Tool to Resolve Distortions in Elemental and Isotopic Imaging.

Nanoscale secondary ion mass spectrometry (NanoSIMS) makes it possible to visualize elements and isotopes in a wide range of samples at a high resolution. However, the fidelity and quality of NanoSIMS images often suffer from distortions because of a requirement to acquire and integrate multiple image frames. We developed an optical flow-based algorithm tool, NanoSIMS Stabilizer, for all-channel postacquisition registration of images. The NanoSIMS Stabilizer effectively deals with the distortions and artifacts, resulting in a high-resolution visualization of isotope and element distribution. It is open source with an easy-to-use ImageJ plugin and is accompanied by a Python version with GPU acceleration.

Penetration of oils into hair.

OBJECTIVE: The objective of this work was to understand how triglyceride plant oils can deliver strength and softness benefits to hair by their penetration. These plant oils are complex mixtures of TAGs, so the initial studies performed were with pure TAGs and then these data compared to plant oils and their measured TAG compositions. METHODS: LC-MS was used to identify the di and triglycerides in coconut oil, Camellia oleifera oil and safflower seed oil. Penetration of these plant oils and pure individual triglycerides was measured by a differential extraction method. Cross-sections of oils treated with 13C-labelled triolein were studied by NanoSIMS to visualize location of triglyceride inside hair. Fatigue strength was measured using constant stress to generate a survival distribution. Models of the lipid-rich cell membrane complex (CMC) were created with the equimolar ratio of 18-methyl-eicosanoic acid (MEAS), palmitic acid (C16:0) and oleic acid (C18:1). RESULTS: Penetration of the individual pure TAGs was confirmed for all chain lengths and degree of unsaturation tested with higher penetration for shorter chain lengths and unsaturated fatty acids. Detailed compositional analysis of selected plant oils showed a wide variety of TAGs and penetration was also demonstrated for these oils. NanoSIMS and modelling confirmed these TAGs are penetrating the lipid-rich CMC of hair and are interacting with the fatty acids that make up the CMC. All plant oils delivered a fatigue strength improvement by penetration into the CMC and it is proposed that these oils prevent formation and/or propagation of flaws in the CMC network that leads to breakage. CONCLUSIONS: Many plant oils with a wide range of triglyceride compositions can penetrate into hair and NanoSIMS data confirmed these oils partition into the lipid-rich cell membrane complex. Penetration studies of individual TAGs shown to be present in these oils confirmed TAGs of varying chain length can penetrate and there is a correlation between increased penetration efficacy and shorter chain lengths and presence of unsaturation in the fatty acid chains. All the oils studied delivered single fibre fatigue strength benefits.

OBJECTIF: L'objectif de ce travail était de comprendre comment les huiles végétales à base de triglycérides peuvent apporter aux cheveux des bienfaits en termes de résistance et de douceur grâce à leur pénétration. Ces huiles végétales sont des mélanges complexes de TAG, donc les études réalisées initiales ont porté sur des TAG purs et ces données ont été comparées à des huiles végétales et leurs compositions en TAG mesurées. MÉTHODES: La LC­MS a été utilisée pour identifier les di­ et triglycérides dans l'huile de noix de coco, l'huile de Camellia oleifera et l'huile de graines de carthame. La pénétration de ces huiles végétales et des triglycérides individuels purs a été mesurée par une méthode d'extraction différentielle. Des coupes transversales d'huiles traitées avec de la trioléine marquée au C13 ont été étudiées par NanoSIMS pour visualiser l'emplacement des triglycérides à l'intérieur des cheveux. La résistance à la fatigue a été mesurée à l'aide d'une sollicitation constante pour générer une distribution de la survie. Des modèles du complexe de membrane cellulaire riche en lipides (CMC) ont été créés avec le rapport équimolaire en acide 18­méthyleicosanoïque (MEAS), acide palmitique (C16:0) et acide oléique (C18:1). RÉSULTATS: La pénétration des TAG purs individuels a été confirmée pour toutes les longueurs de chaîne et le degré d'insaturation a été testé avec une pénétration plus élevée pour les chaînes plus courtes et les acides gras insaturés. Une analyse détaillée de la composition de certaines huiles végétales a montré une grande variété de TAG et la pénétration a également été démontrée pour ces huiles. Le NanoSIMS et la modélisation ont confirmé que ces TAG pénètrent dans la CMC riche en lipides des cheveux et interagissent avec les acides gras qui composent le CMC. Toutes les huiles végétales ont produit une amélioration de la résistance à la fatigue par pénétration dans le CMC et il est proposé que ces huiles préviennent la formation et/ou la propagation de défauts dans le réseau CMC qui entraînent une rupture. CONCLUSIONS: De nombreuses huiles végétales avec un large éventail de compositions de triglycérides peuvent pénétrer dans les cheveux et les données du NanoSIMS ont confirmé que ces huiles se divisent en complexe de membrane cellulaire riche en lipides. Les études de pénétration des TAG individuels qui se sont avérés présents dans ces huiles ont confirmé que les TAG de longueur de chaîne variable peuvent pénétrer et il existe une corrélation entre l'augmentation de l'efficacité de pénétration et les longueurs de chaîne plus courtes et la présence d'une insaturation dans les chaînes d'acides gras. Toutes les huiles étudiées ont montré des bienfaits en matière de résistance à la fatigue pour une seule fibre.

