Structure and mechanism of a eukaryotic ceramide synthase complex.

Ceramide synthases (CerS) catalyze ceramide formation via N-acylation of a sphingoid base with a fatty acyl-CoA and are attractive drug targets for treating numerous metabolic diseases and cancers. Here, we present the cryo-EM structure of a yeast CerS complex, consisting of a catalytic Lac1 subunit and a regulatory Lip1 subunit, in complex with C26-CoA substrate. The CerS holoenzyme exists as a dimer of Lac1-Lip1 heterodimers. Lac1 contains a hydrophilic reaction chamber and a hydrophobic tunnel for binding the CoA moiety and C26-acyl chain of C26-CoA, respectively. Lip1 interacts with both the transmembrane region and the last luminal loop of Lac1 to maintain the proper acyl chain binding tunnel. A lateral opening on Lac1 serves as a potential entrance for the sphingoid base substrate. Our findings provide a template for understanding the working mechanism of eukaryotic ceramide synthases and may facilitate the development of therapeutic CerS modulators.

Proteolytic stability and aggregation in a key metabolic enzyme of bacteria.

Proteins that are kinetically stable are thought to be less prone to both aggregation and proteolysis. We demonstrate that the classical lac system of Escherichia coli can be leveraged as a model system to study this relation. ß-galactosidase (LacZ) plays a critical role in lactose metabolism and is an extremely stable protein that can persist in growing cells for multiple generations after expression has stopped. By attaching degradation tags to the LacZ protein, we find that LacZ can be transiently degraded during lac operon expression but once expression has stopped functional LacZ is protected from degradation. We reversibly destabilize its tetrameric assembly using α-complementation, and show that unassembled LacZ monomers and dimers can either be degraded or lead to formation of aggregates within cells, while the tetrameric state protects against proteolysis and aggregation. We show that the presence of aggregates is associated with cell death, and that these proteotoxic stress phenotypes can be alleviated by attaching an ssrA tag to LacZ monomers which leads to their degradation. We unify our findings using a biophysical model that enables the interplay of protein assembly, degradation, and aggregation to be studied quantitatively in vivo. This work may yield approaches to reversing and preventing protein-misfolding disease states, while elucidating the functions of proteolytic stability in constant and fluctuating environments.

Circulating N-lactoyl-amino acids and N-formyl-methionine reflect mitochondrial dysfunction and predict mortality in septic shock.

INTRODUCTION: Sepsis is a highly morbid condition characterized by multi-organ dysfunction resulting from dysregulated inflammation in response to acute infection. Mitochondrial dysfunction may contribute to sepsis pathogenesis, but quantifying mitochondrial dysfunction remains challenging. OBJECTIVE: To assess the extent to which circulating markers of mitochondrial dysfunction are increased in septic shock, and their relationship to severity and mortality. METHODS: We performed both full-scan and targeted (known markers of genetic mitochondrial disease) metabolomics on plasma to determine markers of mitochondrial dysfunction which distinguish subjects with septic shock (n = 42) from cardiogenic shock without infection (n = 19), bacteremia without sepsis (n = 18), and ambulatory controls (n = 19) - the latter three being conditions in which mitochondrial function, proxied by peripheral oxygen consumption, is presumed intact. RESULTS: Nine metabolites were significantly increased in septic shock compared to all three comparator groups. This list includes N-formyl-L-methionine (f-Met), a marker of dysregulated mitochondrial protein translation, and N-lactoyl-phenylalanine (lac-Phe), representative of the N-lactoyl-amino acids (lac-AAs), which are elevated in plasma of patients with monogenic mitochondrial disease. Compared to lactate, the clinical biomarker used to define septic shock, there was greater separation between survivors and non-survivors of septic shock for both f-Met and the lac-AAs measured within 24 h of ICU admission. Additionally, tryptophan was the one metabolite significantly decreased in septic shock compared to all other groups, while its breakdown product kynurenate was one of the 9 significantly increased. CONCLUSION: Future studies which validate the measurement of lac-AAs and f-Met in conjunction with lactate could define a sepsis subtype characterized by mitochondrial dysfunction.

Development of inducible promoters for regulating gene expression in Clostridium tyrobutyricum for biobutanol production.

