Transfection of hypoxia-inducible factor-1α mRNA upregulates the expression of genes encoding angiogenic growth factors.

Hypoxia-Inducible Factor-1α (HIF-1α) has presented a new direction for ischemic preconditioning of surgical flaps to promote their survival. In a previous study, we demonstrated the effectiveness of HIF-1a DNA plasmids in this application. In this study, to avoid complications associated with plasmid use, we sought to express HIF-1α through mRNA transfection and determine its biological activity by measuring the upregulation of downstream angiogenic genes. We transfected six different HIF-1a mRNAs-one predominant, three variant, and two novel mutant isoforms-into primary human dermal fibroblasts using Lipofectamine, and assessed mRNA levels using RT-qPCR. At all time points examined after transfection (3, 6, and 10 h), the levels of HIF-1α transcript were significantly higher in all HIF-1α transfected cells relative to the control (all p < 0.05, unpaired Student's T-test). Importantly, the expression of HIF-1α transcription response genes (VEGF, ANG-1, PGF, FLT1, and EDN1) was significantly higher in the cells transfected with all isoforms than with the control at six and/or ten hours post-transfection. All isoforms were transfected successfully into human fibroblast cells, resulting in the rapid upregulation of all five downstream angiogenic targets tested. These findings support the potential use of HIF-1α mRNA for protecting ischemic dermal flaps.

The interaction between Epstein-Barr virus and multiple sclerosis genetic risk loci: insights into disease pathogenesis and therapeutic opportunities.

Multiple sclerosis (MS) is a chronic neurodegenerative autoimmune disease, characterised by the demyelination of neurons in the central nervous system. Whilst it is unclear what precisely leads to MS, it is believed that genetic predisposition combined with environmental factors plays a pivotal role. It is estimated that close to half the disease risk is determined by genetic factors. However, the risk of developing MS cannot be attributed to genetic factors alone, and environmental factors are likely to play a significant role by themselves or in concert with host genetics. Epstein-Barr virus (EBV) infection is the strongest known environmental risk factor for MS. There has been increasing evidence that leaves little doubt that EBV is necessary, but not sufficient, for developing MS. One plausible explanation is EBV may alter the host immune response in the presence of MS risk alleles and this contributes to the pathogenesis of MS. In this review, we discuss recent findings regarding how EBV infection may contribute to MS pathogenesis via interactions with genetic risk loci and discuss possible therapeutic interventions.

Non-coding RNAs and glioma: Focus on cancer stem cells.

Glioblastoma and gliomas can have a wide range of histopathologic subtypes. These heterogeneous histologic phenotypes originate from tumor cells with the distinct functions of tumorigenesis and self-renewal, called glioma stem cells (GSCs). GSCs are characterized based on multi-layered epigenetic mechanisms, which control the expression of many genes. This epigenetic regulatory mechanism is often based on functional non-coding RNAs (ncRNAs). ncRNAs have become increasingly important in the pathogenesis of human cancer and work as oncogenes or tumor suppressors to regulate carcinogenesis and progression. These RNAs by being involved in chromatin remodeling and modification, transcriptional regulation, and alternative splicing of pre-mRNA, as well as mRNA stability and protein translation, play a key role in tumor development and progression. Numerous studies have been performed to try to understand the dysregulation pattern of these ncRNAs in tumors and cancer stem cells (CSCs), which show robust differentiation and self-regeneration capacity. This review provides recent findings on the role of ncRNAs in glioma development and progression, particularly their effects on CSCs, thus accelerating the clinical implementation of ncRNAs as promising tumor biomarkers and therapeutic targets.

KARAJ: An Efficient Adaptive Multi-Processor Tool to Streamline Genomic and Transcriptomic Sequence Data Acquisition.

Here we developed KARAJ, a fast and flexible Linux command-line tool to automate the end-to-end process of querying and downloading a wide range of genomic and transcriptomic sequence data types. The input to KARAJ is a list of PMCIDs or publication URLs or various types of accession numbers to automate four tasks as follows; firstly, it provides a summary list of accessible datasets generated by or used in these scientific articles, enabling users to select appropriate datasets; secondly, KARAJ calculates the size of files that users want to download and confirms the availability of adequate space on the local disk; thirdly, it generates a metadata table containing sample information and the experimental design of the corresponding study; and lastly, it enables users to download supplementary data tables attached to publications. Further, KARAJ provides a parallel downloading framework powered by Aspera connect which reduces the downloading time significantly.