Site-2 Protease Slr1821 Regulates Carbon/Nitrogen Homeostasis during Ammonium Stress Acclimation in Cyanobacterium Synechocystis sp. PCC 6803.

Excess ammonium imposes toxicity and stress response in cyanobacteria. How cyanobacteria acclimate to NH4+ stress is so far poorly understood. Here, Synechocystis sp. PCC6803 S2P homolog Slr1821 was identified as the essential regulator through physiological characterization and transcriptomic analysis of its knockout mutant. The proper expression of 60% and 67% of the NH4+ activated and repressed genes, respectively, were actually Slr1821-dependent since they were abolished or reversed in ∆slr1821. Synechocystis 6803 suppressed nitrogen uptake and assimilation, ammonium integration and mobilization of other nitrogen sources upon NH4+ stress. Opposite regulation on genes for assimilation of nitrogen and carbon, such as repression of nitrogen regulatory protein PII, PII interactive protein PirC and activation of carbon acquisition regulator RcbR, demonstrated that Synechocystis 6803 coordinated regulation to maintain carbon/nitrogen homeostasis under increasing nitrogen, while functional Slr1821 was indispensable for most of this coordinated regulation. Additionally, slr1821 knockout disrupted the proper response of regulators and transporters in the ammonium-specific stimulon, and resulted in defective photosynthesis as well as compromised translational and transcriptional machinery. These results provide new insight into the coordinated regulation of nutritional fluctuation and the functional characterization of S2Ps. They also provide new targets for bioengineering cyanobacteria in bioremediation and improving ammonium tolerance in crop plants.

Topological Ordering of Memory Glass on Extended Length Scales.

Identifying ordering in non-crystalline solids has been a focus of natural science since the publication of Zachariasen's random network theory in 1932, but it still remains as a great challenge of the century. Literature shows that the hierarchical structures, from the short-range order of first-shell polyhedra to the long-range order of translational periodicity, may survive after amorphization. Here, in a piece of AlPO4, or berlinite, we combine X-ray diffraction and stochastic free-energy surface simulations to study its phase transition and structural ordering under pressure. From reversible single crystals to amorphous transitions, we now present an unambiguous view of the topological ordering in the amorphous phase, consisting of a swarm of Carpenter low-symmetry phases with the same topological linkage, trapped in a metastable intermediate stage. We propose that the remaining topological ordering is the origin of the switchable "memory glass" effect. Such topological ordering may hide in many amorphous materials through disordered short atomic displacements.

Proteasome Dysfunction Leads to Suppression of the Hypoxic Response Pathway in Arabidopsis.

