In Vivo DYSF Gene Viral Delivery Provides a Histoprotective Effect in Skeletal Muscle Tissue in Dysferlin-Deficient Mice.

We studied the effects of a dual-vector DYSF gene delivery system based on adeno-associated virus serotype 9 capsids on pathological manifestations of dysferlinopathy in skeletal muscles of Bla/J mice lacking DYSF expression. The mice received intravenous injection of 3×1013 genomic copies of the virus containing the dual-vector system. M. gastrocnemius, m. psoas major, m. vastus lateralis, and m. gluteus superficialis were isolated for histological examination in 3, 6, and 12 weeks after treatment. Healthy wild-type (C57BL/6) mice served as positive control and were sacrificed 3 weeks after injection of 150 µl of 0.9% NaCl into the caudal vein. To detect dysferlin in muscle cryosections, immunohistochemical analysis with diagnostic antibodies was performed; paraffin sections were stained with hematoxylin and eosin for morphometric analysis. After administration of gene-therapeutic constructs, muscle fibers with membrane or cytoplasmic dysferlin location were detected in all examined muscles. The proportion of necrotic muscle fibers decreased, the number of muscle fibers with central location of the nucleus increased, and the mean cross-section area of the muscle fibers decreased.

[Editing of Phage Genomes - Recombineering-Assisted SpCas9 Modification of Model Coliphages T7, T5, and T3].

Bacteriophages-viruses that infect bacterial cells - are the most abundant biological entities on Earth. The use of phages in fundamental research and industry requires tools for precise manipulation of their genomes. Yet, compared to bacterial genome engineering, modification of phage genomes is challenging because of the lack of selective markers and thus requires laborious screenings of recombinant/mutated phage variants. The development of the CRISPR-Cas technologies allowed to solve this issue by the implementation of negative selection that eliminates the parental phage genomes. In this manuscript, we summarize current methods of phage genome engineering and their coupling with CRISPR-Cas technologies. We also provide examples of our successful application of these methods for introduction of specific insertions, deletions, and point mutations in the genomes of model Escherichia coli lytic phages T7, T5, and T3.

[Metastatic basal cell carcinoma with a SMO gene mutation. A case report]. / Metastaticheskaya forma bazal'no-kletochnoi kartsinomy s mutatsiei v gene SMO. Opisanie nablyudeniya.

The authors describe a clinical case of a patient with metastatic basal cell carcinoma and secondary resistance to vismodegib. A SMO G416E mutation of unknown clinical significance was found in the gene encoding the transmembrane receptor protein Smoothened (SMO) protein, which suggests a defect in the Sonic Hedgehog (SH) pathway and may cause tumor resistance to the prescribed drug. Comprehensive genomic profiling and in silico analysis have been used to diagnose the tumor.

[New possibilities in antibacterial treatment of intra-abdominal infections caused by multiresistant microflora]. / Novye vozmozhnosti antibakterial'noi terapii intraabdominal'nykh infektsii, vyzvannykh polirezistentnoi mikrobnoi floroi.

AIM: To determine the place of new drugs with activity against multidrug resistant strains of microorganisms in the treatment of complicated intraabdominal infections. MATERIAL AND METHODS: The incidence and distribution of pathogens isolated from intra-abdominal specimens in patients with intra-abdominal infections are analyzed. RESULTS: The current situation on the growth of resistant strains among pathogens causing intra-abdominal infections is rewied. New combined drugs for the treatment of multidrug resistant infections - ceftolozane/tazobactam and ceftazidim/avibactam plus metronidazole, has been suggested. Their potential role in empiric and targeted antibacterial treatment of complicated intraabdominal infections is defined. CONCLUSION: Taking into consideration local monitoring data and risk factors of multi resistant strains Ceftolozane/tazobactam in combination with metronidazole can be used in empiric regime of treatment. Due to the high activity on carbapenem resistant strains of Klebsiella pneumonia and the lack of alternatives, it is advisable to use Ceftazidim/avibactam for the targeted therapy.