PeakCNV: A multi-feature ranking algorithm-based tool for genome-wide copy number variation-association study.

Copy Number Variation (CNV) refers to a type of structural genomic alteration in which a segment of chromosome is duplicated or deleted. To date, many CNVs have been identified as causative genetic elements for several diseases and phenotypes. However, performing a CNV-based genome-wide association study is challenging due to inconsistency in length and occurrence of CNVs across different individuals under investigation. One of the most efficient strategies to address this issue is building CNV regions (genomic regions in which CNVs are overlapping - CNVRs). However, this approach is susceptible to a high false positive rate due to overlapping and co-occurring of confounding CNVRs with true positive CNVRs. Here, we develop PeakCNV that differentiates false-positive CNVRs from true positives by calculating a new metric, independence ranking score, (IR-score) via a feature ranking approach. We compared the performance of PeakCNV with other current existing tools by carrying out two case studies one using the CNV genotype data for individuals with prostate cancer (194 cases and 2,392 healthy individuals) and the second one for individuals with neurodevelopmental disorders (19,642 cases and 6,451 healthy individuals). Crucially, our benchmarking analyses on prostate cancer cohort indicated that PeakCNV identifies a fewer risk candidate CNVRs with shorter lengths compared to other tools. Importantly, these CNVRs cover a greater proportion of case over healthy individuals compared to other tools. The accuracy of PeakCNV in identifying relevant candidate CNVRs was reproducible in the case study on neurodevelopmental disorders. Using data from the FANTOM5 expression atlas and the Clinical Genomic Database, we show that the candidate CNVRs identified by PeakCNV for neurodevelopmental disorders overlap with a greater number of genes with the brain-enriched expression, and a greater number of genes that are associated with neurological conditions compared to candidate CNVRs identified by other tools. Taken together, PeakCNV outperformed current existing CNV association study tools by identifying more biologically meaningful CNVRs relevant to the phenotype of interest. PeakCNV is publicly available for the analysis of CNV-associated diseases and is accessible from https://rdrr.io/github/mahdieh1/PeakCNV.

Interplays between non-coding RNAs and chemokines in digestive system cancers.

Within tumors, chemokines and their cognate receptors are expressed by infiltrated leukocytes, cancerous cells, and related cells of stroma, like tumor-associated fibroblasts and tumor-associated macrophages. In malignancies, the altered expression of chemokines/chemokine receptors governs leukocyte infiltration and activation, epithelial-mesenchymal transition (EMT), cancer cell proliferation, angiogenesis, and metastasis. Non-coding RNAs (ncRNAs) contribute to multiple physiological and pathophysiological processes. Some miRNAs can exert anti-tumorigenic activity in digestive system malignancies by repressing the expression of tumor-promoting chemokines/chemokine receptors or by upregulating tumor-suppressing chemokines/chemokine receptors. However, many miRNAs exert pro-tumorigenic activity by suppressing the expression of chemokines/chemokine receptors or by upregulating tumor-promoting chemokines/chemokine receptors. LncRNA and circRNAs also exert pro- and anti-tumorigenic effects by targeting downstream miRNAs influencing the expression of tumor-promoting and tumor-suppressor chemokines/chemokine receptors. On the other side, some chemokines influence the expression of ncRNAs affecting tumor formation. The current review explains the communications between ncRNAs and chemokines/chemokine receptors in certain digestive system malignancies, such as gastric, colorectal, and pancreatic cancers and hepatocellular carcinoma to gain better insights into their basic crosstalk as well as possible therapeutic modalities.

The low abundance of CpG in the SARS-CoV-2 genome is not an evolutionarily signature of ZAP.

The zinc finger antiviral protein (ZAP) is known to restrict viral replication by binding to the CpG rich regions of viral RNA, and subsequently inducing viral RNA degradation. This enzyme has recently been shown to be capable of restricting SARS-CoV-2. These data have led to the hypothesis that the low abundance of CpG in the SARS-CoV-2 genome is due to an evolutionary pressure exerted by the host ZAP. To investigate this hypothesis, we performed a detailed analysis of many coronavirus sequences and ZAP RNA binding preference data. Our analyses showed neither evidence for an evolutionary pressure acting specifically on CpG dinucleotides, nor a link between the activity of ZAP and the low CpG abundance of the SARS-CoV-2 genome.

