Whole-cell Extracts Research Articles

HERC5 downregulation in non-small cell lung cancer is associated with altered energy metabolism and metastasis

BackgroundMetastasis is the leading cause of cancer-related death in non-small cell lung cancer (NSCLC) patients. We previously showed that low HERC5 expression predicts early tumor dissemination and a dismal prognosis in NSCLC patients. Here, we performed functional studies to unravel the mechanism underlying the “metastasis-suppressor” effect of HERC5, with a focus on mitochondrial metabolism pathways.MethodsWe assessed cell proliferation, colony formation potential, anchorage-independent growth, migration, and wound healing in NSCLC cell line models with HERC5 overexpression (OE) or knockout (KO). To study early tumor cell dissemination, we used these cell line models in zebrafish experiments and performed intracardial injections in nude mice. Mass spectrometry (MS) was used to analyze protein changes in whole-cell extracts. Furthermore, electron microscopy (EM) imaging, cellular respiration, glycolytic activity, and lactate production were used to investigate the relationships with mitochondrial energy metabolism pathways.ResultsUsing different in vitro NSCLC cell line models, we showed that NSCLC cells with low HERC5 expression had increased malignant and invasive properties. Furthermore, two different in vivo models in zebrafish and a xenograft mouse model showed increased dissemination and metastasis formation (in particular in the brain). Functional enrichment clustering of MS data revealed an increase in mitochondrial proteins in vitro when HERC5 levels were high. Loss of HERC5 leads to an increased Warburg effect, leading to improved adaptation and survival under prolonged inhibition of oxidative phosphorylation.ConclusionsTaken together, these results indicate that low HERC5 expression increases the metastatic potential of NSCLC in vitro and in vivo. Furthermore, HERC5-induced proteomic changes influence mitochondrial pathways, ultimately leading to alterations in energy metabolism and demonstrating its role as a new potential metastasis suppressor gene.

A Knockout of Poly(ADP-Ribose) Polymerase 1 in a Human Cell Line: An Influence on Base Excision Repair Reactions in Cellular Extracts.

Base excision repair (BER) is the predominant pathway for the removal of most forms of hydrolytic, oxidative, and alkylative DNA lesions. The precise functioning of BER is achieved via the regulation of each step by regulatory/accessory proteins, with the most important of them being poly(ADP-ribose) polymerase 1 (PARP1). PARP1's regulatory functions extend to many cellular processes including the regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of BER proteins and at the level of their mRNAs. Systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells are extremely limited. In this study, a CRISPR/Cas9-based technique was used to knock out the PARP1 gene in the human HEK 293FT line. The obtained cell clones with the putative PARP1 deletion were characterized by several approaches including PCR analysis of deletions in genomic DNA, Sanger sequencing of genomic DNA, quantitative PCR analysis of PARP1 mRNA, Western blot analysis of whole-cell-extract (WCE) proteins with anti-PARP1 antibodies, and PAR synthesis in WCEs. A quantitative PCR analysis of mRNAs coding for BER-related proteins-PARP2, uracil DNA glycosylase 2, apurinic/apyrimidinic endonuclease 1, DNA polymerase β, DNA ligase III, and XRCC1-did not reveal a notable influence of the PARP1 knockout. The corresponding WCE catalytic activities evaluated in parallel did not differ significantly between the mutant and parental cell lines. No noticeable effect of poly(ADP-ribose) synthesis on the activity of the above WCE enzymes was revealed either.

THU407 CRISPR/Cas9-based Knockout Of CFTR Alters Mechano-sensitivity And Proteomic Profile In An Osteocyte Model