[Compression anastomoses with nikelid-titanium rings in peritonitis]. / Kompressionnye anastomozy nikelid-titanovymi kol'tsami pri peritonite.

AIM: To introduce into clinical practice compression anastomosis with nikelid-titanium rings (NTR) with shape memory in advanced peritonitis patients who need for intestinal resections. MATERIAL AND METHODS: Seventy-six procedures of interintestinal anastomoses with NTR (Günter-Ziganshin implant) have been analyzed for the period 2011-2017. Interventions were carried out for widespread peritonitis. In all cases a unified technique of anastomosis was used by applying an implant in the form of a 'clerical clip'. There were following procedures: gastroenterostomy resection for its failure - 5; small intestine resection followed by interntestinal anastomosis - 36; ileotransverso- or colocolostomy - 35. RESULTS: Mean time of enteroenterostomy was 2.6±0.98 min. If elective re-laparomy was supposed, laparostomy was formed followed by sanation within 48 hours. In 55% of patients 3-5-fold elective sanations were performed. Adequacy of compression anastomoses was assessed clinically according to the nature of drainage output and visually during redo surgery. There were 11 (14.5%) deaths: 9 (12%) patients had APACHE-II score over 20. Macro- and microscopic analysis of intestinal specimens and interintestinal anastomoses with nikelid-titanium rings showed complete impermeability and no visual defects. Microscopically neutrophilic leukocyte infiltration and increased number of fibroblasts were observed as the evidence of regenerative processes activation. Implant rejection time was 7.8±1.4 days. CONCLUSION: The use of compression anastomoses in case of advanced peritonitis reduces the risk of anastomosis failure to 3% and decreases time of surgery that reduces intraoperative aggression and improves outcomes in patients with prognostically unfavorable outcomes.

Unconsidered sporadic sources of carbon dioxide emission from soils in taiga forests.

Long-term monitoring in the Russian taiga zone has shown that all known extreme destructive effects resulting in the weakening and death of tree stands (windfalls, pest attacks, drought events, etc.) can be sporadic, but significant sources of CO2 soil emission. Among them are (i) a recently found effect of the multiyear CO2 emission from soil at the bottom of deadwood of spruce trees that died due to climate warming and subsequent pest outbreaks, (ii) increased soil CO2 emissions due to to the fall of tree trunks during massive windfalls, and (iii) pulse CO2 emission as a result of the so-called Birch effect after drought events in the taiga zone. According to the modeling, while depending on the spatial and temporal scales of their manifestation, the impact of these sporadic effects on the regional and global soil respiration fluxes could be significant and should be taken into consideration. This is due to continuing Climate Change, and further increase of local, regional and Global human impacts on the atmospheric greenhouse gases balance, and land use, as well.

[First experience with total laparoscopic reconstructive operations in occlusive lesion of the aortoiliac arterial segment]. / Pervyi opyt total'nykh laparoskopicheskikh rekonstruktivnykh operatsii pri okkliuzionnom porazhenii aortopodvzdoshnogo arterial'nogo segmenta.

