Harbour H2L2 transgenic mice immunized with Spike receptor binding domain (RBD) (1). Representative antibodies from genetically-distinct families were evaluated for inhibition of replication-competent VSV expressing SARS-CoV-2 Spike (rcVSV-S) in place of VSV-G. One mAb (denoted FG-10A3) inhibited infection of all rcVSV-S variants; its therapeutically-modified version, STI-9167, inhibited infection of all tested SARS-CoV-2 variants, including Omicron BA.1 and BA.2, and limited virus proliferation in vivo (1). To characterize the binding specificity and epitope of FG-10A3, we generated mAb-resistant rcVSV-S virions and performed structural analysis of the antibody/antigen complex using cryo-EM. FG-10A3/STI-9167 is a Class 1 antibody that prevents Spike-ACE2 binding by engaging a region within the Spike receptor binding motif (RBM). Sequencing of mAb-resistant rcVSV-S virions identified F486 as a critical residue for mAb neutralization, with structural analysis revealing that both the variable heavy and light chains of STI-9167 bound the disulfide-stabilized 470-490 loop at the Spike RBD tip. Interestingly, substitutions at position 486 were later observed in emerging variants of concern BA.2.75.2 and XBB. This work provides a predictive modeling strategy to define the neutralizing capacity and limitations of mAb therapeutics against emerging SARS-CoV-2 variants. Importance: The COVID-19 pandemic remains a significant public health concern for the global population; development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as SARS-CoV-2 variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread with the caveat that they interact with the circulating variants. The epitope and binding specificity of a broadly neutralizing anti-SARS-CoV-2 Spike RBD antibody clone against many SARS-CoV-2 VOC was characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

Na/K-ATPase signaling tonically inhibits sodium reabsorption in the renal proximal tubule.

Through its classic ATP-dependent ion-pumping function, basolateral Na/K-ATPase (NKA) generates the Na+ gradient that drives apical Na+ reabsorption in the renal proximal tubule (RPT), primarily through the Na+ /H+ exchanger (NHE3). Accordingly, activation of NKA-mediated ion transport decreases natriuresis through activation of basolateral (NKA) and apical (NHE3) Na+ reabsorption. In contrast, activation of the more recently discovered NKA signaling function triggers cellular redistribution of RPT NKA and NHE3 and decreases Na+ reabsorption. We used gene targeting to test the respective contributions of NKA signaling and ion pumping to the overall regulation of RPT Na+ reabsorption. Knockdown of RPT NKA in cells and mice increased membrane NHE3 and Na+ /HCO3 - cotransporter (NBCe1A). Urine output and absolute Na+ excretion decreased by 65%, driven by increased RPT Na+ reabsorption (as indicated by decreased lithium clearance and unchanged glomerular filtration rate), and accompanied by elevated blood pressure. This hyper reabsorptive phenotype was rescued upon crossing with RPT NHE3-/- mice, confirming the importance of NKA/NHE3 coupling. Hence, NKA signaling exerts a tonic inhibition on Na+ reabsorption by regulating key apical and basolateral Na+ transporters. This action, lifted upon NKA genetic suppression, tonically counteracts NKA's ATP-driven function of basolateral Na+ reabsorption. Strikingly, NKA signaling is not only physiologically relevant but it also appears to be functionally dominant over NKA ion pumping in the control of RPT reabsorption.

Cheese-whey permeate improves the fitness of Escherichia coli cells during recombinant protein production.

BACKGROUND: Escherichia coli cells are the most frequently used hosts in recombinant protein production processes and mainly require molecules such as IPTG or pure lactose as inducers of heterologous expression. A possible way to reduce the production costs is to replace traditional inducers with waste materials such as cheese whey permeate (CWP). CWP is a secondary by-product generated from the production of the valuable whey proteins, which are obtained from ultrafiltration of cheese whey, a main by-product of the dairy industry, which is rich in lactose. RESULTS: The effects of CWP collected from an Italian plant were compared with those of traditional inducers on the production of two model proteins (i.e., green fluorescent protein and the toxic Q55 variant of ataxin-3), in E. coli BL21 (DE3) cells. It was found that the high lactose content of CWP (165 g/L) and the antioxidant properties of its micronutrients (vitamins, cofactors and osmolytes) sustain production yields similar to those obtained with traditional inducers, accompanied by the improvement of cell fitness. CONCLUSIONS: CWP has proven to be an effective and low-cost alternative inducer to produce recombinant proteins. Its use thus combines the advantage of exploiting a waste product with that of reducing the production costs of recombinant proteins.

