New anticoagulant protein from medicinal leech.

The saliva of the medicinal leech contains various anticoagulants. Some of them, such as hirudin, are well known. However, it is reasonable to believe that not all anticoagulant proteins from medicinal leech saliva have been identified. We previously performed a comprehensive study of the transcriptome, genome, and proteome of leech salivary gland cells, which led to the discovery of several previously unknown hypothetical proteins that may have anticoagulant properties. Subsequently, we obtained a series of recombinant proteins and investigated their impact on coagulation in in vitro assays. We identified a previously undescribed protein that exhibited a high ability to suppress coagulation. The His-tagged recombinant protein was expressed in Escherichia coli and purified using metal chelate chromatography. To determine its activity, commonly used coagulation methods were used: activated partial thromboplastin time, prothrombin time, and thrombin inhibition clotting assay. Clotting and chromogenic assays for factor Xa inhibition were performed to evaluate anti-Xa activity. We used recombinant hirudin as a control anticoagulant protein in all experiments. The new protein showed significantly greater inhibition of coagulation than hirudin at the same molar concentrations in the activated partial thrombin time assay. However, hirudin demonstrated better results in the direct thrombin inhibition test, although the tested protein also exhibited the ability to inhibit thrombin. The chromogenic analysis of factor Xa inhibition revealed no activity, whereas the clotting test for factor Xa showed the opposite result. Thus, a new powerful anticoagulant protein has been discovered in the medicinal leech. This protein is homologous to antistatin, with 28 % identical amino acid residues. The recombinant protein was expressed in E. coli. This protein is capable of directly inhibiting thrombin, and based on indirect evidence, other proteases of the blood coagulation cascade have been identified.

Upregulation of YciM Expression Reduces Endotoxin Contamination of Recombinant Proteins Produced in Escherichia coli Cells.

Recombinant proteins produced in Escherichia coli are often contaminated with endotoxins, which can be a serious problem for their further application. One of the possible solutions is the use of modified strains with reduced lipopolysaccharide (LPS) levels. We compared two approaches to engineering such strains. The first commonly known approach was modification of LPS biosynthesis pathway by knocking out seven genes in the E. coli genome. The second approach, which has not been previously used, was to increase expression of E. coli protein YciM. According to the published data, elevated expression of YciM leads to the reduction in the amount of the LpxC enzyme involved in LPS biosynthesis. We investigated the impact of YciM coexpression with eGFP on the content of endotoxins in the purified recombinant eGFP samples. Both approaches provided similar outcomes, i.e., decreased the endotoxin levels in the purified protein samples.

TACSTD2 in gelatinous drop-like corneal dystrophy: variant functional analysis and expression in the cornea after limbal stem cell transplantation.

Gelatinous drop-like corneal dystrophy (GDLD) is a rare autosomal recessive eye disease. GDLD is characterized by the loss of barrier function in corneal epithelial cells (CECs) and amyloid deposition due to pathogenic variants in the TACSTD2 gene. Limbal stem cell transplantation (LSCT) has been suggested as an effective therapeutic alternative for patients with GDLD. However, despite LSCT, amyloid deposition recurs in some patients. The pathogenesis of recurrence is poorly studied. We present the case of a patient with GDLD. Genetic analysis revealed a homozygous deletion, NM_002353.3:c.653del, in the TACSTD2 gene. Functional analysis in a cell model system revealed the loss of the transmembrane domain and subcellular protein mislocalization. The patient with GDLD underwent direct allogeneic LSCT with epithelial debridement followed by deep anterior lamellar keratoplasty 10 months later due to amyloid deposition and deterioration of vision. Taken together, the results of transcriptome analysis and immunofluorescence staining of post-LSCT corneal sample with amyloid deposits obtained during keratoplasty demonstrated complete restoration of wild-type TACSTD2 expression, indicating that donor CECs replaced host CECs. Our study provides experimental evidence that amyloid deposition can recur after LSCT despite complete restoration of wild-type TACSTD2 expression.