According to the TASC II and the Russian National Guidelines on management of patients with lower limb arterial diseases, in patients with type C and D lesions of the arterial bed performing aortofemoral bifurcation bypass grafting is preferable. Laparoscopic technique makes it possible to combine advantages of minimally invasive surgery with well-known remote results of open reconstructive operations on the aortoiliac segment. The study included a total of 54 patients undergoing treatment at the Cardiosurgical Department of Clinic No 1 of the Volgograd State Medical University over the period from January 2012 to September 2015. The examined patients were subdivided into two groups. Group One was composed of the initial 26 patients operated on during the period from January 2012 to April 2014. Group Two comprised the remaining 28 patients operated on during the period from April 2014 to September 2015. Safety of performing the intervention was determined by prediction of the postoperative lethality and complications rates by means of the V-POSSUM scale. The predicted lethality and complications rates for both groups amounted to 2.3 and 23.5%, respectively. We carried out a comparative analysis of intraoperative data such as duration of the operative intervention, duration of mobilization of the aortoiliac segment, time of aortic cross-clamping, volume of intraoperative blood loss, intraoperative complications rate, conversion in the open access. In the early postoperative period we analysed the level of lethality, complications rate and indices of postoperative rehabilitation of the patient. For demonstrativeness of the dynamics of alterations of intraoperative indices, as well as for plotting the 'learning curve' the moving average method was used. Analysing the obtained findings revealed that implementation of total laparoscopic aortofemoral reconstructive operations was not accompanied by either high lethality or great number of complications, not exceeding the predicted indices by the V-POSSUM scale. The average duration of the operation in Group I amounted to 346±18.3 min, and in Group II to 316±13.3 min, with the time of aortic cross-clamping averaging 80±10.3 and 61±4.2 min and the volume of blood loss 898±23.5 ml, respectively. As experience was gained in performing laparoscopic reconstructive operations in Group Two patients the 'learning curve' demonstrated a statistically significant decrease of these intraoperative values, as well as a decrease in the complication rate and parameters of the patient's state in the postoperative period. Due to absence of extensive laparotomic or retroperitoneal accesses, the early postoperative period was characterised by rapid restoration of the passage along the intestine, early activation of patients, short length of stay in the intensive care unit and hospital stay.

Peroxy-Titanium Complex-based inks for low temperature compliant anatase thin films.

Stable highly crystalline titanium dioxide colloids are of paramount importance for the establishment of a solution-processable library of materials that could help in bringing the advantages of digital printing to the world of photocatalysis and solar energy conversion. Nano-sized titanium dioxide in the anatase phase was synthesized by means of hydrothermal methods and treated with hydrogen peroxide to form Peroxy-Titanium Complexes (PTCs). The influence of hydrogen peroxide on the structural, optical and rheological properties of titanium dioxide and its colloidal solutions were assessed and a practical demonstration of a low temperature compliant digitally printed anatase thin film given.

First experience of hematopoietic stem cell transplantation treatment of Shwachman-Diamond syndrome using unaffected HLA-matched sibling donor produced through preimplantation HLA typing.

The only proven cure for Shwachman-Diamond syndrome (SDS) bone marrow failure is allogeneic hematopoietic stem cell transplantation (HSCT). However HSCT with donors other than HLA-identical siblings is associated with high mortality and unfavorable prognosis. This paper presents the first experience of HSCT treatment of SDS using an unaffected HLA-identical sibling produced through preimplantation genetic diagnosis (PGD). The patient was a 6-year-old blood transfusion-dependent SDS baby girl with secondary myelodysplastic syndrome, for whom no HLA-identical donor was available. As a result of PGD, two unaffected HLA matched embryos were identified; one of them was randomly selected for transfer, resulting in a clinical pregnancy and birth of an apparently healthy child. The patient underwent allogeneic transplantation of cord blood hematopoietic stem cells, together with bone marrow from this sibling, resulting in complete hemopoietic recovery. The patient was no longer transfusion-dependent and had normal blood values 160 days after transplantation.

[Five-year results of treating patients with chronic lower limb ischaemia by means of gene engineering]. / Pyatiletnie rezul'taty lecheniya bol'nykh khronicheskoi ishemiei nizhnikh konechnostei s ispol'zovaniem gennoi terapii.

The authors share their experience in comprehensive conservative treatment of patients presenting with chronic lower limb ischaemia (CLLI) associated with atherosclerosis of peripheral arteries by means of the first Russian registered gene therapeutic agent "Neovasculgen" (plasmid with the vegf165 gene), analysing the long-term outcomes of treating a total of 45 patients with stage II and III CLLI according to the classification of Pokrovsky-Fontain. The patients were followed up for 5 years. Efficacy of treatment was assessed by registering the dynamics of the pain-free walking distance (PFWD), linear blood velocity (LBV), ankle-brachial index (ABI), as well as the limb salvage rate and survival of patients. All patients showed good tolerance of treatment, with neither side effects nor complications noted. Clinical improvement in stage IIB CLLI was observed in 91% of patients with complete stabilization of the clinical course during 5 years. The limb salvage rate in this group amounted to 95%, with the survival rate equalling 82%. In patients with stage III CLLI, improvement was noted in 78% of cases, manifesting itself by a decrease of its degree to stage IIB (44.4%) and to stage IIA (33.3%). Progression of CLLI followed by amputation was registered in 22% of cases, with the survival rate of 78%. Hence, the use of a single course of combined treatment including the gene therapeutic agent "Neovasculgen" in patients with stage II and III CLLI resulted in a persistent positive effect in a considerable majority of patients in the remote period of not less than 5 years.