Genetic and transcriptomic analyses support a switch to lytic phase in Epstein Barr virus infection as an important driver in developing Systemic Lupus Erythematosus.

To investigate the molecular mechanisms through which Epstein-Barr virus (EBV) may contribute to Systemic Lupus Erythematosus (SLE) pathogenesis, we interrogated SLE genetic risk loci for signatures of EBV infection. We first compared the gene expression profile of SLE risk genes across 459 different cell/tissue types. EBV-infected B cells (LCLs) had the strongest representation of highly expressed SLE risk genes. By determining an SLE risk allele effect on gene expression (expression quantitative trait loci, eQTL) in LCLs and 16 other immune cell types, we identified 79 SLE risk locus:gene pairs putatively interacting with EBV infection. A total of 10 SLE risk genes from this list (CD40, LYST, JAZF1, IRF5, BLK, IKZF2, IL12RB2, FAM167A, PTPRC and SLC15A) were targeted by the EBV transcription factor, EBNA2, differentially expressed between LCLs and B cells, and the majority were also associated with EBV DNA copy number, and expression level of EBV encoded genes. Our final gene network model based on these genes is suggestive of a nexus involving SLE risk loci and EBV latency III and B cell proliferation signalling pathways. Collectively, our findings provide further evidence to support the interaction between SLE risk loci and EBV infection that is in part mediated by EBNA2. This interplay may increase the tendency towards EBV lytic switching dependent on the presence of SLE risk alleles. These results support further investigation into targeting EBV as a therapeutic strategy for SLE.

Gender and the Sex Hormone Estradiol Affect Multiple Sclerosis Risk Gene Expression in Epstein-Barr Virus-Infected B Cells.

Multiple Sclerosis (MS) is a complex immune-mediated disease of the central nervous system. Treatment is based on immunomodulation, including specifically targeting B cells. B cells are the main host for the Epstein-Barr Virus (EBV), which has been described as necessary for MS development. Over 200 genetic loci have been identified as increasing susceptibility to MS. Many MS risk genes have altered expression in EBV infected B cells, dependent on the risk genotype, and are themselves regulated by the EBV transcription factor EBNA2. Females are 2-3 times more likely to develop MS than males. We investigated if MS risk loci might mediate the gender imbalance in MS. From a large public dataset, we identified gender-specific associations with EBV traits, and MS risk SNP/gene pairs with gender differences in their associations with gene expression. Some of these genes also showed gender differences in correlation of gene expression level with Estrogen Receptor 2. To test if estrogens may drive these gender specific differences, we cultured EBV infected B cells (lymphoblastoid cell lines, LCLs), in medium depleted of serum to remove the effects of sex hormones as well as the estrogenic effect of phenol red, and then supplemented with estrogen (100 nM estradiol). Estradiol treatment altered MS risk gene expression, LCL proliferation rate, EBV DNA copy number and EBNA2 expression in a sex-dependent manner. Together, these data indicate that there are estrogen-mediated gender-specific differences in MS risk gene expression and EBV functions. This may in turn contribute to gender differences in host response to EBV and to MS susceptibility.

The interaction of Epstein-Barr virus encoded transcription factor EBNA2 with multiple sclerosis risk loci is dependent on the risk genotype.

BACKGROUND: Epstein-Barr virus (EBV) infection may be necessary for the development of Multiple sclerosis (MS). Earlier we had identified six MS risk loci that are co-located with binding sites for the EBV transcription factor Epstein-Barr Nuclear Antigen 2 (EBNA2) in EBV-infected B cells (lymphoblastoid cell lines - LCLs). METHODS: We used an allele-specific chromatin immunoprecipitation PCR assay to assess EBNA2 allelic preference. We treated LCLs with a peptide inhibitor of EBNA2 (EBNA2-TAT), reasoning that inhibiting EBNA2 function would alter gene expression at these loci if it was mediated by EBNA2. FINDINGS: We found that EBNA2 binding was dependent on the risk allele for five of these six MS risk loci (p < 0·05). Treatment with EBNA2-TAT significantly altered the expression of TRAF3 (p < 0·05), CD40 (p < 0·001), CLECL1 (p <0·0001), TNFAIP8 (p < 0·001) and TNFRSF1A (p < 0·001). INTERPRETATION: These data suggest that EBNA2 can enhance or reduce expression of the gene depending on the risk allele, likely promoting EBV infection. This is consistent with the concept that these MS risk loci affect MS risk through altering the response to EBNA2. Together with the extensive data indicating a pathogenic role for EBV in MS, this study supports targeting EBV and EBNA2 to reduce their effect on MS pathogenesis. FUNDING: Funding was provided by grants from MS Research Australia, National Health and Medical Research Council of Australia, Australian Government Research Training Program, Multiple Sclerosis International Federation, Trish Multiple Sclerosis Research Foundation.