Clostridium tyrobutyricum is an anaerobe known for its ability to produce short-chain fatty acids, alcohols, and esters. We aimed to develop inducible promoters for fine-tuning gene expression in C. tyrobutyricum. Synthetic inducible promoters were created by employing an Escherichia coli lac operator to regulate the thiolase promoter (PCathl) from Clostridium acetobutylicum, with the best one (LacI-Pto4s) showing a 5.86-fold dynamic range with isopropyl ß- d-thiogalactoside (IPTG) induction. A LT-Pt7 system with a dynamic range of 11.6-fold was then created by combining LacI-Pto4s with a T7 expression system composing of RNA polymerase (T7RNAP) and Pt7lac promoter. Furthermore, two inducible expression systems BgaR-PbgaLA and BgaR-PbgaLB with a dynamic range of ~40-fold were developed by optimizing a lactose-inducible expression system from Clostridium perfringens with modified 5' untranslated region (5' UTR) and ribosome-binding site (RBS). BgaR-PbgaLB was then used to regulate the expressions of a bifunctional aldehyde/alcohol dehydrogenase encoded by adhE2 and butyryl-CoA/acetate Co-A transferase encoded by cat1 in C. tyrobutyricum wild type and Δcat1::adhE2, respectively, demonstrating its efficient inducible gene regulation. The regulated cat1 expression also confirmed that the Cat1-catalyzed reaction was responsible for acetate assimilation in C. tyrobutyricum. The inducible promoters offer new tools for tuning gene expression in C. tyrobutyricum for industrial applications.

CUREs for high-level Galectin-3 expression.

Galectins are a large and diverse protein family defined by the presence of a carbohydrate recognition domain (CRD) that binds ß-galactosides. They play important roles in early development, tissue regeneration, immune homeostasis, pathogen recognition, and cancer. In many cases, studies that examine galectin biology and the effect of manipulating galectins are aided by, or require the ability to express and purify, specific members of the galectin family. In many cases, E. coli is employed as a heterologous expression system, and galectin expression is induced with isopropyl ß-galactoside (IPTG). Here, we show that galectin-3 recognizes IPTG with micromolar affinity and that as IPTG induces expression, newly synthesized galectin can bind and sequester cytosolic IPTG, potentially repressing further expression. To circumvent this putative inhibitory feedback loop, we utilized an autoinduction protocol that lacks IPTG, leading to significantly increased yields of galectin-3. Much of this work was done within the context of a course-based undergraduate research experience, indicating the ease and reproducibility of the resulting expression and purification protocols.

Efficient heterologous expression of cellobiose 2-epimerase gene in Escherichia coli under the control of T7 lac promoter without addition of IPTG and lactose.

In this study, the cellobiose 2-epimerase gene csce from Caldicellulosiruptor saccharolyticus was expressed in Escherichia coli using TB medium containing yeast extract Oxoid and tryptone Oxoid. Interesting, it was found that when the concentration of isopropyl-beta-d-thiogalactopyranoside (IPTG) and lactose was 0 (no addition), the activity of cellobiose 2-epimerase reached 5.88 U/mL. It was 3.70-fold higher than the activity observed when 1.0 mM IPTG was added. When using M9 medium without yeast extract Oxoid and tryptone Oxoid, cellobiose 2-epimerase gene could not be expressed without IPTG and lactose. However, cellobiose 2-epimerase gene could be expressed when yeast extract Oxoid or tryptone Oxoid was added, indicating that these supplements contained inducers for gene expression. In the absence of IPTG and lactose, the addition of soy peptone Angel-1 or yeast extract Angel-1 to M9 medium significantly upregulated the expression of cellobiose 2-epimerase gene in E. coli BL21 pET28a-csce, and these inductions led to higher expression levels compared to tryptone Oxoid or yeast extract Oxoid. The relative transcription level of csce was consistent with its expression level in E. coli BL21 pET28a-csce. In the medium TB without IPTG and lactose and containing yeast extract Angel-1 and soy peptone Angel-1, the activity of cellobiose 2-epimerase reached 6.88 U/mL, representing a 2.2-fold increase compared to previously reported maximum activity in E. coli. The significance of this study lies in its implications for efficient heterologous expression of recombinant enzyme proteins in E. coli without the need for IPTG and lactose addition.

How the xerophytic moss Pogonatum inflexum tolerates desiccation.