Abstract Disclosure: W. Du: None. J. Chen: None. Y. Ruan: None. Osteocytes, the most abundant bone cell type with recognized endocrine functions, play essential roles in mechano-signal transduction in the skeletal system required for bone modelling/remodelling and mineral homeostasis, although the underlying molecular mechanisms are not fully understood. Bone problems are present in cystic fibrosis (CF), a genetic disease resulted from mutations in CF transmembrane conductance regulator (CFTR), which is a Cl− channel gene recently revealed to be expressed in osteocytes with its exact roles largely unexplored. In the present study, CRISPR/Cas9 technology was used to knock out CFTR (CFTR-KO) in MLO-Y4, a commonly used osteocyte model, which resulted in a significant decrease (by 80.7 ± 7.4%) in shear force-induced Ca2+ oscillations (measured by Fura-2 imaging) as compared to wild-type (WT) cells (n = 205 (WT) and 114 (KO) cells, t-test, p < 0.001). However, the CFTR-KO cells did not show, in comparison with WT cells, any reduction in expressing (measured by quantitative PCR, n = 5) Piezo1 or TRPV4, two reported key mechano-sensitive Ca2+ channels in osteocytes, nor change in cell membrane stiffness (measured by atom force microscope, n = 58 (WT) and 64 (KO) cells, t-test, p > 0.05). Moreover, the shear-force-stimulated Ca2+ oscillations in WT cells were observed to be dependent on extracellular Cl−, which together suggested possibly a direct role of CFTR, through its Cl- channel function, in osteocyte mechano-sensitivity. We next performed proteomic analysis by mass spectrometry detection of whole-cell protein extracts from the MLO-Y4 cells, which revealed downregulation of genes related to focal adhesion, gap junction and actin cytoskeleton regulation in CFTR-KO cells. These results, therefore, have suggested a crucial involvement of CFTR in osteocyte function, providing new insights into mechanistic understanding of the mechano-transduction in the skeletal system. This work was supported in part by Health and Medical Research Fund of Hong Kong (No.18191361), Theme-based Research Scheme of Hong Kong (No. T13-402/17N), and Areas of Excellence Scheme of Hong Kong (AoE/M-402/20). Presentation: Thursday, June 15, 2023

Resveratrol Mitigates Metabolism in Human Microglia Cells.

The recognition of the role of microglia cells in neurodegenerative diseases has steadily increased over the past few years. There is growing evidence that the uncontrolled and persisting activation of microglial cells is involved in the progression of diseases such as Alzheimer's or Parkinson's disease. The inflammatory activation of microglia cells is often accompanied by a switch in metabolism to higher glucose consumption and aerobic glycolysis. In this study, we investigate the changes induced by the natural antioxidant resveratrol in a human microglia cell line. Resveratrol is renowned for its neuroprotective properties, but little is known about its direct effect on human microglia cells. By analyzing a variety of inflammatory, neuroprotective, and metabolic aspects, resveratrol was observed to reduce inflammasome activity, increase the release of insulin-like growth factor 1, decrease glucose uptake, lower mitochondrial activity, and attenuate cellular metabolism in a 1H NMR-based analysis of whole-cell extracts. To this end, studies were mainly performed by analyzing the effect of exogenous stressors such as lipopolysaccharide or interferon gamma on the metabolic profile of microglial cells. Therefore, this study focuses on changes in metabolism without any exogenous stressors, demonstrating how resveratrol might provide protection from persisting neuroinflammation.

Identification of endometrial biofilm-specific proteins for in vivo diagnostics

Bacteria enter the uterus in a free-floating planktonic state, which makes them vulnerable to the host-immune system as well as exposure to effective antimicrobials. If not eliminated in a timely fashion, the bacteria will adhere to the endometrial surface and may form micro-colonies, develop a complex matrix, and switch into a biofilm community. The ability of bacteria to shift from planktonic to biofilm phenotypes has been described for both Gram-negative and Gram-positive bacteria in human mucosal tissue, indwelling catheters, and implants as well as other organs, including the equine endometrium. Unfortunately, there is currently no available assay to diagnose the condition in vivo. Literature in human medicine suggests that biofilm matrices contain both secretory and non-secretory proteins, and while some of the proteins are common among all biofilms, others may be specific for bacterial species. Therefore, the objective of this research was to identify specific proteins from biofilm-producing bacteria from mares with endometritis. Whole-cell extracts from potentially biofilm-forming bacterial isolates of Streptococcus zooepidemicus (SEZ) (n=3), Pseudomonas aeruginosa (n=1), and Escherichia coli (n=4) were analyzed by 2-D PAGE. Differentially expressed proteins were selected by overlapping 2-D PAGE biofilm proteins with planktonic culture proteins.In-gel analysis of differences (with a 3-fold cut-off)identified a total of 96 proteins for SEZ, 83 proteins for P. aeruginosa, and 69 proteins for E. coli as potential biofilm markers. Select proteins were further identified by MALDI-TOF/TOF. In SEZ, glutathione-disulfide reductase (48.8 kDa, PI 5.6), which catalyzes the reduction of glutathione disulfide to glutathione, a major cellular antioxidant, and TIGR00266 (25.3 kDa, PI 5.4) were identified as proteins of interest. For P. aeruginosa, thioredoxin A (11.8 kDa, PI 4.7), which participates in various redox reactions, osmotically inducible protein C (15.6 kDa, PI 5.9), important for cellular defense mechanism against oxidative stress, and bacterial extracellular solute-binding s, 3 family protein (33 kDa, PI 8.3) serve as potential targets. Potential biomarker candidates for E. coli include the serine protease pet autotransporter (139 kDa, PI 6.5) and outer membrane protein X (18 kDa, PI 6.6), a protein involved in numerous physiological processes including invasion, signal transduction, and binding external proteins. In conclusion, we identified biofilm-specific proteins that may serve as in vivo diagnostic markers for biofilms in uterine lavage from mares with biofilm-associated bacterial endometritis. Future research will pursue these proteins of interest for their use as diagnostic tools in the field.