Alpha-1-antitrypsin and its variant-dependent role in COVID-19 pathogenesis.

Rationale: SARS-CoV-2 entry into host cells is facilitated by endogenous and exogenous proteases that proteolytically activate the spike glycoprotein and antiproteases inhibiting this process. Understanding the key actors in viral entry is crucial for advancing knowledge of virus tropism, pathogenesis, and potential therapeutic targets. Objectives: We aimed to investigate the role of naïve serum and alpha-1-antitrypsin (AAT) in inhibiting protease-mediated SARS-CoV-2 entry and explore the implications of AAT deficiency on susceptibility to different SARS-CoV-2 variants. Findings: Our study demonstrates that naïve serum exhibits significant inhibition of SARS-CoV-2 entry, with AAT identified as the major serum protease inhibitor potently restricting entry. Using pseudoparticles, replication-competent pseudoviruses, and authentic SARS-CoV-2, we show that AAT inhibition occurs at low concentrations compared with those in serum and bronchoalveolar tissues, suggesting physiological relevance. Furthermore, sera from subjects with an AAT-deficient genotype show reduced ability to inhibit entry of both Wuhan-Hu-1 (WT) and B.1.617.2 (Delta) but exhibit no difference in inhibiting B.1.1.529 (Omicron) entry. Conclusions: AAT may have a variant-dependent therapeutic potential against SARS-CoV-2. Our findings highlight the importance of further investigating the complex interplay between proteases, antiproteases, and spike glycoprotein activation in SARS-CoV-2 and other respiratory viruses to identify potential therapeutic targets and improve understanding of disease pathogenesis.

Neutralizing activity of Sputnik V vaccine sera against SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected at least 180 million people since its identification as the cause of the current COVID-19 pandemic. The rapid pace of vaccine development has resulted in multiple vaccines already in use worldwide. The contemporaneous emergence of SARS-CoV-2 'variants of concern' (VOC) across diverse geographic locales underscores the need to monitor the efficacy of vaccines being administered globally. All WHO designated VOC carry spike (S) polymorphisms thought to enable escape from neutralizing antibodies. Here, we characterize the neutralizing activity of post-Sputnik V vaccination sera against the ensemble of S mutations present in alpha (B.1.1.7) and beta (B.1.351) VOC. Using de novo generated replication-competent vesicular stomatitis virus expressing various SARS-CoV-2-S in place of VSV-G (rcVSV-CoV2-S), coupled with a clonal 293T-ACE2 + TMPRSS2 + cell line optimized for highly efficient S-mediated infection, we determine that only 1 out of 12 post-vaccination serum samples shows effective neutralization (IC90) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralize S from B.1.1.7 and exhibit only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines.

Neutralizing activity of Sputnik V vaccine sera against SARS-CoV-2 variants.

The novel pandemic betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected at least 120 million people since its identification as the cause of a December 2019 viral pneumonia outbreak in Wuhan, China1,2. Despite the unprecedented pace of vaccine development, with six vaccines already in use worldwide, the emergence of SARS-CoV-2 'variants of concern' (VOC) across diverse geographic locales have prompted re-evaluation of strategies to achieve universal vaccination3. All three officially designated VOC carry Spike (S) polymorphisms thought to enable escape from neutralizing antibodies elicited during initial waves of the pandemic4-8. Here, we characterize the biological consequences of the ensemble of S mutations present in VOC lineages B.1.1.7 (501Y.V1) and B.1.351 (501Y.V2). Using a replication-competent EGFP-reporter vesicular stomatitis virus (VSV) system, rcVSV-CoV2-S, which encodes S from SARS coronavirus 2 in place of VSV-G, and coupled with a clonal HEK-293T ACE2 TMPRSS2 cell line optimized for highly efficient S-mediated infection, we determined that only 1 out of 12 serum samples from a cohort of recipients of the Gamaleya Sputnik V Ad26 / Ad5 vaccine showed effective neutralization (IC90) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralized S from B.1.1.7 and showed only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines.

Neutralizing activity of Sputnik V vaccine sera against SARS-CoV-2 variants.