KEY MESSAGE: Desiccation-tolerant process of xerophytic moss Pogonatum inflexum were identified through de novo transcriptome assembly , morphological structure and physiology analysis. Pogonatum inflexum (Lindb.) Lac. is a typical xerophytic moss and have been widely used in gardening and micro-landscape. However, the mechanisms underlying desiccation tolerance are still unclear. In this study, morphological,  physiological and trancriptomic analyses of P. inflexum to tolerate desiccation were carried out. Our results indicate that P. inflexum increase osmoregulation substances, shut down photosynthesis, and alter the content of membrane lipid fatty acids in response to desiccation, and the genes involved in these biological processes were changes in expression after desiccation. 12 h is the threshold for P. inflexum to tolerate desiccation and its photosynthesis has not been damaged within 12 h of desiccation and can still recover after rewater. We also proved that the gametocyte of P. inflexum has the ability to absorb and transport water, and contains lignin-synthesis genes in response to tolerant desiccation. Our findings not only explain the mechanisms of P. inflexum during desiccation, but also provide some attractive candidate genes for genetic breeding.

Lactate dynamics in paediatric patients with severe sepsis: insights from a prospective cohort study.

BACKGROUND: Sepsis is an infection-related systemic inflammatory response that often leads to elevated lactate levels. Monitoring lactate levels during severe sepsis is vital for influencing clinical outcomes. The aim of this study was to assess the association between plasma lactate levels and mortality in children with severe sepsis or septic shock. METHODS: The current prospective study was conducted in the PICU of University Children's Hospital. The International Paediatric Sepsis Consensus Conference criteria for Definitions of Sepsis and Organ Failure in 2005 were used to diagnose patients with sepsis. We measured plasma lactate levels upon admission (Lac H0) and 6 h later (Lac H6). The static indices included the absolute lactate values (Lac H0 and Lac H6), while the dynamic indices included the delta-lactate level (ΔLac) and the 6-hour lactate clearance. The 6-hour lactate clearance was calculated using the following formula: [(Lac H0-Lac H6)100/Lac H0]. ΔLac was calculated as the difference between the Lac H0 and Lac H6 levels. Patient survival or death after a PICU stay was the primary outcome. RESULTS: A total of 46 patients were included in this study: 25 had septic shock, and 21 had severe sepsis. The mortality rate was 54.3%. The Lac H0 did not significantly differ between survivors and nonsurvivors. In contrast, the survivors had significantly lower Lac H6 levels, higher ΔLac levels, and higher 6-hour lactate clearance rates than nonsurvivors. Lactate clearance rates below 10%, 20%, and 30% were significantly associated with mortality. The best cut-off values for the lactate clearance rate and Lac H6 for the prediction of mortality in the PICU were < 10% and ≥ 4 mmol/L, respectively. Patients with higher Lac H6 levels and lower lactate clearance rates had significantly higher PICU mortality based on Kaplan-Meier survival curve analysis. CONCLUSIONS: This study highlights the significance of lactate level trends over time for the prediction of mortality in the PICU in patients with severe sepsis or septic shock. Elevated lactate levels and decreased lactate clearance six hours after hospitalisation are associated with a higher mortality rate.

Deep representation learning improves prediction of LacI-mediated transcriptional repression.

Recent progress in DNA synthesis and sequencing technology has enabled systematic studies of protein function at a massive scale. We explore a deep mutational scanning study that measured the transcriptional repression function of 43,669 variants of the Escherichia coli LacI protein. We analyze structural and evolutionary aspects that relate to how the function of this protein is maintained, including an in-depth look at the C-terminal domain. We develop a deep neural network to predict transcriptional repression mediated by the lac repressor of Escherichia coli using experimental measurements of variant function. When measured across 10 separate training and validation splits using 5,009 single mutations of the lac repressor, our best-performing model achieved a median Pearson correlation of 0.79, exceeding any previous model. We demonstrate that deep representation learning approaches, first trained in an unsupervised manner across millions of diverse proteins, can be fine-tuned in a supervised fashion using lac repressor experimental datasets to more effectively predict a variant's effect on repression. These findings suggest a deep representation learning model may improve the prediction of other important properties of proteins.

Efficient degradation and detoxification of structurally different dyes and mixed dyes by LAC-4 laccase purified from white-rot fungi Ganoderma lucidum.