Therapy-induced secretion of spliceosomal components mediates pro-survival crosstalk between ovarian cancer cells.

Ovarian cancer often develops resistance to conventional therapies, hampering their effectiveness. Here, using ex vivo paired ovarian cancer ascites obtained before and after chemotherapy and in vitro therapy-induced secretomes, we show that molecules secreted by ovarian cancer cells upon therapy promote cisplatin resistance and enhance DNA damage repair in recipient cancer cells. Even a short-term incubation of chemonaive ovarian cancer cells with therapy-induced secretomes induces changes resembling those that are observed in chemoresistant patient-derived tumor cells after long-term therapy. Using integrative omics techniques, we find that both ex vivo and in vitro therapy-induced secretomes are enriched with spliceosomal components, which relocalize from the nucleus to the cytoplasm and subsequently into the extracellular vesicles upon treatment. We demonstrate that these molecules substantially contribute to the phenotypic effects of therapy-induced secretomes. Thus, SNU13 and SYNCRIP spliceosomal proteins promote therapy resistance, while the exogenous U12 and U6atac snRNAs stimulate tumor growth. These findings demonstrate the significance of spliceosomal network perturbation during therapy and further highlight that extracellular signaling might be a key factor contributing to the emergence of ovarian cancer therapy resistance.

Discovery of novel antimicrobial peptides: A transcriptomic study of the sea anemone Cnidopus japonicus.

As essential conservative component of the innate immune systems of living organisms, antimicrobial peptides (AMPs) could complement pharmaceuticals that increasingly fail to combat various pathogens exhibiting increased resistance to microbial antibiotics. Among the properties of AMPs that suggest their potential as therapeutic agents, diverse peptides in the venoms of various predators demonstrate antimicrobial activity and kill a wide range of microorganisms. To identify potent AMPs, the study reported here involved a transcriptomic profiling of the tentacle secretion of the sea anemone Cnidopus japonicus. An in silico search algorithm designed to discover toxin-like proteins containing AMPs was developed based on the evaluation of the properties and structural peculiarities of amino acid sequences. The algorithm revealed new proteins of the anemone containing antimicrobial candidate sequences, and 10 AMPs verified using high-throughput proteomics were synthesized. The antimicrobial activity of the candidate molecules was experimentally estimated against Gram-positive and -negative bacteria. Ultimately, three peptides exhibited antimicrobial activity against bacterial strains, which suggests that the method can be applied to reveal new AMPs in the venoms of other predators as well.

Corrigendum: Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery.

[This corrects the article on p. 2 in vol. 7, PMID: 28144586.].

Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery.

The only recognized virulence factor of enterotoxigenic Bacteroides fragilis (ETBF) that accompanies bloodstream infections is the zinc-dependent non-lethal metalloprotease B. fragilis toxin (BFT). The isolated toxin stimulates intestinal secretion, resulting in epithelial damage and necrosis. Numerous publications have focused on the interrelation of BFT with intestinal inflammation and colorectal neoplasia, but nothing is known about the mechanism of its secretion and delivery to host cells. However, recent studies of gram-negative bacteria have shown that outer membrane vesicles (OMVs) could be an essential mechanism for the spread of a large number of virulence factors. Here, we show for the first time that BFT is not a freely secreted protease but is associated with OMVs. Our findings indicate that only outer surface-exposed BFT causes epithelial cell contact disruption. According to our in silico models confirmed by Trp quenching assay and NMR, BFT has special interactions with outer membrane components such as phospholipids and is secreted during vesicle formation. Moreover, the strong cooperation of BFT with polysaccharides is similar to the behavior of lectins. Understanding the molecular mechanisms of BFT secretion provides new perspectives for investigating intestinal inflammation pathogenesis and its prevention.

Complete Genome Sequence of an Enterotoxigenic Bacteroides fragilis Clinical Isolate.

Here we present the complete genome sequence of Bacteroides fragilis isolate BOB25. It is an enterotoxigenic isolate that was obtained from a stool sample of a patient with dysbiosis.