Contrasting the phospholipid profiles of two neoplastic cell lines reveal a high PC:PE ratio for SH-SY5Y cells relative to A431 cells

Lipids have been implicated in Parkinson's Disease (PD). We therefore studied the lipid profile of the neuroblastoma SH-SY5Y cell line, which is used extensively in PD research and compared it to that of the A431 epithelial cancer cell line. We have isolated whole cell extracts (WC) and plasma membrane (PM) fractions of both cell lines. The isolates were analyzed with 31P NMR. We observed a significant higher abundance of phosphatidylcholine (PC) for SH-SY5Y cells for both WC (55 ± 4.1%) and PM (63.3 ± 3.1%) compared to WC (40.5 ± 2.2%) and PM (43.4 ± 1.3%) of A431. Moreover, a higher abundance of phosphatidylethanolamine was detected for the WC of A431 compared to the SH-SY5Y. Using LC-MS/MS, we also determined the relative abundance of fatty acid (FA) moieties for each phospholipid class, finding that SH-SY5Y had high polyunsaturated FA levels, including arachidonic acid compared to A431 cells. When comparing our results to reported compositions of brain and neural tissues, we note the much higher PC levels, as well as very low levels of docosahexaenoic acid. However, relative levels of arachidonic acid and other polyunsaturated fatty acids were elevated, in line with what is desirable for a neural model system.

Serine 970 of RNA helicase MOV10 is phosphorylated and controls unfolding activity and fate of mRNAs targeted for AGO2-mediated silencing

MOV10 is an RNA helicase required for organismal development and is highly expressed in postnatal brain. MOV10 is an AGO2-associated protein that is also necessary for AGO2-mediated silencing. AGO2 is the primary effector of the miRNA pathway. MOV10 has been shown to be ubiquitinated, leading to its degradation and release from bound mRNAs, but no other posttranslational modifications with functional implications have been described. Using mass spectrometry, we show that MOV10 is phosphorylated in cells at the C-terminus, specifically at serine 970 (S970). Substitution of S970 to phospho-mimic aspartic acid (S970D) blocked unfolding of an RNA G-quadruplex, similar to when the helicase domain was mutated (K531A). In contrast, the alanine substitution (S970A) of MOV10 unfolded the model RNA G-quadruplex. To examine its role in cells, our RNA-seq analysis showed that the expression of S970D causes decreased expression of MOV10 enhanced Cross-Linking Immunoprecipitation targets compared to WT. Introduction of S970A had an intermediate effect, suggesting that S970 was protective of mRNAs. In whole-cell extracts, MOV10 and its substitutions bound AGO2 comparably; however, knockdown of AGO2 abrogated the S970D-induced mRNA degradation. Thus, MOV10 activity protects mRNA from AGO2; phosphorylation of S970 restricts this activity resulting in AGO2-mediated mRNA degradation. S970 is positioned C-terminal to the defined MOV10-AGO2 interaction site and is proximal to a disordered region thatlikely modulates AGO2 interaction with target mRNAs upon phosphorylation. In summary, we provide evidence whereby MOV10 phosphorylation facilitates AGO2 association with the 3'UTR of translating mRNAs that leads to their degradation.