The purpose of this study is to evaluate the decolorization ability and detoxification effect of LAC-4 laccase on various types of single and mixed dyes, and lay a good foundation for better application of laccase in the efficient treatment of dye pollutants. The reaction system of the LAC-4 decolorizing single dyes (azo, anthraquinone, triphenylmethane, and indigo dyes, 17 dyes in total) were established. To explore the decolorization effect of the dye mixture by LAC-4, two dyes of the same type or different types were mixed at the same concentration (100 mg/L) in the reaction system containing 0.5 U laccase, and time-course decolorization were performed on the dye mixture. The combined dye mixtures consisted of azo + azo, azo + anthraquinone, azo + indigo, azo + triphenylmethane, indigo + triphenylmethane, and triphenylmethane + triphenylmethane. The results obtained in this study were as follows. Under optimal conditions of 30 °C and pH 5.0, LAC-4 (0.5 U) can efficiently decolorize four different types of dyes. The 24-hour decolorization efficiencies of LAC-4 for 800 mg/L Orange G and Acid Orange 7 (azo), Remazol Brilliant Blue R (anthraquinone), Bromophenol Blue and Methyl Green (triphenylmethane), and Indigo Carmine (indigo) were 75.94%, 93.30%, 96.56%, 99.94%, 96.37%, and 37.23%, respectively. LAC-4 could also efficiently decolorize mixed dyes with different structures. LAC-4 can achieve a decolorization efficiency of over 80% for various dye mixtures such as Orange G + Indigo Carmine (100 mg/L+100 mg/L), Reactive Orange 16 + Methyl Green (100 mg/L+100 mg/L), and Remazol Brilliant Blue R + Methyl Green (100 mg/L+100 mg/L). During the decolorization process of the mixed dyes by laccase, four different interaction relationships were observed between the dyes. Decolorization efficiencies and rates of the dyes that were difficult to be degraded by laccase could be greatly improved when mixed with other dyes. Degradable dyes could greatly enhance the ability of LAC-4 to decolorize extremely difficult-to-degrade dyes. It was also found that the decolorization efficiencies of the two dyes significantly increased after mixing. The possible mechanisms underlying the different interaction relationships were further discussed. Free, but not immobilized, LAC-4 showed a strong continuous batch decolorization ability for single dyes, two-dye mixtures, and four-dye mixtures with different structures. LAC-4 exhibited high stability, sustainable degradability, and good reusability in the continuous batch decolorization. The LAC-4-catalyzed decolorization markedly reduced or fully abolished the toxic effects of single dyes (azo, anthraquinone, and indigo dye) and mix dyes (nine dye mixtures containing four structural types of dyes) on plants. Our findings indicated that LAC-4 laccase had significant potential for use in bioremediation due to its efficient degradation and detoxification of single and mixed dyes with different structural types.

Environmental Dyeing and Functionalization of Silk Fabrics with Natural Dye Extracted from Lac.

Most traditional synthetic dyes and functional reagents used in silk fabrics are not biodegradable and lack green environmental protection. Natural dyes have attracted more and more attention because of their coloring, functionalization effects, and environmental benefits. In this study, natural dyes were extracted from lac and used for coloring and functionalization in silk fabrics without any other harmful dyes. The extraction conditions were studied and analyzed by the univariate method. The optimal extraction process was that the volume ratio of ethanol to water was 60:40 with a solid-liquid ratio of 1:10, and reacting under the neutrality condition for 1 h at 70 °C. Silk fabric can be dyed dark owing to the certain lifting property of lac. After being dyed by Al3+ post-medium, the levels of the washing fastness, light fastness, and friction fastness of silk fabric are all above four with excellent fastness. The results show that the dyed silk fabrics have good UV protection, antioxidation, and antibacterial properties. The UV protection coefficient UPF is 42.68, the antioxidant property is 98.57%, and the antibacterial property can reach more than 80%. Therefore, the dyeing and functionalization of silk fabrics by utilizing naturally lac dyes show broad prospects in terms of the application of green sustainable dyeing and functionalization.

A practical guide to mechanistic systems modeling in biology using a logic-based approach.