Proteasome is a large proteolytic complex that consists of a 20S core particle (20SP) and 19S regulatory particle (19SP) in eukaryotes. The proteasome degrades most cellular proteins, thereby controlling many key processes, including gene expression and protein quality control. Proteasome dysfunction in plants leads to abnormal development and reduced adaptability to environmental stresses. Previous studies have shown that proteasome dysfunction upregulates the gene expression of proteasome subunits, which is known as the proteasome bounce-back response. However, the proteasome bounce-back response cannot explain the damaging effect of proteasome dysfunction on plant growth and stress adaptation. To address this question, we focused on downregulated genes caused by proteasome dysfunction. We first confirmed that the 20SP subunit PBE is an essential proteasome subunit in Arabidopsis and that PBE1 mutation impaired the function of the proteasome. Transcriptome analyses showed that hypoxia-responsive genes were greatly enriched in the downregulated genes in pbe1 mutants. Furthermore, we found that the pbe1 mutant is hypersensitive to waterlogging stress, a typical hypoxic condition, and hypoxia-related developments are impaired in the pbe1 mutant. Meanwhile, the 19SP subunit rpn1a mutant seedlings are also hypersensitive to waterlogging stress. In summary, our results suggested that proteasome dysfunction downregulated the hypoxia-responsive pathway and impaired plant growth and adaptability to hypoxia stress.

Metabolic engineering of a robust Escherichia coli strain with a dual protection system.

Considerable attention has been given to the development of robust fermentation processes, but microbial contamination and phage infection remain deadly threats that need to be addressed. In this study, a robust Escherichia coli BL21(DE3) strain was successfully constructed by simultaneously introducing a nitrogen and phosphorus (N&P) system in combination with a CRISPR/Cas9 system. The N&P metabolic pathways were able to express formamidase and phosphite dehydrogenase in the host cell, thus enabled cell growth in auxotrophic 3-(N-morpholino)propanesulfonic acid medium with formamide and phosphite as nitrogen and phosphorus sources, respectively. N&P metabolic pathways also allowed efficient expression of heterologous proteins, such as green fluorescent protein (GFP) and chitinase, while contaminating bacteria or yeast species could hardly survive in this medium. The host strain was further engineered by exploiting the CRISPR/Cas9 system to enhance the resistance against phage attack. The resultant strain was able to grow in the presence of T7 phage at a concentration of up to 2 × 107 plaque-forming units/ml and produce GFP with a yield of up to 30 µg/109 colony-forming units, exhibiting significant advantages over conventional engineered E. coli. This newly engineered, robust E. coli BL21(DE3) strain therefore shows great potential for future applications in industrial fermentation.

Identification and Characterization of MiRNAs in Coccomyxa subellipsoidea C-169.

Coccomyxa subellipsoidea C-169 (C-169) is an oleaginous microalga which is promising for renewable biofuel production. MicroRNAs (miRNAs), as the pivotal modulators of gene expression at post-transcriptional level, are prospective candidates for bioengineering practice. However, so far, no miRNA in C-169 has been reported and its potential impact upon CO2 supplementation remains unclear. High-throughput sequencing of small RNAs from C-169 cultured in air or 2% CO2 revealed 124 miRNAs in total, including 118 conserved miRNAs and six novel ones. In total, 384 genes were predicted as their potential target genes, 320 for conserved miRNAs and 64 for novel miRNAs. The annotated target genes were significantly enriched in six KEGG pathways, including pantothenate and CoA biosynthesis, C5-branched dibasic acid metabolism, 2-oxocarboxylic acid metabolism, butanoate metabolism, valine, leucine and isoleucine biosynthesis and alpha-linolenic acid metabolism. The miRNAs' target genes were enriched in lipid metabolism as well as RNA-interacting proteins involved in translation, transcription and rRNA processing. The pioneering identification of C-169 miRNAs and analysis of their putative target genes lay the foundation for further miRNA research in eukaryotic algae and will contribute to the development of C-169 as an oleaginous microalga through bioengineering in the future.

Pathogenic features and characteristics of food borne pathogens biofilm: Biomass, viability and matrix.