The novel pandemic betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected at least 120 million people since its identification as the cause of a December 2019 viral pneumonia outbreak in Wuhan, China. Despite the unprecedented pace of vaccine development, with six vaccines already in use worldwide, the emergence of SARS-CoV-2 'variants of concern' (VOC) across diverse geographic locales suggests herd immunity may fail to eliminate the virus. All three officially designated VOC carry Spike (S) polymorphisms thought to enable escape from neutralizing antibodies elicited during initial waves of the pandemic. Here, we characterize the biological consequences of the ensemble of S mutations present in VOC lineages B.1.1.7 (501Y.V1) and B.1.351 (501Y.V2). Using a replication-competent EGFP-reporter vesicular stomatitis virus (VSV) system, rcVSV-CoV2-S, which encodes S from SARS coronavirus 2 in place of VSV-G, and coupled with a clonal HEK-293T ACE2 TMPRSS2 cell line optimized for highly efficient S-mediated infection, we determined that only 1 out of 12 serum samples from a cohort of recipients of the Gamaleya Sputnik V Ad26 / Ad5 vaccine showed effective neutralization (IC90) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralized S from B.1.1.7 and showed only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines.

The hypoxic transcription factor KlMga2 mediates the response to oxidative stress and influences longevity in the yeast Kluyveromyces lactis.

Hypoxia is defined as the decline of oxygen availability, depending on environmental supply and cellular consumption rate. The decrease in O2 results in reduction of available energy in facultative aerobes. The response and/or adaptation to hypoxia and other changing environmental conditions can influence the properties and functions of membranes by modifying lipid composition. In the yeast Kluyveromyces lactis, the KlMga2 gene is a hypoxic regulatory factor for lipid biosynthesis-fatty acids and sterols-and is also involved in glucose signaling, glucose catabolism and is generally important for cellular fitness. In this work we show that, in addition to the above defects, the absence of the KlMGA2 gene caused increased resistance to oxidative stress and extended lifespan of the yeast, associated with increased expression levels of catalase and SOD genes. We propose that KlMga2 might also act as a mediator of the oxidative stress response/adaptation, thus revealing connections among hypoxia, glucose signaling, fatty acid biosynthesis and ROS metabolism in K. lactis.

Unsaturated fatty acids-dependent linkage between respiration and fermentation revealed by deletion of hypoxic regulatory KlMGA2 gene in the facultative anaerobe-respiratory yeast Kluyveromyces lactis.

In the yeast Kluyveromyces lactis, the inactivation of structural or regulatory glycolytic and fermentative genes generates obligate respiratory mutants which can be characterized by sensitivity to the mitochondrial drug antimycin A on glucose medium (Rag(-) phenotype). Rag(-) mutations can occasionally be generated by the inactivation of genes not evidently related to glycolysis or fermentation. One such gene is the hypoxic regulatory gene KlMGA2. In this work, we report a study of the many defects, in addition to the Rag(-) phenotype, generated by KlMGA2 deletion. We analyzed the fermentative and respiratory metabolism, mitochondrial functioning and morphology in the Klmga2Δ strain. We also examined alterations in the regulation of the expression of lipid biosynthetic genes, in particular fatty acids, ergosterol and cardiolipin, under hypoxic and cold stress and the phenotypic suppression by unsaturated fatty acids of the deleted strain. Results indicate that, despite the fact that the deleted mutant strain had a typical glycolytic/fermentative phenotype and KlMGA2 is a hypoxic regulatory gene, the deletion of this gene generated defects linked to mitochondrial functions suggesting new roles of this protein in the general regulation and cellular fitness of K. lactis. Supplementation of unsaturated fatty acids suppressed or modified these defects suggesting that KlMga2 modulates membrane functioning or membrane-associated functions, both cytoplasmic and mitochondrial.

High cell density culture with S. cerevisiae CEN.PK113-5D for IL-1ß production: optimization, modeling, and physiological aspects.

Saccharomyces cerevisiae CEN.PK113-5D, a strain auxotrophic for uracil belonging to the CEN.PK family of the yeast S. cerevisiae, was cultured in aerated fed-batch reactor as such and once transformed to express human interleukin-1ß (IL-1ß), aiming at obtaining high cell densities and optimizing IL-1ß production. Three different exponentially increasing glucose feeding profiles were tested, all of them "in theory" promoting respiratory metabolism to obtain high biomass/product yield. A non-structured non-segregated model was developed to describe the performance of S. cerevisiae CEN.PK113-5D during the fed-batch process and, in particular, its capability to metabolize simultaneously glucose and ethanol which derived from the precedent batch growth. Our study showed that the proliferative capacity of the yeast population declined along the fed-batch run, as shown by the exponentially decreasing specific growth rates on glucose. Further, a shift towards fermentative metabolism occurred. This shift took place earlier the higher was the feed rate and was more pronounced in the case of the recombinant strain. Determination of some physiological markers (acetate production, intracellular ROS accumulation, catalase activity and cell viability) showed that neither poor oxygenation nor oxidative stress was responsible for the decreased specific growth rate, nor for the shift to fermentative metabolism.