Influence of Poly(ADP-ribose)polymerase 1 Level on the Status of Base Excision Repair in Human Cells

The base excision repair (BER) system is aimed at repair of the largest group of DNA lesions, namely of damaged bases. The main steps in BER are: recognition and removal of the aberrant base, cutting the DNA sugar-phosphate backbone, gap processing (including dNMP insertion), and DNA ligation. The precise functioning of BER depends on the regulation of each step of the process by regulatory/accessory proteins, the most important of which is poly(ADP-ribose)polymerase 1 (PARP1). PARP1 plays an important role in various processes of DNA repair, maintenance of genome integrity, and regulation of mRNA stability and decay. In this regard, PARP1 can affect BER both at the level of proteins involved in the process and at the level of expression of the mRNAs encoding them. There are no systematic data on the effect of various amounts of PARP1 on the activity of key BER proteins and the levels of mRNAs encoding them in human cells. In our work, using whole-cell extracts and RNA preparations obtained from the parental HEK293T cell line and its derivative HEK293T/P1-KD cell line with reduced PARP1 expression (shPARP1-expressing cells, PARP1 knockdown), we assessed the levels of mRNA encoding BER proteins: PARP1, PARP2, uracil DNA glycosylase (UNG2), AP endonuclease 1 (APE1), DNA polymerase β (POLβ), DNA ligase III (LIG3), and XRCC1. In parallel, the catalytic activity of these enzymes was evaluated. No significant effect of the PARP1 amount of on the mRNA levels of UNG2, APE1, POLβ, LIG3, and XRCC1 was found. At the same time, in HEK293T/P1-KD cells, the amount of PARP2 mRNA was reduced by 2 times. The activities of these enzymes in whole-cell extracts of HEK293T and HEK293T/P1-KD cells also did not differ significantly. Under the conditions of poly(ADP-ribose) synthesis, the efficiency of the reactions catalyzed by UNG2, APE1, POLβ, and LIG3 also did not change significantly. In addition, it was shown that a reduced amount of PARP1 in the extract of HEK293T/P1-KD cells does not cause fundamental changes in the nature of DNA PARylation compared to the extract of the parental HEK293T cell line.

Influence of the Poly(ADP-Ribose) Polymerase 1 Level on the Status of Base Excision Repair in Human Cells

Base excision repair (BER) is aimed at repair of damaged bases, which are the largest group of DNA lesions. The main steps of BER are recognition and removal of the aberrant base, cutting of the DNA sugar-phosphate backbone, gap processing (including dNMP insertion), and DNA ligation. The precise function of BER depends on the regulation of each step by regulatory/accessory proteins, the most important of which is poly(ADP-ribose) (PAR) polymerase 1 (PARP1). PARP1 plays an important role in DNA repair, maintenance of genome integrity, and regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of BER proteins and at the level of their mRNAs. There is no systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells. Whole-cell extracts and RNA preparations obtained from the parental HEK293T cell line and its derivative HEK293T/P1-KD cell line with reduced PARP1 expression (shPARP1-expressing cells, a PARP1 knockdown) were used to assess the levels of mRNAs coding for BER proteins: PARP1, PARP2, uracil DNA glycosylase (UNG2), AP endonuclease 1 (APE1), DNA polymerase β (POLβ), DNA ligase III (LIG3), and XRCC1. Catalytic activities of the enzymes were evaluated in parallel. No significant effect of the PARP1 content was observed for the mRNA levels of UNG2, APE1, POLβ, LIG3, and XRCC1. The amount of the PARP2 mRNA proved to be reduced two times in HEK293T/P1-KD cells. Activities of these enzymes in whole-cell extracts did not differ significantly between HEK293T and HEK293T/P1-KD cells. No significant change was observed in the efficiencies of the reactions catalyzed by UNG2, APE1, POLβ, and LIG3 in conditions of PAR synthesis. A DNA PARylation pattern did not dramatically change in a HEK293T/P1-KD cell extract with a reduced PARP1 content as compared with an extract of the parental HEK293T cell line.