Biofilm is a ubiquitous growth pattern of bacterial species survival but is notorious for its threat on public health and food contamination. Extensive studies of the biofilm structure, formation, quantification, quorum sensing system and underlying control strategies have been reported during the past decades. Insightful elucidation of the pathogenic features and characteristic of bacterial biofilm can facilitate in devising appropriate control strategies for biofilm eradication. Therefore, this review mainly summarized the pathogenic features of biofilms from food borne microorganisms, including the biomass (which could be quantified using crystal violet and fluorogenic dye Syto9 assays), viability (which could be determined by tetrazolium salts, fluorescein diacetate, resazurin staining and alamar blue assays) and matrix (which are commonly detected by dimethyl methylene blue and wheat germ agglutinin assays). In addition, three features were further compared with its particular benefits in specific application.

Global Metabolic Regulation of the Snow Alga Chlamydomonas nivalis in Response to Nitrate or Phosphate Deprivation by a Metabolome Profile Analysis.

In the present work, Chlamydomonas nivalis, a model species of snow algae, was used to illustrate the metabolic regulation mechanism of microalgae under nutrient deprivation stress. The seed culture was inoculated into the medium without nitrate or phosphate to reveal the cell responses by a metabolome profile analysis using gas chromatography time-of-flight mass spectrometry (GC/TOF-MS). One hundred and seventy-one of the identified metabolites clustered into five groups by the orthogonal partial least squares discriminant analysis (OPLS-DA) model. Among them, thirty of the metabolites in the nitrate-deprived group and thirty-nine of the metabolites in the phosphate-deprived group were selected and identified as "responding biomarkers" by this metabolomic approach. A significant change in the abundance of biomarkers indicated that the enhanced biosynthesis of carbohydrates and fatty acids coupled with the decreased biosynthesis of amino acids, N-compounds and organic acids in all the stress groups. The up- or down-regulation of these biomarkers in the metabolic network provides new insights into the global metabolic regulation and internal relationships within amino acid and fatty acid synthesis, glycolysis, the tricarboxylic acid cycle (TCA) and the Calvin cycle in the snow alga under nitrate or phosphate deprivation stress.

Identification of Specific Variations in a Non-Motile Strain of Cyanobacterium Synechocystis sp. PCC 6803 Originated from ATCC 27184 by Whole Genome Resequencing.

Cyanobacterium Synechocystis sp. PCC 6803 is a widely used model organism in basic research and biofuel biotechnology application. Here, we report the genomic sequence of chromosome and seven plasmids of a glucose-tolerant, non-motile strain originated from ATCC 27184, GT-G, in use at Guangzhou. Through high-throughput genome re-sequencing and verification by Sanger sequencing, eight novel variants were identified in its chromosome and plasmids. The eight novel variants, especially the five non-silent mutations might have interesting effects on the phenotype of GT-G strains, for example the truncated Sll1895 and Slr0322 protein. These resequencing data provide background information for further research and application based on the GT-G strain and also provide evidence to study the evolution and divergence of Synechocystis 6803 globally.

Nutraceuticals and functional foods in the management of hyperlipidemia.

Hyperlipidemia is one of the major risk factor for the development of cardiovascular disease. Hypolipidemic nutraceuticals and functional foods help improve serum lipid profiles as reducing total cholesterol, triglyceride, and low-density lipoprotein cholesterol, while elevating high-density lipoprotein cholesterol. The effectiveness of omega-3 polyunsaturated fatty acid, phytosterols, dietary fiber, and tea catechin in management of hyperlipidemia has been clearly demonstrated in epidemiological and interventional trials. Studies on mechanism reveal that they act as inhibitor or activator of critical enzyme, agonist or inhibitor of transcription factor, competitor of transporter, and sequestrant of bile acid to modulate lipid homeostasis. Hypolipidemic effects are also claimed in dietary proteins, many polyphenols, other phytochemicals, raw extract, or even whole food. This review attempts to give an overview of lipid homeostasis and summarize recent findings of hypolipidemic nutraceuticals and functional foods according to their active ingredients, focusing on the efficacy and underlying mechanisms.

Hypolipidemic activity of okra is mediated through inhibition of lipogenesis and upregulation of cholesterol degradation.

