Cyanobacterial bioactive metabolites—A review of their chemistry and biology

Cyanobacterial blooms occur when algal densities exceed baseline population concentrations. Cyanobacteria can produce a large number of secondary metabolites. Odorous metabolites affect the smell and flavor of aquatic animals, whereas bioactive metabolites cause a range of lethal and sub-lethal effects in plants, invertebrates, and vertebrates, including humans. Herein, the bioactivity, chemistry, origin, and biosynthesis of these cyanobacterial secondary metabolites were reviewed. With recent revision of cyanobacterial taxonomy by Anagnostidis and Komárek as part of the Süβwasserflora von Mitteleuropa volumes 19(1–3), names of many cyanobacteria that produce bioactive compounds have changed, thereby confusing readers. The original and new nomenclature are included in this review to clarify the origins of cyanobacterial bioactive compounds.Due to structural similarity, the 157 known bioactive classes produced by cyanobacteria have been condensed to 55 classes. This review will provide a basis for more formal procedures to adopt a logical naming system. This review is needed for efficient management of water resources to understand, identify, and manage cyanobacterial harmful algal bloom impacts.     

Identification and characterization of a differentially expressed protein (CAPZB) in skeletal muscle between Meishan and Large White pigs

Actin capping protein beta (CAPZB) protein was identified with considerable differences in the longissimus dorsi muscle between Large White and Meishan pigs using proteomics approach. However, in pigs, the information on CAPZB is very limited. In this study, we cloned and characterized the porcine actin capping protein beta (CAPZB) gene. In addition, we present two novel porcine CAPZB splice variants CAPZB1 and CAPZB2. CAPZB1 was expressed in all twenty tissues. However, CAPZB2 was predominantly expressed in the skeletal muscle and heart. In addition, the two isoforms had different expression profiles during the skeletal muscle development and between breeds. Moreover, the SNP T394G was identified in the coding region of the CAPZB gene, which was significantly associated with the carcass traits including the LFW, CFW, SFT and LEA. Data presented in our study suggests that the CAPZB gene may be a candidate gene of meat production trait and provides useful information for further studies on its roles in porcine skeletal muscle.     

Conjugation of genetically engineered protein phosphatases to magnetic particles for okadaic acid detection

This work presents the functional characterisation of a protein phosphatase 2A (PP2A) catalytic subunit obtained by genetic engineering and its conjugation to magnetic particles (MPs) via metal coordination chemistry for the subsequent development of assays for diarrheic lipophilic marine toxins. Colorimetric assays with free enzyme have allowed the determination of the best enzyme activity stabiliser, which is glycerol at 10%. They have also demonstrated that the recombinant enzyme can be as sensitive towards okadaic acid (OA) (LOD = 2.3 μg/L) and dinophysistoxin-1 (DTX-1) (LOD = 15.2 μg/L) as a commercial PP2A and, moreover, it has a higher operational stability, which makes possible to perform the protein phosphatase inhibition assay (PPIA) with a lower enzyme amount. Once conjugated to MPs, the PP2A catalytic subunit still retains its enzyme activity and it can also be inhibited by OA (LOD = 30.1 μg/L).     

Suitable reference genes for the analysis of direct hyperplasia in mice

The liver is capable of undergoing a proliferative growth, known as direct hyperplasia, in which the naïve liver increases in size due to stimulation with primary mitogens. To produce accurate gene expression data, housekeeping genes (HKGs) that are stably expressed need to be determined. In the present study, liver regeneration was promoted via the direct hyperplasia mode by inducing mice with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. Gene expression levels of nine commonly used HKGs were analyzed in the liver of different timing during the regeneration. The stability of gene expression was assessed using two different analysis programs, geNorm and NormFinder. Using these analyses, we identified that PPIA and RPL4 showed the most stable expression regardless of the status of the liver regeneration. In conclusion, the present study demonstrated that the use of PPIA and RPL4 were the most optimal in providing reliable normalization of gene expression when assessing liver regeneration attributed to direct hyperplasia.     