Critical point for membrane bilayer formation

Unilamellar liposomes often are employed in investigations of lipid-protein interactions and the delivery of drugs in therapies for disease. Also, related lipid-containing nanoparticles have been developed as elements of a new class of mRNA vaccines. We show that only unilamellar films form in equilibrium lipid dispersions, at temperature values {T*} that depend on the identities of the lipids (e.g., T* ≈ 29 °C for DMPC). Thermodynamic analysis confirms that films at air-water surfaces can be used to monitor the properties of the lipid vesicles that form in the dispersion. When T > T*, critical exponents describing film properties as T approaches T* are μ ≈ 1.4 and ν ≈ 0.7, which are close to values for the interfacial tension and the correlation length of density fluctuations at fluid interfaces. These results, and observations that within the bilayer the lateral diffusion of fluorescent lipid probes demonstrates increases at T*, suggest that unilamellar vesicles at T* are a transition state between two different multilamellar structures. We generalize the thermodynamic arguments to explain the linkage between lipid structures in the surface and bulk dispersion within more complex samples, showing that dispersions containing total lipid extracts of cell membranes have properties similar to those in dispersions containing single lipids. Information from various independent studies indicates that T* noted for bilayer membranes of a population of cells is identical to the temperature at which the growth or gestation of the cells occurs in vivo. Examples include whole-cell lipid extracts obtained from bacteria, and poikilothermic and homeothermic animals.

In Vitro Assay to Measure APE1 Enzymatic Activity on Ribose Monophosphate Abasic Site.

APE1 (apurinic/apyrimidinic endodeoxyribonuclease 1) is a central enzyme of the base excision repair (BER) pathway playing a pivotal role in protecting mammaliancells against genotoxins and in safeguarding genome stability. Recently, we demonstrated the APE1 ability to process abasic ribonucleotides embedded in DNA. Here, we provide a pipeline of protocols to quantify endodeoxyribonuclease activity by APE1 on these substrates, by using recombinant protein and whole-cell extracts. The repair capacity is measured by using fluorescent oligonucleotide substrates, which are then separated by polyacrylamide gel electrophoresis and detected by imaging scanning. The specificity of APE1 action is demonstrated using specific APE1 enzymatic inhibitors.

Antibacterial Effects of ZnO Nanodisks: Shape Effect of the Nanostructure on the Lethality in Escherichia coli.

The role of the shape of the nanostructure on the antibacterial effects of ZnO nanodisks has been investigated by detailed mass spectrometry-based proteomics along with other spectroscopic and microscopic studies on E. coli. The primary interaction study of the E. coli cells in the presence of ZnO nanodisks showed rigorous cell surface damage disrupting the cell wall/membrane components detected by microscopic and ATR-FTIR studies. Protein profiling of whole-cell extracts in the presence and absence of ZnO nanodisks identified several proteins that are upregulated and downregulated under the stress of the nanodisks. This suggests that the bacterial response to the primary stress leads to a secondary impact of ZnO nanodisk toxicity via regulation of the expression of specific proteins. Results showed that the ZnO nanodisks lead to the over-expression of peptidyl-dipeptidase Dcp, Transketolase-1, etc., which are important to maintaining the osmotic balance in the cell. The abrupt change in osmotic pressure leads to mechanical injury to the membrane, and nutritional starvation conditions, which is revealed from the expression of the key proteins involved in membrane-protein assembly, maintaining membrane integrity, cell division processes, etc. Thus, indicating a deleterious effect of ZnO nanodisk on the protective layer of E. coli. ZnO nanodisks seem to primarily affect the protective membrane layer, inducing cell death via the development of osmotic shock conditions, as one of the possible reasons for cell death. These results unravel a unique behavior of the disk-shaped ZnO nanostructure in executing lethality in E. coli, which has not been reported for other known shapes or morphologies of ZnO nanoforms.

A Missense Mutation (c.1037 G > C, p. R346P) in PAPSS2 Gene Results in Autosomal Recessive form of Brachyolmia Type 1 (Hobaek Form) in A Consanguineous Family.

Brachyolmia is a skeletal disorder with an autosomal mode of inheritance (both dominant and recessive) in which the patients have a short height, scoliosis and a reduced trunk size. From the Muzaffargarh District in Pakistan, a consanguineous family with multiple Brachyolmia-affected subjects were enrolled in the present study. Basic epidemiological data and radiographs were collected for the subjects. Whole exome sequencing (WES) which was followed by Sanger sequencing was applied to report the geneticbasic of Brachyolmia. The WES identified a missense mutation (c.1037 G > C, p. R346P) in exon 9 of the PAPSS2 gene that was confirmed by the Sanger sequencing in the enrolled subjects. The mutation followed a Mendalian pattern with an autosomal recessive inheritance mode. Multiple sequence alignment by Clustal Omega indicated that the PAPSS2 mutation-containing domain is highly conserved. The HEK293T whole-cell extract that was transfected with the Myc-tagged PCMV6-PAPSS2 of both the wild and mutant constructs were resolved by SDS-PAGE as well as by a Western blot, which confirmed that there are different PAPSS2 protein expression patterns when they were compared between the control and Brachyolmia patients. This difference between the normal and mutated protein was not evident when the three-dimensional computational structures were generated using homology modeling. We report a missense mutation (c.1037 G > C, p. R346P) in the PAPSS2 gene that caused Brachyolmia in a consanguineous Pakistani family.