Little is known about the hypolipidemic activity of okra; therefore, we investigated the hypolipidemic activity of okra and its interaction with gene expression of several key components involved in lipid homeostasis. Male C57BL/6 mice were randomly divided into three groups and fed with hyperlipidemic diet or two hyperlipidemic diets supplemented with 1% or 2% okra powder for eight weeks. Results demonstrated that okra dose-dependently decreased serum and hepatic total cholesterol and triglyceride, and enhanced fecal excretion of bile acids. Gene expression analysis revealed that okra upregulated cholesterol 7α-hydroxylase (CYP7A1) expression, downregulated expression of sterol regulatory element-binding protein 1c (SREBP1c) and fatty acid synthase (FAS), with no effect on sterol regulatory element-binding protein 2 (SREBP2), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), low-density lipoprotein receptor (LDLR) and carnitine palmitoyltransferase-1A (CPT1A). It was suggested that hypolipidemic activity of okra was mediated most likely by upregulation of cholesterol degradation through CYP7A1 and by inhibition of lipogenesis through SREBP1c and FAS. Okra raw and fractionated polysaccharide showed strong bile acid binding capacity in vitro, which may contribute to the hypolipidemic activity observed. In conclusion, okra has potential application in the management of hyperlipidemia and its associated metabolic disorders.

Sll0528, a Site-2-Protease, Is Critically Involved in Cold, Salt and Hyperosmotic Stress Acclimation of Cyanobacterium Synechocystis sp. PCC 6803.

Site-2-proteases (S2Ps) mediated proteolysis of transmembrane transcriptional regulators is a conserved mechanism to regulate transmembrane signaling. The universal presence of S2P homologs in different cyanobacterial genomes suggest conserved and fundamental functions, though limited data has been available. Here we provide the first evidence that Sll0528, a site-2-protease in Synechocystis sp. PCC 6803 is crucial for salt, cold and hyperosmotic stress acclimation. Remarkable induction of sll0528 gene expression was observed under salt, cold and hyperosmotic stress, much higher than induction of the other three S2Ps. Knock-out of sll0528 gene in wild type Synechocystis sp. PCC 6803 increased their sensitivity to salt, cold and hyperosmotic stress, as revealed by retarded growth, reduced pigments and disrupted photosystems. The sll0528 gene was induced to a much smaller extent by high light and mixotrophic growth with glucose. Similar growth responses of the sll0528 knockout mutant and wild type under high light and mixotrophic growth indicated that sll0528 was dispensable for these conditions. Recombinant Sll0528 protein could cleave beta-casein into smaller fragments. These results together suggest that the Sll0528 metalloprotease plays a role in the stress response and lays the foundation for further investigation of its mechanism, as well as providing hints for the functional analysis of other S2Ps in cyanobacteria.

Diffusion-based deep learning method for augmenting ultrastructural imaging and volume electron microscopy.

Electron microscopy (EM) revolutionized the way to visualize cellular ultrastructure. Volume EM (vEM) has further broadened its three-dimensional nanoscale imaging capacity. However, intrinsic trade-offs between imaging speed and quality of EM restrict the attainable imaging area and volume. Isotropic imaging with vEM for large biological volumes remains unachievable. Here, we developed EMDiffuse, a suite of algorithms designed to enhance EM and vEM capabilities, leveraging the cutting-edge image generation diffusion model. EMDiffuse generates realistic predictions with high resolution ultrastructural details and exhibits robust transferability by taking only one pair of images of 3 megapixels to fine-tune in denoising and super-resolution tasks. EMDiffuse also demonstrated proficiency in the isotropic vEM reconstruction task, generating isotropic volume even in the absence of isotropic training data. We demonstrated the robustness of EMDiffuse by generating isotropic volumes from seven public datasets obtained from different vEM techniques and instruments. The generated isotropic volume enables accurate three-dimensional nanoscale ultrastructure analysis. EMDiffuse also features self-assessment functionalities on predictions' reliability. We envision EMDiffuse to pave the way for investigations of the intricate subcellular nanoscale ultrastructure within large volumes of biological systems.

Comparative effectiveness of immunotherapy and chemotherapy in patients with metastatic colorectal cancer stratified by microsatellite instability status.

