Prokaryotic Argonaute nuclease cooperates with co-encoded RNase to acquire guide RNAs and target invader DNA.

Argonautes are an evolutionary conserved family of programmable nucleases that identify target nucleic acids using small guide oligonucleotides. In contrast to eukaryotic Argonautes (eAgos) that act on RNA, most studied prokaryotic Argonautes (pAgos) recognize DNA targets. Similarly to eAgos, pAgos can protect prokaryotic cells from invaders, but the biogenesis of guide oligonucleotides that confer them specificity to their targets remains poorly understood. Here, we have identified a new group of RNA-guided pAgo nucleases and demonstrated that a representative pAgo from this group, AmAgo from the mesophilic bacterium Alteromonas macleodii, binds guide RNAs of varying lengths for specific DNA targeting. Unlike most pAgos and eAgos, AmAgo is strictly specific to hydroxylated RNA guides containing a 5'-adenosine. AmAgo and related pAgos are co-encoded with a conserved RNA endonuclease from the HEPN superfamily (Ago-associated protein, Agap-HEPN). In vitro, Agap cleaves RNA between guanine and adenine nucleotides producing hydroxylated 5'-A guide oligonucleotides bound by AmAgo. In vivo, Agap cooperates with AmAgo in acquiring guide RNAs and counteracting bacteriophage infection. The AmAgo-Agap pair represents the first example of a pAgo system that autonomously produces RNA guides for DNA targeting and antiviral defense, which holds promise for programmable DNA targeting in biotechnology.

DNA-targeting short Argonautes complex with effector proteins for collateral nuclease activity and bacterial population immunity.

Two prokaryotic defence systems, prokaryotic Argonautes (pAgos) and CRISPR-Cas, detect and cleave invader nucleic acids using complementary guides and the nuclease activities of pAgo or Cas proteins. However, not all pAgos are active nucleases. A large clade of short pAgos bind nucleic acid guides but lack nuclease activity, suggesting a different mechanism of action. Here we investigate short pAgos associated with a putative effector nuclease, NbaAgo from Novosphingopyxis baekryungensis and CmeAgo from Cupriavidus metallidurans. We show that these pAgos form a heterodimeric complex with co-encoded effector nucleases (short prokaryotic Argonaute, DNase and RNase associated (SPARDA)). RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA by plasmids or phages results in degradation of cellular DNA and cell death or dormancy, conferring target-specific population protection and expanding the range of known prokaryotic immune systems.

Bacterial Argonaute nucleases reveal different modes of DNA targeting in vitro and in vivo.

Prokaryotic Argonaute proteins (pAgos) are homologs of eukaryotic Argonautes (eAgos) and are also thought to play a role in cell defense against invaders. However, pAgos are much more diverse than eAgos and little is known about their functional activities and target specificities in vivo. Here, we describe five pAgos from mesophilic bacteria that act as programmable DNA endonucleases and analyze their ability to target chromosomal and invader DNA. In vitro, the analyzed proteins use small guide DNAs for precise cleavage of single-stranded DNA at a wide range of temperatures. Upon their expression in Escherichia coli, all five pAgos are loaded with small DNAs preferentially produced from plasmids and chromosomal regions of replication termination. One of the tested pAgos, EmaAgo from Exiguobacterium marinum, can induce DNA interference between homologous sequences resulting in targeted processing of multicopy plasmid and genomic elements. EmaAgo also protects bacteria from bacteriophage infection, by loading phage-derived guide DNAs and decreasing phage DNA content and phage titers. Thus, the ability of pAgos to target multicopy elements may be crucial for their protective function. The wide spectrum of pAgo activities suggests that they may have diverse functions in vivo and paves the way for their use in biotechnology.

Specific targeting of plasmids with Argonaute enables genome editing.

Prokaryotic Argonautes (pAgos) are programmable nucleases involved in cell defense against invading DNA. In vitro, pAgos can bind small single-stranded guide DNAs to recognize and cleave complementary DNA. In vivo, pAgos preferentially target plasmids, phages and multicopy genetic elements. Here, we show that CbAgo nuclease from Clostridium butyricum can be used for genomic DNA engineering in bacteria. We demonstrate that CbAgo loaded with plasmid-derived guide DNAs can recognize and cleave homologous chromosomal loci, and define the minimal length of homology required for this targeting. Cleavage of plasmid DNA at an engineered site of the I-SceI meganuclease increases guide DNA loading into CbAgo and enhances processing of homologous chromosomal loci. Analysis of guide DNA loading into CbAgo also reveals off-target sites of I-SceI in the Escherichia coli genome, demonstrating that pAgos can be used for highly sensitive detection of double-stranded breaks in genomic DNA. Finally, we show that CbAgo-dependent targeting of genomic loci with plasmid-derived guide DNAs promotes homologous recombination between plasmid and chromosomal DNA, depending on the catalytic activity of CbAgo. Specific targeting of plasmids with Argonautes can be used to integrate plasmid-encoded sequences into the chromosome thus enabling genome editing.

Strong temperature effects on the fidelity of target DNA recognition by a thermophilic pAgo nuclease.

Prokaryotic Argonaute (pAgo) proteins are programmable nucleases with great promise in genetic engineering and biotechnology. Previous studies identified several DNA-targeting pAgo nucleases from mesophilic and thermophilic prokaryotic species that are active in various temperature ranges. However, the effects of temperature on the specificity of target recognition and cleavage by pAgos have not been studied. Here, we describe a thermostable pAgo nuclease from the thermophilic bacterium Thermobrachium celere, TceAgo. We show that TceAgo preferentially uses 5'-phosphorylated small DNA guides and can perform specific cleavage of both single-stranded and double-stranded DNA substrates in a wide range of temperatures. Single-nucleotide mismatches between guide and target molecules differently change the reaction efficiency depending on the mismatch position, with the fidelity of target recognition greatly increased at elevated temperatures. Thus, TceAgo can serve as a tool to allow specific detection and cleavage of DNA targets in a temperature-dependent manner. The results demonstrate that the specificity of programmable nucleases can be strongly affected by the reaction conditions, which should be taken into account when using these nucleases in various in vitro and in vivo applications.