World's First Clinical Case of Gene-Activated Bone Substitute Application.

Treatment of patients with large bone defects is a complex clinical problem. We have initiated the first clinical study of a gene-activated bone substitute composed of the collagen-hydroxyapatite scaffold and plasmid DNA encoding vascular endothelial growth factor. The first patient with two nonunions of previously reconstructed mandible was enrolled into the study. Scar tissues were excised; bone defects (5-14 mm) between the mandibular fragments and nonvascularized rib-bone autograft were filled in with the gene-activated bone substitute. No adverse events were observed during 12 months of follow-up. In 3 months, the average density of newly formed tissues within the implantation zone was 402.21 ± 84.40 and 447.68 ± 106.75 HU in the frontal and distal regions, respectively, which correlated with the density of spongy bone. Complete distal bone defect repair with vestibular and lingual cortical plates formation was observed in 6 and 12 months after surgery; thereby the posterior nonunion was successfully eliminated. However, there was partial resorption of the proximal edge of the autograft entailed to relapse of the anterior nonunion. Thus, the first clinical data on the safety and efficacy of the gene-activated bone substitute were obtained. Given a high complexity of the clinical situation the treatment, results might be considered as promising. NCT02293031.

[Role of correction of the syndrome of intestinal failure and abdominal hypertension in the prevention of infection of pancreatic necrosis]. / Rol' korrektsii sindromov kishechnoi nedostatochnosti i vnutribryushnoi gipertenzii v profilaktike infitsirovaniya pankreonekroza.

UNLABELLED: The number of patients with acute pancreatitis and pancreatic necrosis has been steadily increasing. Mortality in infected pancreatic necrosis remains high. AIM: To develop measures to prevent infection of pancreatic necrosis by timely correction of intra-abdominal hypertension and the syndrome of intestinal failure. MATERIAL AND METHODS: Developed a package of measures, consisting of early intestinal lavage and enterosorption, intravenous highdoses of octreotide, epidural blockade, adequate detox and the start of effective antimicrobial therapy. Comparative evaluation of clinical, laboratory and instrumental data in the primary (n=50) and control (n=50) groups. RESULTS: In the main group pancreatogenic sepsis occurred in 10%, control 18%. Mortality, respectively, was 8 and 16%.

[PAD prevalence in Russian Federation]. / Rasprostranennost' simptomnoi ishemii nizhnikh konechnostei v Rossiiskoi Federatsii.

BACKGROUND: Currently there is no data on peripheral arterial disease prevalence in Russia. AIM: To estimate prevalence of patients with symptomatic low limb ischemia (LLI) in Russian Federation on 2014. PATIENTS AND METHODS: «League for patient' protection¼ had sent questionnaires for all regional health departments asking to name prevalence of ICD-10 codes I70.2 (atherosclerosis of arteries of extremities) and I73.1 (thromboangiitis obliterans (Buerger)) on 2014. The figures were listed separately for ischemia I-IIa and III-IV (critical limb ischemia, CLI) (Pokrovsky, 1976). Also were mentioned major amputations and mortality. RESULTS: Total number of received questionnaires was 44, we've got information from all RF' federal regions with a population of appr. 71 million people. There were 174 125 cases of symptomatic LLI. Prevalence of symptomatic LLI among Russian population aged more than 40 years was 0.51%. Prevalence of intermittent claudication was 0.36% and of critical limb ischemia - 0.13%. Rate of major amputations was 6.9%. CONCLUSION: World PAD prevalence among people older than 25 years is 8.3%. The same figure in our study was only 0.5%. This is due to the fact that we considered only symptomatic LLI and only with codes I70.2 and I73.1. Prevalence of symptomatic patients among all PAD patients is 10-35%. So the real number of PAD patients in Russian might be 1.5-5%. We can conclude that PAD is still unrevealed in Russia. Another conclusion could be that the treatment results are less than satisfactory. Finally there is urgent necessity for special PAD register in RF.