BACKGROUND: Immunotherapy have demonstrated promising outcomes in patients with high microsatellite instability (MSI) (MSI-H) metastatic colorectal cancer. However, the comparative effectiveness of Immunotherapy and chemotherapy for patients with low MSI (MSI-L), and microsatellite stable (MSS) metastatic colorectal cancer remains unclear. AIM: To investigate immunotherapy vs chemotherapy for treatment of MSI-L/MSS metastatic colorectal cancer, and to evaluate the success of immunotherapy against chemotherapy in managing MSI-H metastatic colorectal cancer during a follow-up of 50 months. METHODS: We conducted a retrospective cohort study using the National Cancer Database (NCDB) to evaluate the overall survival (OS) of patients with metastatic colorectal cancer treated with immunotherapy or chemotherapy. The study population was stratified by MSI status (MSI-H, MSI-L, and MSS). Multivariable Cox proportional hazard models were used to assess the association between treatment modality and OS, adjusting for potential confounders. RESULTS: A total of 21951 patients with metastatic colorectal cancer were included in the analysis, of which 2358 were MSI-H, and 19593 were MSI-L/MSS. In the MSI-H cohort, immunotherapy treatment (n = 142) was associated with a significantly improved median OS compared to chemotherapy (n = 860). After adjusting for potential confounders, immunotherapy treatment remained significantly associated with better OS in the MSI-H cohort [adjusted hazard ratio (aHR): 0.57, 95% confidence interval (95%CI): 0.43-0.77, P < 0.001]. In the MSS cohort, no significant difference in median OS was observed between immunotherapy treatment and chemotherapy (aHR: 0.94, 95%CI: 0.69-1.29, P = 0.715). CONCLUSION: In this population-based study using the NCDB, immunotherapy treatment was associated with significantly improved OS compared to chemotherapy in patients with MSI-H metastatic colorectal cancer, but not in those with MSI-L/MSS metastatic colorectal cancer. Further studies are warranted to determine the optimal therapeutic approach for patients with MSI-L/MSS metastatic colorectal cancer.

Effects of Cold Plasma Treatment Conditions on the Lipid Oxidation Kinetics of Tilapia Fillets.

This study investigated the effects of different cold plasma treatment conditions on the lipid oxidation kinetics of tilapia fillets. The results indicated that increasing the voltage and prolonging the treatment time of cold plasma could cause an increase in the peroxide value and thiobarbituric acid-reactive substance values of the fillets. The changes in the primary and secondary oxidation rates of the lipids in the fillets under different treatment conditions were consistent with zero-order reaction kinetics. The analysis of the fitting of the Arrhenius equation showed that the effect of treatment voltage on the activation energy of lipid oxidation was higher than that of treatment time. When the voltage was higher than 64.71 kV, the activation energy of the primary oxidation of lipids was greater than that of secondary oxidation. Within 0-5 min, the activation energy of primary oxidation first increased then decreased, and was always greater than that of secondary oxidation. Therefore, the primary lipid oxidation of tilapia was more sensitive to the treatment conditions of cold plasma.

Comparison of early and late intervention for necrotizing pancreatitis: A systematic review and meta-analysis.

OBJECTIVES: Postponed open necrosectomy or minimally invasive intervention has become the treatment option for necrotizing pancreatitis. Nevertheless, several studies point to the safety and efficacy of early intervention for necrotizing pancreatitis. Therefore, we conducted a systematic review and meta-analysis to compare clinical outcomes of acute necrotizing pancreatitis between early and late intervention. METHODS: Literature search was performed in multiple databases for articles that compared the safety and clinical outcomes of early (<4 weeks from the onset of pancreatitis) versus late intervention (≥4 weeks from the onset of pancreatitis) for necrotizing pancreatitis published up to August 31, 2022. The meta-analysis was performed to determine pooled odds ratio (OR) of mortality rate and procedure-related complications. RESULTS: Fourteen studies were included in the final analysis. For open necrosectomy intervention, the overall pooled OR of mortality rate with the late intervention compared with early intervention was 7.09 (95% confidence interval [CI] 2.33-21.60; I2 = 54%; P = 0.0006). For minimally invasive intervention, the overall pooled OR of mortality rate with the late intervention compared with early intervention was 1.56 (95% CI 1.11-2.20; I2 = 0%; P = 0.01). The overall pooled OR of pancreatic fistula with the late minimally invasive intervention compared with early intervention was 2.49 (95% CI 1.75-3.52; I2 = 0%; P < 0.00001). CONCLUSION: These results showed the benefit of late interventions in patients with necrotizing pancreatitis in both minimally invasive procedures and open necrosectomy. Late intervention is preferred in the management of necrotizing pancreatitis.