Successful Surgical Treatment of a Patient with Synchronic Intraductal Papillary Mucinous Carcinoma and Solid Pseudopapillary Neoplasm of the Pancreas.

Intraductal papillary mucinous neoplasm (IPMN) and solid pseudopapillary neoplasm (SPN) are uncommon cystic tumors of the pancreas. The simultaneous occurrence of these 2 pancreatic neoplasms is extremely rare. We present a case of synchronous solid SPN and IPMN - carcinoma in a 44-year-old female patient. An uncommon surgical treatment was performed - pancreatoduodenectomy with the resection of the pancreas body, resection of the mesentericoportal trunk + distal pancreatectomy, and splenectomy. Part of the pancreatic tissue was preserved.

Two-stage combined treatment of leiomyosarcoma of iliac vein using robotic surgery.

Vascular leiomyosarcoma is a rare tumor originating from the vascular smooth muscle cells. Leiomyosarcomas of the external iliac vein are extremely rare. Only single observations of this tumor are reported. We describe a 48-year-old woman who underwent a two-step combined treatment: robot-assisted removal of the extraperitoneal tumor and removal of the lower part of the tumor from the open femoral approach. There have been no signs of tumor recurrence during the 34-month follow-up period.

Surgical treatment of liver echinococcosis and alveococcosis.

Parasitic liver zoonoses are endemic to some regions of Russia as well as to Mediterranean countries, Australia, New Zealand, South America, and the Indian subcontinent. However, the available data on the surgical treatment of patients with parasitic liver diseases are often contradictory, and such treatments remain a difficult task today. The effectiveness of surgical treatment was analyzed in 628 patients with echinococcosis and 58 patients with liver alveococcosis managed at the Republican Clinical Hospital during 1998-2018. The most commonly performed surgery for echinococcosis was closed echinococcectomy of different types, which was applied in 428 patients. Surgical outcomes were evaluated according to the type of surgery. Although there were no differences in outcomes in the immediate postoperative period, long-term postoperative outcomes differed with the type of surgery, compelling us to reconsider the surgical methods employed, with a preference for implementing cystectomy with omentoplasty of the residual cavity of the liver. In fact, the best outcomes in terms of recurrence and complications were obtained with minimal but adequate procedures, namely actual echinococcectomy without the resection of the residual cavity or liver. Thus procedures with low-traumatic access should be performed in cases with specific indications, such as the presence of easily accessible and well-visualized parasitic cysts.

Proinsulin-Secreting Neuroendocrine Tumors of the Pancreas: A Single-Centre Experience.

BACKGROUND: Proinsulinoma is a neuroendocrine tumor (NET) of the pancreas that produces prohormone proinsulin. These tumors are very rare. In the literature, they are most often presented in the form of case reports. MATERIALS AND METHODS: We studied 177 patients with NET of the pancreas who underwent surgical treatment in the A.V. Vishnevsky National Medical Research Centre of Surgery from January 2007 to December 2018. RESULTS: Of 81 patients with organic hyperinsulinism caused by functioning NETs of the pancreas during the study period, 3 (3.7%) had a proinsulinoma; 2 were female; and 1 was male. None of them admitted to weight gain during this period, and their BMI was normal. All patients presented with Whipple's triad during the 72-h fast. Tumor-enucleating surgery was performed: one robot assisted, two laparotomies. A normal glucose level after treatment was achieved in all cases. CONCLUSION: In cases where clinical hypoglycemia is present, but the serum insulin level is within the normal range or even decreased, proinsulinoma should be suspected. For now, surgical resection remains the only effective method of treatment. Further investigation of pro-insulinomas is needed.

Changes in cardiovascular risk factors among adolescents from 1995 to 2004 in the Republic of Karelia, Russia.

BACKGROUND: In Russia, cardiovascular mortality is among the highest in the world. Behaviours related to the development of cardiovascular disease are usually adopted in childhood and adolescence. Very little information exists on prevalence and trends of risk factors among Russian youth. This study aims to investigate changes in the prevalence of cardiovascular risk factors among adolescents in the Republic of Karelia, Russia, from 1995 to 2004. METHODS: Cross-sectional surveys on chronic disease risk factors were carried out among 15-year-old adolescents in Pitkäranta region, in the Republic of Karelia. The surveys were conducted in all 10 secondary schools in the Pitkäranta region. All ninth-grade students in 1995 (N = 385, response rate 95%) and in 2004 (N = 395, response rate 85%) were included in the survey samples. RESULTS: Systolic blood pressure decreased statistically significantly among boys (from 119 to 116 mmHg). Diastolic blood pressure decreased statistically significantly among both girls (from 64 to 59 mmHg) and boys (from 62 to 59 mmHg). Total cholesterol increased statistically significantly only among girls (from 3.9 to 4.1 mmol/l). Body mass index did not exhibit any significant changes in both genders. Daily smoking rate doubled statistically significantly among girls from 7% to 15%. CONCLUSION: The study results show changes in the prevalence of cardiovascular disease risk factors among adolescents in the Republic of Karelia occurring over the last decade. Active measures need to be taken to prevent the increase in smoking prevalence, especially among girls, and to avert the unfavourable development of other risk factors in the future.