Lack of Sir2 increases acetate consumption and decreases extracellular pro-aging factors.

Yeast chronological aging is regarded as a model for aging of mammalian post-mitotic cells. It refers to changes occurring in stationary phase cells over a relatively long period of time. How long these cells can survive in such a non-dividing state defines the chronological lifespan. Several factors influence cell survival including two well known normal by-products of yeast glucose fermentation such as ethanol and acetic acid. In fact, the presence in the growth medium of these C2 compounds has been shown to limit the chronological lifespan. In the chronological aging paradigm, a pro-aging role has also emerged for the deacetylase Sir2, the founding member of the Sirtuin family, whose loss of function increases the depletion of extracellular ethanol by an unknown mechanism. Here, we show that lack of Sir2 strongly influences carbon metabolism. In particular, we point out a more efficient acetate utilization which in turn may have a stimulatory effect on ethanol catabolism. This correlates with an enhanced glyoxylate/gluconeogenic flux which is fuelled by the acetyl-CoA produced from the acetate activation. Thus, when growth relies on a respiratory metabolism such as that on ethanol or acetate, SIR2 inactivation favors growth. Moreover, in the chronological aging paradigm, the increase in the acetate metabolism implies that sir2Δ cells avoid acetic acid accumulation in the medium and deplete ethanol faster; consequently pro-aging extracellular signals are reduced. In addition, an enhanced gluconeogenesis allows replenishment of intracellular glucose stores which may be useful for better long-term cell survival.

Effect of auxotrophies on yeast performance in aerated fed-batch reactor.

A systematic investigation on the effects of auxotrophies on the performance of yeast in aerated fed-batch reactor was carried out. Six isogenic strains from the CEN.PK family of Saccharomyces cerevisiae, one prototroph and five auxotrophs, were grown in aerated fed-batch reactor using the same operative conditions and a proper nutritional supplementation. The performance of the strains, in terms of final biomass decreased with increasing the number of auxotrophies. Auxotrophy for leucine exerted a profound negative effect on the performance of the strains. Accumulation of reactive oxygen species (ROS) in the cells of the strain carrying four auxotrophies and its significant viability loss, were indicative of an oxidative stress response induced by exposure of cells to the environmental conditions. The mathematical model was fundamental to highlight how the carbon flux, depending on the number and type of auxotrophies, was diverted towards the production of increasingly large quantities of energy for maintenance.

Nutritional modulation of CK2 in Saccharomyces cerevisiae: regulating the activity of a constitutive enzyme.

CK2 is a highly conserved protein kinase involved in different cellular processes, which shows a higher activity in actively proliferating mammalian cells and in various types of cancer and cancer cell lines. We recently demonstrated that CK2 activity is strongly influenced by growth rate in yeast cells as well. Here, we extend our previous findings and show that, in cells grown in either glucose or ethanol-supplemented media, CK2 presents no alteration in K(m) for both the ATP and the peptide substrate RRRADDSDDDDD, while a significant increase in V (max) is observed. In chemostat-grown cells, no difference of CK2 activity was observed in cells grown at the same dilution rate in media supplemented with either ethanol or glucose, excluding the contribution of carbon metabolism on CK2 activity. By using the eIF2ß-derived peptide, which can be phosphorylated by the holoenzyme but not by the free catalytic subunits, we show that the holoenzyme activity requires the concurrent presence of both ß and ß' encoding genes. Finally, conditions of nitrogen deprivation leading to a G0-like arrest result in a decrease of total CK2 activity, but have no effect on the activity of the holoenzyme. These findings newly indicate a regulatory role of ß and ß' subunits of CK2 in the nutrient response.

Deletion or overexpression of mitochondrial NAD+ carriers in Saccharomyces cerevisiae alters cellular NAD and ATP contents and affects mitochondrial metabolism and the rate of glycolysis.