Promoter variants at AP2 box region of Hsp70.1 affect thermal stress response and milk production traits in Frieswal cross bred cattle

Heat shock proteins (Hsp) are known to play major role in protection of cells from thermal stress. Nucleotide polymorphisms within the promoter of Hsp affect degree of expression and inducibility of Hsp mRNA. The present study aimed to investigate the effect of polymorphism within promoter region on the cellular expression of Hsp70.1 mRNA and association of identified polymorphisms with the physiological parameters during summer stress and milk production traits in dairy cattle. Two hundred Frieswal cows were genotyped using double PCR-RFLP to identify deletion of cytosine within the Hsp70.1 promoter AP2 box at base position 895. Homozygous wild type genotypes (CC) were found in lower frequency (39.29, n = 78) than heterozygous cytosine deletion mutant genotypes (C −) (60.71, n = 122). In the observed physiological parameters (rectal temperature, respiration rate and heat tolerance coefficient), cows that were homozygous wild types had better significant (P < 0.05) summer tolerance than the heterozygous deletion genotypes. Cytosine deletion mutation in the promoter region negatively affected (P < 0.01) the expression of Hsp70.1 mRNA in peripheral bovine mononuclear cells (PBMC) subjected to in vitro heat stress. Further association of observed polymorphism with the milk production traits was significant as the heterozygous cytosine deletion cows had lower total milk yield, peak yield, yield at 300 days, protein% (P < 0.01) and fat% (P < 0.05) than the native wild type promoter cows. The results from the present study suggest that the promoter region of bovine hsp70.1 gene is polymorphic and may be useful in selection of dairy cows for relatively better thermotolerance and higher milk production.     

Direct detection of RNA in vitro and in situ by target-primed RCA: The impact of E. coli RNase III on the detection efficiency of RNA sequences distanced far from the 3′-end

We improved the target RNA-primed RCA technique for direct detection and analysis of RNA in vitro and in situ. Previously we showed that the 3′ → 5′ single-stranded RNA exonucleolytic activity of Phi29 DNA polymerase converts the target RNA into a primer and uses it for RCA initiation. However, in some cases, the single-stranded RNA exoribonucleolytic activity of the polymerase is hindered by strong double-stranded structures at the 3′-end of target RNAs. We demonstrate that in such hampered cases, the double-stranded RNA-specific Escherichia coli RNase III efficiently assists Phi29 DNA polymerase in converting the target RNA into a primer. These observations extend the target RNA-primed RCA possibilities to test RNA sequences distanced far from the 3′-end and customize this technique for the inner RNA sequence analysis.     

Pentoxifylline administration changes protein expression profile of coronary artery disease patients

Increased level of inflammatory mediators plays a central role in the features of coronary artery diseases. As pentoxifylline could suppress the inflammatory process and has shown some promising beneficial effects in inflammatory diseases, we evaluated the effect of two months pentoxifylline administration in proteome of PBMCs of patients with coronary artery disease (CAD). A randomized placebo-controlled study was used. Fourteen CAD patients were randomized to 2 months of pentoxifyline treatment (1200 mg/day) (n = 7) or placebo treatment (n = 7). Blood samples were obtained before and after treatment. A comparative 2 dimensional gel electrophoresis was performed, and gels were silver-stained. Differentially expressed protein spots were detected and were identified by MALDI-TOF spectrometry. Six differentially expressed proteins were identified as HSP70, PPIA and α-Enolase, (all up-regulated) S100-A9, PIMT and β-5 tubulin (all down-regulated), most of which had previously been shown to play a potential role in the pathogenesis of atherosclerosis. As the blood mononuclear cell proteome responds to pentoxifylline with changes in a number of atherosclerosis-relevant proteins, it seems that pentoxifylline could be a good choice for future studies for prevention of cardiovascular events.     

Analysis of SARS-CoV-2 infection associated cell entry proteins ACE2, CD147, PPIA,and PPIB in datasets from non SARS-CoV-2 infected neuroblastoma patients,as potential prognostic and infection biomarkers in neuroblastoma