Mechanistic computational models enable the study of regulatory mechanisms implicated in various biological processes. These models provide a means to analyze the dynamics of the systems they describe, and to study and interrogate their properties, and provide insights about the emerging behavior of the system in the presence of single or combined perturbations. Aimed at those who are new to computational modeling, we present here a practical hands-on protocol breaking down the process of mechanistic modeling of biological systems in a succession of precise steps. The protocol provides a framework that includes defining the model scope, choosing validation criteria, selecting the appropriate modeling approach, constructing a model and simulating the model. To ensure broad accessibility of the protocol, we use a logical modeling framework, which presents a lower mathematical barrier of entry, and two easy-to-use and popular modeling software tools: Cell Collective and GINsim. The complete modeling workflow is applied to a well-studied and familiar biological process-the lac operon regulatory system. The protocol can be completed by users with little to no prior computational modeling experience approximately within 3 h.

Portrait of autosomal recessive diseases in the French-Canadian founder population of Saguenay-Lac-Saint-Jean.

The population of the Saguenay-Lac-Saint-Jean (SLSJ) region, located in the province of Quebec, Canada, is recognized as a founder population, where some rare autosomal recessive diseases show a high prevalence. Through the clinical and molecular study of 82 affected individuals from 60 families, this study outlines 12 diseases identified as recurrent in SLSJ. Their carrier frequency was estimated with the contribution of 1059 healthy individuals, increasing the number of autosomal recessive diseases with known carrier frequency in this region from 14 to 25. We review the main clinical and molecular features previously reported for these disorders. Five of the studied diseases have a potential lethal effect and three are associated with intellectual deficiency. Therefore, we believe that the provincial program for carrier screening should be extended to include these eight disorders. The high-carrier frequency, together with the absence of consanguinity in most of these unrelated families, suggest a founder effect and genetic drift for the 12 recurrent variants. We recommend further studies to validate this hypothesis, as well as to extend the present study to other regions in the province of Quebec, since some of these disorders could also be present in other French-Canadian families.

Preferences of pregnant individuals to undergo labor after one cesarean in southern China.

BACKGROUND: Labor after cesarean (LAC) remains an optional delivery method among healthy pregnant individuals. Exploring women's attitudes, preferences, reasons for previous cesarean delivery, and the incentives underlying pregnant individuals' preferences could help us understand their choice of delivery mode. In this study we evaluated the preferences and attitudes of eligible pregnant women regarding participation in a LAC in Foshan, China. METHODS: A cross-sectional survey was conducted among 438 pregnant individuals with one prior cesarean delivery (CD) who attended their antenatal examination at a tertiary hospital in southern China, between November 1, 2018, and October 31, 2019. Information on demographic characteristics, obstetric data, preferences for LAC, and incentives for LAC were analyzed. RESULTS: Overall, 85.4% (374/438) of women preferred LAC if they did not have contraindications before delivery, whereas 12.3% (54/438) refused and 2.3% (10/438) were unsure. Participants reported that the most important factors affecting their willingness to undergo LAC were safety indicators (i.e., "ability of hospitals to perform emergency cesarean delivery" [score of 9.28 ± 1.86]), followed by accessibility indicators (i.e., "priority bed arrangements" [score of 9.17 ± 1.84]). Logistic regression analysis indicated that neonatal wellbeing with the prior CD was an independent influencing factor (OR = 2.235 [95%CI: 1.115-4.845], p = 0.024) affecting willingness to access LAC in the subsequent pregnancy. CONCLUSIONS: We found a high preference for LAC among pregnant individuals without contraindications before delivery in southern China. Healthcare providers need to ensure access to LAC and increase pregnant individuals' LAC willingness through high-quality shared decsision-making in alignment with patient preferences.

Simplified and effective RNA interference and CRISPR-Cas9 systems for Cryptococcus neoformans.