Quantitative neurogenetics: applications in understanding disease.

Neurodevelopmental and neurodegenerative disorders (NNDs) are a group of conditions with a broad range of core and co-morbidities, associated with dysfunction of the central nervous system. Improvements in high throughput sequencing have led to the detection of putative risk genetic loci for NNDs, however, quantitative neurogenetic approaches need to be further developed in order to establish causality and underlying molecular genetic mechanisms of pathogenesis. Here, we discuss an approach for prioritizing the contribution of genetic risk loci to complex-NND pathogenesis by estimating the possible impacts of these loci on gene regulation. Furthermore, we highlight the use of a tissue-specificity gene expression index and the application of artificial intelligence (AI) to improve the interpretation of the role of genetic risk elements in NND pathogenesis. Given that NND symptoms are associated with brain dysfunction, risk loci with direct, causative actions would comprise genes with essential functions in neural cells that are highly expressed in the brain. Indeed, NND risk genes implicated in brain dysfunction are disproportionately enriched in the brain compared with other tissues, which we refer to as brain-specific expressed genes. In addition, the tissue-specificity gene expression index can be used as a handle to identify non-brain contexts that are involved in NND pathogenesis. Lastly, we discuss how using an AI approach provides the opportunity to integrate the biological impacts of risk loci to identify those putative combinations of causative relationships through which genetic factors contribute to NND pathogenesis.

The Interaction of Human and Epstein-Barr Virus miRNAs with Multiple Sclerosis Risk Loci.

Although the causes of Multiple Sclerosis (MS) still remain largely unknown, multiple lines of evidence suggest that Epstein-Barr virus (EBV) infection may contribute to the development of MS. Here, we aimed to identify the potential contribution of EBV-encoded and host cellular miRNAs to MS pathogenesis. We identified differentially expressed host miRNAs in EBV infected B cells (LCLs) and putative host/EBV miRNA interactions with MS risk loci. We estimated the genotype effect of MS risk loci on the identified putative miRNA:mRNA interactions in silico. We found that the protective allele of MS risk SNP rs4808760 reduces the expression of hsa-mir-3188-3p. In addition, our analysis suggests that hsa-let-7b-5p may interact with ZC3HAV1 differently in LCLs compared to B cells. In vitro assays indicated that the protective allele of MS risk SNP rs10271373 increases ZC3HAV1 expression in LCLs, but not in B cells. The higher expression for the protective allele in LCLs is consistent with increased IFN response via ZC3HAV1 and so decreased immune evasion by EBV. Taken together, this provides evidence that EBV infection dysregulates the B cell miRNA machinery, including MS risk miRNAs, which may contribute to MS pathogenesis via interaction with MS risk genes either directly or indirectly.

Prospective validation study of prognostic biomarkers to predict adverse outcomes in patients with COVID-19: a study protocol.