[Remote results of treatment of patients with chronic lower-limb ischaemia by means of indirect revascularization and gene therapy].

Presented herein are comparative remote results of combined surgical treatment of 121 patients with stage IIB-III lower limb chronic ischaemia (LLCI) by means of indirect revascularization (lumbar sympathectomy--LSE and revascularizing osteotrepanation of the tibial bone--ROT) and gene therapy using the first registered Russian gene therapeutic agent Neovasculgen®. Depending on the LLCI degree and the method of treatment, during 3 years we assessed such parameters as the limb salvage rate, pain-free walk distance (PFWD), ankle-brachial index (ABI) and linear blood velocity (LBV). An increase in the PFWD in patients with initial stage IIB LLCI in the group of gene therapy was considerably higher than in other types of treatment (p=0.0001-0.0004). Using indirect methods of revascularization was accompanied and followed by less positive alterations in the PFWD values which by the end of the third year of follow up were observed to decrease. The values of PFWD after ROT at 2 and 3 years were higher than after LSE (p=0.006). During the first year of follow up the highest increment of the ABI was observed after ROT. At two years, the ABI values after ROT and gene therapy became equal. The worst result during 3 years as compared with other methods of treatment was demonstrated by LSE (p=0.006). Changes in ABI after gene therapy were statistically significant at all terms of follow up (p=0.008-0.02). There were no limb amputations in the remote period of follow up in patients with the initial stage IIB of the disease. Patients with initial stage III LLCI also showed a considerably better result by the increment of increased PFWD after gene-therapeutic treatment (p=0.001-0.0005). A small increment of the PFWD after LSE maintained during 1 year and after LSE during 2 years. The ABI values in all periods of follow up were higher after gene therapy (p=0.01-0.003). During the 2- and 3-year period the increment of this parameter after ROT was more significant than after LSE (p=0.046-0.05). Changes in the ABI after gene therapy at all terms also turned out to be more substantial (p=0.03-0.008). An increase in the LBV during the first and second years after ROT turned out more significant as compared to LSE (p=0.05). The limb salvage rate in patients with initial stage III LLCI during the whole period after gene therapy amounted to 78%, after ROT being 54% and after LSE equalling 45%.

Role of Protein L25 and Its Contact with Protein L16 in Maintaining the Active State of Escherichia coli Ribosomes in vivo.

A ribosomal protein of the L25 family specifically binding to 5S rRNA is an evolutionary feature of bacteria. Structural studies showed that within the ribosome this protein contacts not only 5S rRNA, but also the C-terminal region of protein L16. Earlier we demonstrated that ribosomes from the ΔL25 strain of Escherichia coli have reduced functional activity. In the present work, it is established that the reason for this is a fraction of functionally inactive 50S ribosomal subunits. These subunits have a deficit of protein L16 and associate very weakly with 30S subunits. To study the role of the contact of these two proteins in the formation of the active ribosome, we created a number of E. coli strains containing protein L16 with changes in its C-terminal region. We found that some mutations (K133L or K127L/K133L) in this protein lead to a noticeable slowing of cell growth and decrease in the activity of their translational apparatus. As in the case of the ribosomes from the ΔL25 strain, the fraction of 50S subunits, which are deficient in protein L16, is present in the ribosomes of the mutant strains. All these data indicate that the contact with protein L25 is important for the retention of protein L16 within the E. coli ribosome in vivo. In the light of these findings, the role of the protein of the L25 family in maintaining the active state of the bacterial ribosome is discussed.

Ordinary and Activated Bone Grafts: Applied Classification and the Main Features.