The 3,4-dihydroxyphenylalanine (DOPA) melanin is one of the important virulence factors for Cryptococcus neoformans, which may trigger immune responses in the host. While the production of DOPA melanin is catalyzed by laccase that is predominantly encoded by LAC1 gene. Therefore, regulating the genetic expression of C. neoformans is conducive to exploring the impact of interested molecules on the host. In this work, we established two systems that were constructed quickly and easily for the knock-down/knock-out of LAC1 gene: RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats CRISPR-Cas9. The RNAi system was constructed by pSilencer 4.1-CMV neo plasmid and short hairpin RNA to achieve effective transcriptional suppression. The CRISPR-Cas9 system was used the PNK003 vectors to obtain a stable albino mutant strain. The results of phenotype, quantitative real-time polymerase chain reaction, transmission electron microscope, and spectrophotometry were used to assess the ability of melanin production. As a result, the RNAi system displayed attenuation of transcriptional suppression when the transformants continuously passed on new plates. However, the transcriptional suppression of long loop in short hairpin RNA was more powerful and lasted longer. An albino strain produced by CRISPR-Cas9 was completely unable to synthesize melanin. In conclusion, strains with different capacities of melanin production were obtained by RNAi and CRISPR-Cas9 systems, which might be useful for exploring the linear relation between melanin and immunoreaction of the host. In addition, the two systems in this article might be convenient to quickly screen the possible trait-regulating genes of other serotypes of C. neoformans.

Contribution of the LAC Genes in Fruit Quality Attributes of the Fruit-Bearing Plants: A Comprehensive Review.

Laccase genes produce laccase enzymes that play a crucial role in the production of lignin and oxidation reactions within plants. Lignin is a complex polymer that provides structure and toughness to the cell walls of numerous fruit plants. The LAC genes that encode laccase enzymes play vital roles in plant physiology, including the synthesis of pigments like PA that contribute to the colors of fruits, and in defending against pathogens and environmental stresses. They are crucial for fruit development, ripening, structural maintenance in plants, and adaptation to various environmental factors. As such, these genes and enzymes are essential for plant growth and development, as well as for various biotechnological applications in environmental remediation and industrial processes. This review article emphasizes the significance of genes encoding laccase enzymes during fruit growth, specifically pertaining to the strengthening of the endocarp through lignification. This process is crucial for ensuring fruit defense and optimizing seed scattering. The information gathered in this article will aid breeders in producing future fruit-bearing plants that are resistant to disease, cost-effective, and nutrient-rich.

Historical Contingency Causes Divergence in Adaptive Expression of the lac Operon.

Populations of Escherichia coli selected in constant and fluctuating environments containing lactose often adapt by substituting mutations in the lacI repressor that cause constitutive expression of the lac operon. These mutations occur at a high rate and provide a significant benefit. Despite this, eight of 24 populations evolved for 8,000 generations in environments containing lactose contained no detectable repressor mutations. We report here on the basis of this observation. We find that, given relevant mutation rates, repressor mutations are expected to have fixed in all evolved populations if they had maintained the same fitness effect they confer when introduced to the ancestor. In fact, reconstruction experiments demonstrate that repressor mutations have become neutral or deleterious in those populations in which they were not detectable. Populations not fixing repressor mutations nevertheless reached the same fitness as those that did fix them, indicating that they followed an alternative evolutionary path that made redundant the potential benefit of the repressor mutation, but involved unique mutations of equivalent benefit. We identify a mutation occurring in the promoter region of the uspB gene as a candidate for influencing the selective choice between these paths. Our results detail an example of historical contingency leading to divergent evolutionary outcomes.

Characterization and optimization of the LAC4 upstream region for low-leakage expression in Kluyveromyces marxianus.

Kluyveromyces marxianus is a promising host for the production of heterologous proteins, chemicals, and bioethanol. One superior feature of this species is its capacity to assimilate lactose, which is rendered by the LAC12-LAC4 gene pair encoding a lactose permease and a ß-galactosidase enzyme. Little is known about the regulation of LAC4 in K. marxianus. In this study, we showed the presence of weak glucose repression in the regulation of LAC4 and that might contribute to the leaky expression of LAC4 in the glucose medium. In a mutagenesis screen of 1000-bp LAC4 upstream region, one mutant region, named H1, drove low-leakage expression of a URA3 reporter gene in glucose medium. Two mutations inside a polyadenosine stretch (poly(A)) of 5' UTR were major contributors to the low-leakage phenotype of H1. H1 directed low-leakage expression of GFP on a plasmid and that of LAC4 in situ in the glucose medium, which was not due to the reduction of mRNA levels. Meanwhile, H1 did not affect the induction of GFP or LAC4 by lactose. Cre recombinase expressed by H1 caused lower toxicity in the repressive condition and achieved higher yield after induction, compared with that expressed by a wild-type LAC4 upstream region or a strong INU1 promoter. Our study suggested that poly(A) inside 5' UTR played a role in regulating the expression of LAC4 in the repressive condition. Meanwhile, H1 provided a base for the development of a strict inducible system for expressing industrial proteins, especially toxic proteins.