INTRODUCTION: Accurate triage is an important first step to effectively manage the clinical treatment of severe cases in a pandemic outbreak. In the current COVID-19 global pandemic, there is a lack of reliable clinical tools to assist clinicians to perform accurate triage. Host response biomarkers have recently shown promise in risk stratification of disease progression; however, the role of these biomarkers in predicting disease progression in patients with COVID-19 is unknown. Here, we present a protocol outlining a prospective validation study to evaluate the biomarkers' performance in predicting clinical outcomes of patients with COVID-19. METHODS AND ANALYSIS: This prospective validation study assesses patients infected with COVID-19, in whom blood samples are prospectively collected. Recruited patients include a range of infection severity from asymptomatic to critically ill patients, recruited from the community, outpatient clinics, emergency departments and hospitals. Study samples consist of peripheral blood samples collected into RNA-preserving (PAXgene/Tempus) tubes on patient presentation or immediately on study enrolment. Real-time PCR (RT-PCR) will be performed on total RNA extracted from collected blood samples using primers specific to host response gene expression biomarkers that have been previously identified in studies of respiratory viral infections. The RT-PCR data will be analysed to assess the diagnostic performance of individual biomarkers in predicting COVID-19-related outcomes, such as viral pneumonia, acute respiratory distress syndrome or bacterial pneumonia. Biomarker performance will be evaluated using sensitivity, specificity, positive and negative predictive values, likelihood ratios and area under the receiver operating characteristic curve. ETHICS AND DISSEMINATION: This research protocol aims to study the host response gene expression biomarkers in severe respiratory viral infections with a pandemic potential (COVID-19). It has been approved by the local ethics committee with approval number 2020/ETH00886. The results of this project will be disseminated in international peer-reviewed scientific journals.

The interaction of Multiple Sclerosis risk loci with Epstein-Barr virus phenotypes implicates the virus in pathogenesis.

Translating the findings of genome wide association studies (GWAS) to new therapies requires identification of the relevant immunological contexts to interrogate for genetic effects. In one of the largest GWAS, more than 200 risk loci have been identified for Multiple Sclerosis (MS) susceptibility. Infection with Epstein-Barr virus (EBV) appears to be necessary for the development of Multiple Sclerosis (MS). Many MS risk loci are associated with altered gene expression in EBV infected B cells (LCLs). We have interrogated this immunological context to identify interaction between MS risk loci and EBV DNA copy number, intrinsic growth rate and EBV encoded miRNA expression. The EBV DNA copy number was associated with significantly more risk alleles for MS than for other diseases or traits. EBV miRNAs BART4-3p and BART3-5p were highly associated with EBV DNA copy number and MS risk loci. The poliovirus receptor (PVR) risk SNP was associated with EBV DNA copy number, PVR and miRNA expression. Targeting EBV miRNAs BART4-3p and BART3-5p, and the gene PVR, may provide therapeutic benefit in MS. This study also indicates how immunological context and risk loci interactions can be exploited to validate and develop novel therapeutic approaches.

Transcribed B lymphocyte genes and multiple sclerosis risk genes are underrepresented in Epstein-Barr Virus hypomethylated regions.

Epstein-Barr Virus (EBV) infection appears to be necessary for the development of Multiple Sclerosis (MS), although the specific mechanisms are unknown. More than 200 single-nucleotide polymorphisms (SNPs) are known to be associated with the risk of developing MS. About a quarter of these are also highly associated with proximal gene expression in B cells infected with EBV (lymphoblastoid cell lines-LCLs). The DNA of LCLs is hypomethylated compared with both uninfected and activated B cells. Since methylation can affect gene expression, and so cell differentiation and immune evasion, we hypothesised that EBV-driven hypomethylation may affect the interaction between EBV infection and MS. We interrogated an existing dataset comprising three individuals with whole-genome bisulfite sequencing data from EBV transformed B cells and CD40L-activated B cells. DNA methylation surrounding MS risk SNPs associated with gene expression in LCLs (LCLeQTL) was less likely to be hypomethylated than randomly selected chromosomal regions. Differential methylation was independent of genomic features such as promoter regions, but genes preferentially expressed in EBV-infected B cells, including the LCLeQTL genes, were underrepresented in the hypomethylated regions. Our data does not indicate MS genetic risk is affected by EBV hypomethylation.

Evidence from genome wide association studies implicates reduced control of Epstein-Barr virus infection in multiple sclerosis susceptibility.