Protein expression profiling, in silico classification and pathway analysis of cariogenic bacteria Streptococcus mutans under bacitracin stress conditions.

Introduction. Streptococcus mutans is a cariogenic bacterium that causes dental caries as well as being implicated in other dental pathologies and infective endocarditis. Bacitracin is a bactericidal antibiotic that induces cell wall stress in Gram-positive bacteria.Gap Statement. S. mutans is among the most characterized Gram-positive bacteria. However, the transcriptome and proteome of S. mutans have received less attention, and they are actually key in understanding the pathogenesis of any bacteria. In this study, we extracted the whole proteome of S. mutans grown under bacitracin stress. Such a proteome is anticipated to offer deep insights related to physiological dynamic fluctuations and, consequently, it may provide 'proteomic signatures' to be identified as potential targets.Aim. The aim of the study is to explore the general stress response that S. mutans exhibits at the proteome level when cell wall stress is imposed on it.Methodology. A sub-MIC concentration of bacitracin was added to the growth media of S. mutans followed by whole-cell protein extraction. The proteome was then subjected to high-throughput proteomics analysis, i.e. liquid chromatography tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins obtained through LC-MS/MS underwent analyses such as gene ontology, KEGG (Kyoto Encyclopaedia of Genes and Genomes) and DAVID (Database for Annotation, Visualization and Integrated Discovery) analysis, and STRING for functional annotation, pathway enrichment and protein-protein interaction (PPI) networks, respectively. These proteins were also categorized into functional classes using the PANTHER (Protein Annotation Through Evolutionary Relationship) classification system.Result. LC-MS/MS produced data from 321 identified proteins. From these, 41 and 30 were found to be significantly over- (≥2 fold change) and underexpressed (≤0.4 fold change), respectively. In the upregulated proteins we mostly observed sortases and proteins involved in the EPS biosynthesis pathway, whereas among the downregulated proteins the majority related to glycolysis.Conclusion. The sortase family of proteins appear to be potential targets because they regulate various virulence factors and therefore can be targeted to inhibit multiple virulence pathways simultaneously. This study offers an understanding of proteomic fluctuations in response to cell wall stress and can thus help in identifying key players mediating virulence.

Germination screen for microalgae-generated plant growth biostimulants

Greenhouse (controlled environments) horticulture is expected to rise to ~USD 30 billion by 2023. In such climate-controlled systems, crop yields can increase ~10-fold compared with field production, due to higher water and nutrient efficiency and reduced external environmental impacts. The application of plant growth regulators (PGRs) can promote seed germination and plant development, speed up the production cycle and increase production efficiency which is of particular significance for high-value crops produced in controlled environments. Here, a novel in-vivo germination and plant growth assay was developed to rapidly identify microalgae strains that naturally produce PGRs was developed using spinach (Spinacia oleracea L) as test crop. Four newly isolated, fast growing microalgae strains in the genera Chlorococcum, Micractinium, Scenedesmus and Chlorella were screened to evaluate their potential as reliable and cost-effective sources of PGRs. Spinach seeds were primed with soluble and insoluble microalgae fractions from whole-cell and lysed-cell extracts and compared to controls (water and commercial seaweed extract). After a 5-day incubation, algae extracts improved seed germination up to 1.7-fold and 1.6-fold compared to water and seaweed extract, respectively. Green cotyledon emergence improved up to 2-fold (day 6), while seedling biomass improved up to 2.1-fold and 1.9-fold (day 9) compared to water and seaweed extract, respectively. The quantification of cytokinins (trans-Zeatin, DHZR, tZMP, iP, iPA, iPAMP), gibberellins (GA1, GA3, GA4, GA20 and GA29), auxin (IAA) and abscisic acid (ABA) validated that the four uncharacterized microalgal strains produced relevant PGRs. To boost plant propagation, PGR-producing microalgae may offer natural and cost-effective alternative biostimulants to synthetically produced agrochemicals.