T7-lac promoter vectors spontaneous derepression caused by plant-derived growth media may lead to serious expression problems: a systematic evaluation.

BACKGROUND: The widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research that has practical applications in biotechnology industry, including the production of pharmaceutical drugs. Various factors can strongly affect the successful construction and stable maintenance of clones and the resulting biosynthesis levels. These include an appropriate selection of recombinant hosts, expression systems, regulation of promoters, the repression level at an uninduced state, growth temperature, codon usage, codon context, mRNA secondary structure, translation kinetics, the presence/absence of chaperons and others. However, optimization of the growth medium's composition is often overlooked. We systematically evaluate this factor, which can have a dramatic effect on the expression of recombinant proteins, especially those which are toxic to a recombinant host. RESULTS: Commonly used animal tissue- and plant-based media were evaluated using a series of clones in pET vector, containing expressed Open Reading Frames (ORFs) with a wide spectrum of toxicity to the recombinant E. coli: (i) gfpuv (nontoxic); (ii) tp84_28-which codes for thermophilic endolysin (moderately toxic); and (iii) tthHB27IRM-which codes for thermophilic restriction endonuclease-methyltransferase (REase-MTase)-RM.TthHB27I (very toxic). The use of plant-derived peptones (soy peptone and malt extract) in a culture medium causes the T7-lac expression system to leak. We show that the presence of raffinose and stachyose (galactoside derivatives) in those peptones causes premature and uncontrolled induction of gene expression, which affects the course of the culture, the stability of clones and biosynthesis levels. CONCLUSIONS: The use of plant-derived peptones in a culture medium when using T7-lac hybrid promoter expression systems, such as Tabor-Studier, can lead to uncontrolled production of a recombinant protein. These conclusions also extend to other, lac operator-controlled promoters. In the case of proteins which are toxic to a recombinant host, this can result in mutations or deletions in the expression vector and/or cloned gene, the death of the host or highly decreased expression levels. This phenomenon is caused by the content of certain saccharides in plant peptones, some of which (galactosides) may act as T7-lac promoter inducer by interacting with a Lac repressor. Thus, when attempting to overexpress toxic proteins, it is recommended to either not use plant-derived media or to use them with caution and perform a pilot-scale evaluation of the derepression effect on a case-by-case basis.

Sodalis glossinidius and Wolbachia infections in wild population of Glossina morsitans submorsitans caught in the area of Lake Iro in the south of Chad.

Investigations on the bacterial fauna and their association with trypanosome infections in tsetse fly have revealed contrasting results. This study aimed to detect Wolbachia and S. glossinidius in wild populations of G. m. submorsistans and subsequently, understand the influence that these bacteria may have on the vectorial competence of this tsetse species. Tsetse flies were captured in the area of Lake Iro in the south of Chad using biconical traps. After DNA extraction from each tsetse fly, Sodalis glossinidius and Wolbachia were detected using specific primers. Sodalis glossinidius and Wolbachia infection rates were compared and association studies involving trypanosome infections and S. glossinidius or Wolbachia were performed. From 345 G. m. submorsitans analyzed, 9.0% and 14.5% were respectively infected with S. glossinidius and Wolbachia. Only 2.31% of all tsetse flies were co-infected by the 2 bacteria. Of all trypanosome-infected flies, 7.1% and 9.8% harbored, respectively, S. glossinidius and Wolbachia. No association was observed between Wolbachia and trypanosome infections while a significant association (r = 4.992; P = 0.025) was found between S. glossinidius and the presence of trypanosomes. A significant association (r = 3.147; P = 0.043) was also observed between S. glossinidius and T. simiae; and none with T. congolense or T. godfreyi. This study revealed S. glossinidius and Wolbachia in G. m. submorsitans of the area of lake Iro. It showed that co-infections between Wolbachia and S. glossinidius are rare in wild populations of G. m. submorsitans and that the tripartite associations vary according to trypanosome species as well as symbiotic mricroorganisms.