BACKGROUND: Genome wide association studies have identified > 200 susceptibility loci accounting for much of the heritability of multiple sclerosis (MS). Epstein-Barr virus (EBV), a memory B cell tropic virus, has been identified as necessary but not sufficient for development of MS. The molecular and immunological basis for this has not been established. Infected B cell proliferation is driven by signalling through the EBV produced cell surface protein LMP1, a homologue of the MS risk gene CD40. METHODS: We have investigated transcriptomes of B cells and EBV-infected B cells at Latency III (LCLs) and identified MS risk genes with altered expression on infection and with expression levels associated with the MS risk genotype (LCLeQTLs). The association of LCLeQTL genomic burden with EBV phenotypes in vitro and in vivo was examined. The risk genotype effect on LCL proliferation with CD40 stimulation was assessed. RESULTS: These LCLeQTL MS risk SNP:gene pairs (47 identified) were over-represented in genes dysregulated between B and LCLs (p < 1.53 × 10-4), and as target loci of the EBV transcription factor EBNA2 (p < 3.17 × 10-16). Overall genetic burden of LCLeQTLs was associated with some EBV phenotypes but not others. Stimulation of the CD40 pathway by CD40L reduced LCL proliferation (p < 0.001), dependent on CD40 and TRAF3 MS risk genotypes. Both CD40 and TRAF3 risk SNPs are in binding sites for the EBV transcription factor EBNA2, with expression of each correlated with EBNA2 expression dependent on genotype. CONCLUSIONS: These data indicate targeting EBV may be of therapeutic benefit in MS.

The latitude-dependent autoimmune disease risk genes ZMIZ1 and IRF8 regulate mononuclear phagocytic cell differentiation in response to vitamin D.

Epidemiological, molecular and genetic studies have indicated that high serum vitamin D levels are associated with lower risk of several autoimmune diseases. The vitamin D receptor (VDR) binding sites in monocytes and dendritic cells (DCs) are more common in risk genes for diseases with latitude dependence than in risk genes for other diseases. The transcription factor genes Zinc finger MIZ domain-containing protein 1 (ZMIZ1) and interferon regulatory factor 8 (IRF8)-risk genes for many of these diseases-have VDR binding peaks co-incident with the risk single nucleotide polymorphisms (SNPs). We show these genes are responsive to vitamin D: ZMIZ1 expression increased and IRF8 expression decreased, and this response was affected by genotype in different cell subsets. The IL10/IL12 ratio in tolerogenic DCs increased with vitamin D. These data indicate that vitamin D regulation of ZMIZ1 and IRF8 in DCs and monocytes contribute to latitude-dependent autoimmune disease risk.

The Effects of Alpha Boswellic Acid on Reelin Expression and Tau Phosphorylation in Human Astrocytes.

Reelin is an extracellular glycoprotein which contributes to synaptic plasticity and function of memory in the adult brain. It has been indicated that the Reelin signaling cascade participates in Alzheimer's disease (AD). Besides the neurons, glial cells such as astrocytes also express Reelin protein. While functional loss of astrocytes has been reported to be associated with AD, dysfunction of astrocytic Reelin signaling pathway has not received much attention. Therefore, we investigated the effects of α-boswellic acid (ABA) as one of the major component of Boswellia serrata resin on primary fetal human astrocytes under a stress paradigm as a possible model for AD through study on Reelin cascade. For this aim, we used streptozotocin (STZ), in which from an outlook generates Alzheimer's hallmarks in astrocytes, and assayed Reelin expression, Tau and Akt phosphorylation as well as reactive oxygen species (ROS) generation and apoptosis in the presences of ABA. Our results indicated that while STZ (100 µM) down-regulated the expression of Reelin, ABA (25 µM) up-regulated its expression (p < 0.01) for 24 h. ABA efficiently reduced hyperphosphorylated Tau (Ser404) in STZ-treated astrocytes (p < 0.01). Furthermore, STZ-induced apoptosis by increasing cleaved caspase three (p < 0.01) and ROS generation (p < 0.01), a further pathological hallmark of Tauopathy. On the other hand, ABA decreased ROS generation and promoted proliferation of astrocytes through elevating Survivin expression (p < 0.01). These results showed that ABA could be considered as a potent therapeutic agent for prevention and decreasing the progression of Alzheimer's hallmarks in astrocytes; however, more in vivo studies would be needed.

Trichloroacetic acid treatment as a tricky way for rapid purification of 1N/4R tau protein.

Tau protein consists of six different isoforms and each one has particular physiological roles. In order to analyze the specific function of each single isoforms, large quantity of highly purified tau isoforms is essential. Many studies have been done to purify tau isoforms by heat treatment, followed by perchloric acid and glycerol precipitation. We found out that 1N/4R tau is soluble in glycerol, that is why mentioned methods do not work for purifying this isoform. In this study, large amounts of active and highly purified (97%) 1N/4R tau protein has been prepared by utilization of trichloroacetic acid as precipitating agent.