SARS-CoV-2 viral contagion has given rise to a worldwide pandemic. Although most children experience minor symptoms from SARS-CoV-2 infection, some have severe complications including Multisystem Inflammatory Syndrome in Children. Neuroblastoma patients may be at higher risk of severe infection as treatment requires immunocompromising chemotherapy and SARS-CoV-2 has demonstrated tropism for nervous cells. To date, there is no sufficient epidemiological data on neuroblastoma patients with SARS-CoV-2. Therefore, we evaluated datasets of non-SARS-CoV-2 infected neuroblastoma patients to assess for key genes involved with SARS-CoV-2 infection as possible neuroblastoma prognostic and infection biomarkers. We hypothesized that ACE2, CD147, PPIA and PPIB, which are associated with viral-cell entry, are potential biomarkers for poor prognosis neuroblastoma and SARS-CoV-2 infection.We have analysed three publicly available neuroblastoma gene expression datasets to understand the specific molecular susceptibilities that high-risk neuroblastoma patients have to the virus. Gene Expression Omnibus (GEO) GSE49711 and GEO GSE62564 are the microarray and RNA-Seq data, respectively, from 498 neuroblastoma samples published as part of the Sequencing Quality Control initiative. TARGET, contains microarray data from 249 samples and is part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. ACE2, CD147, PPIA and PPIB were identified through their involvement in both SARS-CoV-2 infection and cancer pathogenesis.In-depth statistical analysis using Kaplan-Meier, differential gene expression, and Cox multivariate regression analysis, demonstrated that overexpression of ACE2, CD147, PPIA and PPIB is significantly associated with poor-prognosis neuroblastoma samples. These results were seen in the presence of amplified MYCN, unfavourable tumour histology and in patients older than 18 months of age. Previously, we have shown that high levels of the nerve growth factor receptor NTRK1 together with low levels of the phosphatase PTPN6 and TP53 are associated with increased relapse-free survival of neuroblastoma patients. Interestingly, low levels of expression of ACE2, CD147, PPIA and PPIB are associated with this NTRK1-PTPN6-TP53 module, suggesting that low expression levels of these genes are associated with good prognosis. These findings have implications for clinical care and therapeutic treatment. The upregulation of ACE2, CD147, PPIA and PPIB in poor-prognosis neuroblastoma samples suggests that these patients may be at higher risk of severe SARS-CoV-2 infection. Importantly, our findings reveal ACE2, CD147, PPIA and PPIB as potential biomarkers and therapeutic targets for neuroblastoma.     

The validation of housekeeping genes as a reference in quantitative Real Time PCR analysis : Application in the milk somatic cells and frozen whole blood of goats infected with caprine arthritis encephalitis virus

The validation of housekeeping genes (HKGs) for normalization of RNA expression in Real-Time PCR is crucial to obtain the most reliable results. There is limited information on reference genes used in the study of gene expression in milk somatic cells and the frozen whole blood of goats. Thus, the aim of this study was to propose the most stable housekeeping genes that can be used as a reference in Real-Time PCR analysis of milk somatic cells and whole blood of goats infected with caprine arthritis encephalitis virus (CAEV). Animals were divided into two groups: non-infected (N = 13) and infected with CAEV (N = 13). Biological material (milk somatic cells and whole blood) was collected 4 times during the lactation period (7, 30, 100 and 240 days post-partum). The expression levels of candidate reference genes were analyzed using geNorm and NormFinder software. The stability of candidates for reference gene expression was analyzed for CAEV-free (control) and CAEV-infected groups, and also for both groups together (combined group). The stability of expression of β-actin (ACTB), glyceraldehyde-3P-dehydrogenase (GAPDH), cyclophilin A (PPIA), RNA18S1, ubiquilin (UBQLN1) and ribosomal protein large subunit P0 (RPLP0) was determined in milk somatic cells, while ACTB, PPIA, RPLP0, succinate dehydrogenase complex subunit A (SDHA), zeta polypeptide (YWHAZ), battenin (CLN3), eukaryotic translation initiation factor 3K (EIF3K) and TATA box-binding protein (TBP) were measured in frozen whole blood of goats. PPIA and RPLP0 were considered as the most suitable internal controls as they were stably expressed in milk somatic cells regardless of disease status, according to NormFinder software. Furthermore, geNorm results indicated the expression of PPIA/RPLP0 genes as the best combination under these experimental conditions. The results of frozen whole blood analysis using NormFinder software revealed that the most stable reference gene in control, CAEV-infected and combined groups is YWHAZ, and – according to the geNorm results – the combined expression of PPM/YWHAZ genes is the best reference in the presented experiment. The usefulness in gene expression analysis of whole blood samples frozen immediately in liquid nitrogen and stored at -80 °C was also proved.     

Reference gene selection for real-time RT-PCR in regenerating mouse livers

The liver has an intrinsic ability to undergo active proliferation and recover functional liver mass in response to an injury response. This regenerative process involves a complex yet well orchestrated change in the gene expression profile. To produce accurate and reliable gene expression of target genes during various stages of liver regeneration, the determination of internal control housekeeping genes (HKGs) those are uniformly expressed is required. In the present study, the gene expression of 8 commonly used HKGs, including GAPDH, ACTB, HPRT1, GUSB, PPIA, TBP, TFRC, and RPL4, were studied using mouse livers that were quiescent and actively regenerating induced by partial hepatectomy. The amplification of the HKGs was statistically analyzed by two different mathematical algorithms, geNorm and NormFinder. Using this method, PPIA and TBP gene expression found to be relatively stable regardless of the stages of liver regeneration and would be ideal for normalization to target gene expression.