Slr0643, an S2P homologue, is essential for acid acclimation in the cyanobacterium Synechocystis sp. PCC 6803.

Every cyanobacterial species contains genes encoding site-2-protease (S2P) homologues. The studied prokaryotic S2P homologues play essential roles in regulating stress responses through intramembrane proteolysis of membrane-bound anti-sigma factors. Here, the gene encoding Slr0643, one of four S2P homologues in Synechocystis sp. PCC 6803, was insertionally disrupted to explore its physiological role. Only a partially segregated mutant was obtained, indicating the essentiality of the gene product for growth. A pivotal role of fully functional Slr0643 in acid acclimation was demonstrated by defective acid acclimation to pH 6.5 in the mutant and transient induction of slr0643 in the wild-type after transfer from pH 7.5 to 6.5. DNA microarray and quantitative RT-PCR analyses of mutant and wild-type strains at pH 7.5 versus pH 6.5 identified genes involved in early acid acclimation and revealed genes expressed differentially due to slr0643 disruption. Early acid acclimation to pH 6.5 in the wild-type strain included upregulation of sigH, hik16 and hik35 and downregulation of pcrR and sigG, as well as downregulation of porins and upregulation of inorganic carbon and nitrogen transporters. The inability of the mutant strain to survive at pH 6.5 was found to be related to defective photosynthesis and excess expression of NADH dehydrogenase, together with excessive upregulation of carbon transporter and repression of nitrogen transporter and metabolism genes. Most interestingly, analysis of microarray data revealed the close relationship between slr0643 disruption and expression of the sigH operon. Thus it is suggested that Slr0643/Sll0857/SigH might act through an S2P/anti-Sigma factor/Sigma factor mechanism to play a role in acid acclimation.

EGY2, a chloroplast membrane metalloprotease, plays a role in hypocotyl elongation in Arabidopsis.

Intramembrane proteases control many important processes in a wide variety of organisms through regulated intramembrane proteolysis (RIP). However, very few intramembrane proteases have been characterized in plants. Intriguingly, EGY2 in Arabidopsis belongs to the Site-2 protease (S2P) family that performs RIP. It contains the conserved catalytic motifs, HExxH and NPDG on its multiple transmembrane helices. Four egy2 knockout mutants have significantly shorter hypocotyls and accumulate lower levels of fatty acids in seedlings. Accumulation of fatty acid biosynthesis enzymes in seedlings are also decreased in egy2 knockout mutants. EGY2 protein resides in the chloroplast and EGY2 transcripts are found throughout the plant except root. Recombinant EGY2 protein cleaves ß-casein in an ATP-independent manner. These results together suggest that EGY2 metalloprotease plays a role in hypocotyl elongation likely through a RIP dependent process to regulate the coordinated expression of nuclear- and plastid-encoded genes.

Construction of Escherichia coli strains producing L-serine from glucose.

L-Serine is usually produced from glycine. We have genetically engineered Escherichia coli to produce L-serine from glucose intracellularly. D-3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) in E. coli catalyzes the first committed step in L-serine formation but is inhibited by L-serine. To overcome this feedback inhibition, both the His(344) and Asn(346) residues of PGDH were converted to alanine and the mutated PGDH (PGDH(dr)) became insensitive to L-serine. However, overexpression of PGDH(dr) gave no significant increase of L-serine accumulation but, when L-serine deaminase genes (sdaA, sdaB and tdcG) were deleted, serine accumulated: (1) deletion of sdaA gave up to 0.03 mmol L-serine/g; (2) deletion of both sdaA and sdaB accumulated L-serine up to 0.09 mmol/g; and (3) deletion of sdaA, sdaB and tdcG gave up to 0.13 mmol L-serine/g cell dry wt.