The modification of enzyme cofactor concentrations can be used as a method for both studying and engineering metabolism. We varied Saccharomyces cerevisiae mitochondrial NAD levels by altering expression of its specific mitochondrial carriers. Changes in mitochondrial NAD levels affected the overall cellular concentration of this coenzyme and the cellular metabolism. In batch culture, a strain with a severe NAD depletion in mitochondria succeeded in growing, albeit at a low rate, on fully respiratory media. Although the strain increased the efficiency of its oxidative phosphorylation, the ATP concentration was low. Under the same growth conditions, a strain with a mitochondrial NAD concentration higher than that of the wild type similarly displayed a low cellular ATP level, but its growth rate was not affected. In chemostat cultures, when cellular metabolism was fully respiratory, both mutants showed low biomass yields, indicative of impaired energetic efficiency. The two mutants increased their glycolytic fluxes, and as a consequence, the Crabtree effect was triggered at lower dilution rates. Strikingly, the mutants switched from a fully respiratory metabolism to a respirofermentative one at the same specific glucose flux as that of the wild type. This result seems to indicate that the specific glucose uptake rate and/or glycolytic flux should be considered one of the most important independent variables for establishing the long-term Crabtree effect. In cells growing under oxidative conditions, bioenergetic efficiency was affected by both low and high mitochondrial NAD availability, which suggests the existence of a critical mitochondrial NAD concentration in order to achieve optimal mitochondrial functionality.

CK2 activity is modulated by growth rate in Saccharomyces cerevisiae.

CK2 is a highly conserved protein kinase controlling different cellular processes. It shows a higher activity in proliferating mammalian cells, in various types of cancer cell lines and tumors. The findings presented herein provide the first evidence of an in vivo modulation of CK2 activity, dependent on growth rate, in Saccharomyces cerevisiae. In fact, CK2 activity, assayed on nuclear extracts, is shown to increase in exponential growing batch cultures at faster growth rate, while localization of catalytic and regulatory subunits is not nutritionally modulated. Differences in intracellular CK2 activity of glucose- and ethanol-grown cells appear to depend on both increase in molecule number and k(cat). Also in chemostat cultures nuclear CK2 activity is higher in faster growing cells providing the first unequivocal demonstration that growth rate itself can affect CK2 activity in a eukaryotic organism.

ITAC volume assessment through a Gaussian hidden Markov random field model-based algorithm.

In this paper, a semi-automatic segmentation method for volume assessment of Intestinal-type adenocarcinoma (ITAC) is presented and validated. The method is based on a Gaussian hidden Markov random field (GHMRF) model that represents an advanced version of a finite Gaussian mixture (FGM) model as it encodes spatial information through the mutual influences of neighboring sites. To fit the GHMRF model an expectation maximization (EM) algorithm is used. We applied the method to a magnetic resonance data sets (each of them composed by T1-weighted, Contrast Enhanced T1-weighted and T2-weighted images) for a total of 49 tumor-contained slices. We tested GHMRF performances with respect to FGM by both a numerical and a clinical evaluation. Results show that the proposed method has a higher accuracy in quantifying lesion area than FGM and it can be applied in the evaluation of tumor response to therapy.

Induction by hypoxia of heterologous-protein production with the KlPDC1 promoter in yeasts.

The control of promoter activity by oxygen availability appears to be an intriguing system for heterologous protein production. In fact, during cell growth in a bioreactor, an oxygen shortage is easily obtained simply by interrupting the air supply. The purpose of our work was to explore the possible use of hypoxic induction of the KlPDC1 promoter to direct heterologous gene expression in yeast. In the present study, an expression system based on the KlPDC1 promoter was developed and characterized. Several heterologous proteins, differing in size, origin, localization, and posttranslational modification, were successfully expressed in Kluyveromyces lactis under the control of the wild type or a modified promoter sequence, with a production ratio between 4 and more than 100. Yields were further optimized by a more accurate control of hypoxic physiological conditions. Production of as high as 180 mg/liter of human interleukin-1beta was obtained, representing the highest value obtained with yeasts in a lab-scale bioreactor to date. Moreover, the transferability of our system to related yeasts was assessed. The lacZ gene from Escherichia coli was cloned downstream of the KlPDC1 promoter in order to get beta-galactosidase activity in response to induction of the promoter. A centromeric vector harboring this expression cassette was introduced in Saccharomyces cerevisiae and in Zygosaccharomyces bailii, and effects of hypoxic induction were measured and compared to those already observed in K. lactis cells. Interestingly, we found that the induction still worked in Z. bailii; thus, this promotor constitutes a possible inducible system for this new nonconventional host.