Expression of Deubiquitinating Enzymes in Lung Cancer Cells with Different REST Status

he aims of this study were: (1) to screen a panel of lung cell lines for expression of the proteins UCHL1, USP49, USP15, USP4, and USP11. (2) To investigate the correlation of transcript and protein expression level of these DUBs. (3) To find any proteins that discriminate non-small cell lung cancer (NSCLC) from neuroendocrine (NE) SCLC or benign lung carcinoid. (4) To investigate any correlation of DUB proteins expression with lung cancer sub-types that has different amounts of REST protein. Method: Western blots were performed to detect the expression of DUB expression (USP4, USP11, USP15, USP49, and UCHL1) and REST protein in extracts from normal lung cell lines, SCLC, carcinoid, and NSCLC Results: we found that the transcript level was in general not a good indicator of DUB protein level. There was a significant positive correlation of REST with DUB proteins from whole-cell extracts for USP15, USP11, USP4, and USP49 in normal lung cell lines. The P values were 0.016, 0.002, 0 .018 and 0 .034 respectively. Also in both normal and NSCLC cell lines total USP15, USP11 and USP49 showed a positive correlation with REST. P values were 0.007, 0.029 and 0.001 respectively. However USP11 protein was highly abundant in REST-deficient SCLC. In contrast, both USP11 and USP15 showed a positive correlation with REST in SCLC and NSCLC nuclear extracts. P values were 0.001 and 0.005 respectively. A positive correlation of DUBs with REST would be consistent with their putative role in stabilizing REST.

Enzyme immunoassays (EIA) for serodiagnosis of human leptospirosis: specific IgG3/IgG1 isotyping may further inform diagnosis of acute disease.

The laborious microscopic agglutination test (MAT) is the gold standard serologic test for laboratory diagnosis of leptospirosis. We developed EIA based serologic assays using recombinant proteins (rLigA, rLigB, rLipL32) and whole-cell extracts from eight Leptospira serovars as antigen and assessed the diagnostic performance of the new assay within each class, against MAT positive (MAT+) human sera panels from Portugal/PT (n = 143) and Angola/AO (n = 100). We found that a combination of recombinant proteins rLigA, rLigB and rLipL32 correctly identified antigen-specific IgG from patients with clinical and laboratory confirmed leptospirosis (MAT+) with 92% sensitivity and ~ 97% specificity (AUC 0.974) in serum from the provinces of Luanda (LDA) and Huambo (HBO) in Angola. A combination of whole cell extracts of L. interrogans sv Copenhageni (LiC), L. kirschneri Mozdok (LkM), L. borgpetersenii Arborea (LbA) and L. biflexa Patoc (LbP) accurately identified patients with clinical and laboratory confirmed leptospirosis (MAT+) with 100% sensitivity and ~ 98% specificity for all provinces of Angola and Portugal (AUC: 0.997 for AO/LDA/HBO, 1.000 for AO/HLA, 0.999 for PT/AZ and 1.000 for PT/LIS). Interestingly, we found that MAT+ IgG+ serum from Angola had a significantly higher presence of IgD and that IgG3/IgG1 isotypes were significantly increased in the MAT+ IgG+ serum from Portugal. Given that IgM/IgD class and IgG3/IgG1 specific isotypes are produced in the earliest course of infection, immunoglobulin G isotyping may be used to inform diagnosis of acute leptospirosis. The speed, ease of use and accuracy of EIA tests make them excellent alternatives to the laborious and expensive MAT for screening acute infection in areas where circulating serovars of pathogenic Leptospira are well defined.

Initiator-Directed Transcription: Fission Yeast Nmtl Initiator Directs Preinitiation Complex Formation and Transcriptional Initiation.