Bone grafts are medical devices that are in high demand in clinical practice for substitution of bone defects and recovery of atrophic bone regions. Based on the analysis of the modern groups of bone grafts, the particularities of their composition, the mechanisms of their biological effects, and their therapeutic indications, applicable classification was proposed that separates the bone substitutes into "ordinary" and "activated." The main differential criterion is the presence of biologically active components in the material that are standardized by qualitative and quantitative parameters: growth factors, cells, or gene constructions encoding growth factors. The pronounced osteoinductive and (or) osteogenic properties of activated osteoplastic materials allow drawing upon their efficacy in the substitution of large bone defects.

[Efficacy of using VEGF165 gene in comprehensive treatment of patients with stage 2A-3 lower limb chronic ischaemia].

The authors share their experience in treating a total of 100 patients presenting with stage 2A-3 chronic lower limb ischaemia according to the classification of Pokrovsky-Fontain (the clinical group was composed of 75 patients and the control group comprised 25 subjects), in whom it was impossible to perform surgical revascularization. The clinical group patients received in addition to the conventional vascular therapy local intramuscular injections of Neovasculogen (plasmid genetic construction containing human gene VEGF165) at a course dose of 2.4 mg. The results were assessed after 1 year. It was shown that administration of this gene therapeutic agent is safe with no local or systemic allergic reactions and free form neoplastic processes. Efficacy of treatment was assessed by registering the pain-free walking distance (PFWD), transcutaneous oxygen tension (TCPO2), linear velocity of blood flow, ankle-brachial index (ABI), angiography, and by means of SF-36 questionnaire. It was determined that after 12 months the statistical significance of intergroup and intragroup differences was reached for PFWD (increment 167.2%), TCPO2 (increment 20.4%). The highest clinical response for the PFWD was registered in patients with stage 3 of the disease (547.5%), as well as in those with multi-storey vascular lesions (269.1%). The obtained findings make it possible to consider gene therapy with Neovasculogen as an efficient component of comprehensive treatment of this cohort of patients.

[Therapeutic angiogenesis in treatment of patients with chronic obliterating diseases of lower limb arteries].

The authors share their experience gained in realization of the concept of "therapeutic angiogenesis" by means of the first Russian gene therapeutic agent Neovasculogen in comprehensive treatment of patients with stage II-III chronic obliterating disease of lower limb arteries (CODLLA) according to the Pokrovsky-Fontain classification. A total of 45 patients were treated. The findings were assessed and analyzed depending on the stage of chronic lower limb ischaemia. The clinical results including such measures such as the pain-free walk (PFW), ankle/brachial index (ABI), transcutaneously determined oxygen tension (TDOT), linear blood velocity (LBV) were followed up for 2 years. All patients were found to have good tolerance and at terms up to 2 years, and no side effect or complications were revealed. Patients with stage IIB of the disease after one year demonstrated statistically significant results suggesting improved blood circulation in the ischemized lower limbs after using the agent according to the indices of PFW, TDOT, LBV (p<0.05). In stage III of the disease? significant results were observed for PFD and TODT (p<0.05). Considerable part of patients with stage IIB and III CODDLLA (84.4%) showed increased physical capabilities for ambulation at the expense of considerably increased PFW (p<0.01). It was observed that using the agent was enough for obtaining a persistent satisfactory result preserving for not less than one year, with 50% of patients from stage IIB and 16.7% from stage III transferred to stage IIA of the disease. The effect of treatment persisted during the second year of follow up.

[Worldwide experience and recent trends in gene therapy of ischaemic diseases].

Fifteen odd years have passed since the first application of a gene-therapeutic modality in clinical practice for treatment of lower-limb chronic ischaemia. Over this time, vast experience has been gained worldwide, with not less than one thousand patients treated by gene-based therapies, thus making it possible to generalise the published findings of these clinical trials. Resulting from such an analysis, it should be recognized that the least dangerous gene therapeutic modalities available so far are plasmid ones, with the most efficient being those containing the gene of vascular endothelial growth factor VEGF(165). The most convincing results were obtained while treating chronic ischemia of the lower extremities, whereas gene-based therapy used for treatment of coronary artery disease failed to have yielded, as of yet, clear cut positive results.