The initiator element is a core promoter element encompassing the transcription start site, which is found in yeast, Drosophila, and human promoters. This element is observed in TATA-less promoters. Several studies have defined transcription factor requirements and additional cofactors that are needed for transcription initiation of initiator-containing promoters. However, those studies have been performed with additional core promoters in addition to the initiator. In this work, we have defined the pathway of preinitiation complex formation on the fission yeast nmt1 gene promoter, which contains a functional initiator with striking similarity to the initiator of the human dihydrofolate reductase (hDHFR) gene and to the factor requirement for transcription initiation of the nmt1 gene promoter. The results show that the nmt1 gene promoter possesses an initiator encompassing the transcription start site, and several conserved base positions are required for initiator function. A preinitiation complex formation on the nmt1 initiator can be started by TBP/TFIIA or TBP/TFIIB, but not TBP alone, and afterwards follows the same pathway as preinitiation complex formation on TATA-containing promoters. Transcription initiation is dependent on the general transcription factors TBP, TFIIB, TFIIE, TFIIF, TFIIH, RNA polymerase II, Mediator, and a cofactor identified as transcription cofactor for initiator function (TCIF), which is a high-molecular-weight protein complex of around 500 kDa. However, the TAF subunits of TFIID were not required for the nmt1 initiator transcription, as far as we tested. We also demonstrate that other initiators of the nmt1/hDHFR family can be transcribed in fission yeast whole-cell extracts.

Detection of a target protein (GroEl2) in Mycobacterium tuberculosis using a derivative of 1,2,4-triazolethiols.

Herein, we identified a potent lead compound RRA2, within a series of 54 derivatives of 1,2,4-triazolethiols (exhibit good potency as an anti-mycobacterial agents) against intracellular Mycobacterium tuberculosis (Mtb). Compound RRA2 showed significant mycobactericidal activity against active stage Mycobacterium bovis BCG and Mtb with minimum inhibitory concentration (MIC) values of 2.3 and 2.0µg/mL, respectively. At MIC value, RRA2 compound yielded 0.82 log reduction of colony-forming unit (cfu) against non-replicating Mtb. Furthermore, RRA2 compound was selected for further target identification due to the presence of alkyne group, showing higher selectivity index (> 66.66 ± 0.22, in non-replicating stage). Using "click" chemistry, we synthesized the biotin linker-RRA2 conjugate, purified with HPLC method and confirmed the conjugation of biotin linker-RRA2 complex by HR-MS analysis. Furthermore, we successfully pulled down and identified a specific target protein GroEl2, from Mtb whole-cell extract. Furthermore, computational molecular modeling indicated RRA2 could interact with GroEl2, which explains the structure-activity relationship observed in this study. GroEL-2 identified a potent and specific target protein for RRA 2 compound in whole cell extract of Mtb H37Ra.

Study of Potential Application of Cyanobacteria Leptolyngbya Valderiana in Skin Cosmeceuticals

Skin is the outermost cover and first line of defense of the human body. Skin cosmeceuticals are therapeutic agents used to maintain flawless skin and prevent skin disorders. Considering the ill effects of synthetic active agents, natural metabolites are preferred for skincare. Cyanobacterium was known to produce various secondary metabolites with therapeutic potential. This study focuses on screening whole-cell extract of Leptolyngbya valderiana for skincare. Isolation and identification of cyanobacteria were made. The whole-cell extract of Leptolyngbya valderiana was studied for its metabolite profile, mainly for proteins, carbohydrate, and lipid content. Screening of whole-cell extract was carried out for its (2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) (ABTS) radical scavenging ability and UV absorption potential. The antimicrobial effect of extracts was determined by agar cup and minimum inhibitory concentration (MIC) method using salicylic acid as a standard. Skin whitening potential was determined using tyrosinase inhibition assay. Leptolyngbya valderiana was isolated from the tree bark and selected for further study. The extract of Leptolyngbya valderiana was found to have 25.22% proteins, 36.65 % carbohydrates and 35.97% lipids. The extract was found to have antioxidant potential with 96.55% ABTS radical scavenging potential. Antimicrobial potency of the extract against Staphylococcus aureus ATCC 6538, Propionibacterium acne MTCC 1351, Candida albicans ATCC 10231 was equivalent to salicylic acid. The test extract was found to inhibit the tyrosinase enzyme by 43.46%. The test extract is rich in proteins, carbohydrates, and lipids. The extract has significant antioxidant potential, antimicrobial efficiency, ability to absorb ultraviolet B and ultraviolet A region, tyrosinase inhibition potential indicative photoprotective, and skin whitening potential. The isolate Leptolyngbya valderiana has a promising potential for skin cosmeceuticals.