PpCas9 from Pasteurella pneumotropica - a compact Type II-C Cas9 ortholog active in human cells.

CRISPR-Cas defense systems opened up the field of genome editing due to the ease with which effector Cas nucleases can be programmed with guide RNAs to access desirable genomic sites. Type II-A SpCas9 from Streptococcus pyogenes was the first Cas9 nuclease used for genome editing and it remains the most popular enzyme of its class. Nevertheless, SpCas9 has some drawbacks including a relatively large size and restriction to targets flanked by an 'NGG' PAM sequence. The more compact Type II-C Cas9 orthologs can help to overcome the size limitation of SpCas9. Yet, only a few Type II-C nucleases were fully characterized to date. Here, we characterized two Cas9 II-C orthologs, DfCas9 from Defluviimonas sp.20V17 and PpCas9 from Pasteurella pneumotropica. Both DfCas9 and PpCas9 cleave DNA in vitro and have novel PAM requirements. Unlike DfCas9, the PpCas9 nuclease is active in human cells. This small nuclease requires an 'NNNNRTT' PAM orthogonal to that of SpCas9 and thus potentially can broaden the range of Cas9 applications in biomedicine and biotechnology.

DNA targeting by Clostridium cellulolyticum CRISPR-Cas9 Type II-C system.

Type II CRISPR-Cas9 RNA-guided nucleases are widely used for genome engineering. Type II-A SpCas9 protein from Streptococcus pyogenes is the most investigated and highly used enzyme of its class. Nevertheless, it has some drawbacks, including a relatively big size, imperfect specificity and restriction to DNA targets flanked by an NGG PAM sequence. Cas9 orthologs from other bacterial species may provide a rich and largely untapped source of biochemical diversity, which can help to overcome the limitations of SpCas9. Here, we characterize CcCas9, a Type II-C CRISPR nuclease from Clostridium cellulolyticum H10. We show that CcCas9 is an active endonuclease of comparatively small size that recognizes a novel two-nucleotide PAM sequence. The CcCas9 can potentially broaden the existing scope of biotechnological applications of Cas9 nucleases and may be particularly advantageous for genome editing of C. cellulolyticum H10, a bacterium considered to be a promising biofuel producer.

Diffuse Reflectance Spectroscopy of the Oral Mucosa: In Vivo Experimental Validation of the Precancerous Lesions Early Detection Possibility.

This article is devoted to the experimental validation of the possibility of early detection of precancerous lesions in the oral mucosa in vivo using diffuse reflectance spectroscopy in the wavelength range from 360 to 1000 nm. During the study, a sample of 119 patients with precancerous lesions has been collected and analyzed. As a result of the analysis, the most informative wavelength ranges were determined, in which the maximum differences in the backscattering spectra of lesions and intact tissues were observed, methods for automatic classification of backscattering spectra of the oral mucosa were studied, sensitivity and specificity values, achievable using diffuse reflectance spectroscopy for detecting hyperkeratosis on the tongue ventrolateral mucosa surface and buccal mucosa, were evaluated. As a result of preliminary experimental studies in vivo, the possibility of automatic detection of precancerous lesions of the oral mucosa surface using diffuse reflectance spectroscopy in the wavelength range from 500 to 900 nm with an accuracy of at least 75 percent has been shown.

Responsive Neurostimulation of the Thalamus for the Treatment of Refractory Epilepsy.

Introduction: One-third of patients with epilepsy continue to have seizures despite antiepileptic medications. Some of these refractory patients may not be candidates for surgical resection primarily because the seizure onset zones (SOZs) involve both hemispheres or are located in eloquent areas. The NeuroPace Responsive Neurostimulation System (RNS) is a closed-loop device that uses programmable detection and stimulation to tailor therapy to a patient's individual neurophysiology. Here, we present our single-center experience with the use of RNS in thalamic nuclei to provide long-term seizure control in patients with refractory epilepsy. Methods: We performed a prospective single-center study of consecutive refractory epilepsy patients who underwent RNS system implantation in the anterior (ANT) and centromedian (CM) thalamic nuclei from September 2015 to December 2020. Patients were followed postoperatively to evaluate seizure freedom and complications. Results: Twenty-three patients underwent placement of 36 RNS thalamic leads (CM = 27 leads, ANT = 9 leads). Mean age at implant was 18.8 ± 11.2 years (range 7.8-62 years-old). Two patients (8.7%) developed infections: 1 improved with antibiotic treatments alone, and 1 required removal with eventual replacement of the system to recover the therapeutic benefit. Mean time from RNS implantation to last follow-up was 22.3 months. Based on overall reduction of seizure frequency, 2 patients (8.7%) had no- to <25% improvement, 6 patients (26.1%) had 25-49% improvement, 14 patients (60.9%) had 50-99% improvement, and 1 patient (4.3%) became seizure-free. All patients reported significant improvement in seizure duration and severity, and 17 patients (74%) reported improved post-ictal state. There was a trend for subjects with SOZs located in the temporal lobe to achieve better outcomes after thalamic RNS compared to those with extratemporal SOZs. Of note, seizure etiology was syndromic in 12 cases (52.2%), and 7 patients (30.4%) had undergone resection/disconnection surgery prior to thalamic RNS therapy. Conclusion: Thalamic RNS achieved ≥50% seizure control in ~65% of patients. Infections were the most common complication. This therapeutic modality may be particularly useful for patients affected by aggressive epilepsy syndromes since a young age, those whose seizure foci are located in the mesial temporal lobe, and those who have failed prior surgical interventions.

Percutaneous cephalocaudal implantation of epidural stimulation electrodes over sacral nerve roots--a technical note on the importance of the lateral approach.

OBJECTIVE: Idiopathic overactive bladder, urgency-frequency syndromes, interstitial cystitis, pudendal neuralgia, vulvodynia, prostadynia, and coccygodynia have been effectively treated with sacral nerve root modulation. This is most commonly performed with placement of electrodes via a transforaminal approach, predominately to the S3 foramen. This approach is limited by a high lead migration rate and the limitations of stimulating a single nerve root. Beginning in the 1990s, some centers began pursuing retrograde percutaneous placement from the lumbar spine, but adoption of this technique was limited by the technical difficulty of the approach. METHODS AND MATERIALS: We developed a lateral approach to the epidural space for these procedures, which has improved the learning curve and facilitated the application of the retrograde technique. In this technical note, we describe the "laterograde" approach. RESULTS: A focused description of the technique is presented in this technical report. The epidural space is accessed from lateral to medial with the bevel facing up to keep the approach to the "shingled" lumbar laminae shallow. CONCLUSION: The "laterograde" modification of cephalocaudal approach appears to be technically less difficult method for accomplishing sacral nerve root stimulator electrode placement over multiple roots.

Strength and Fracture Mechanism of an Ultrafine-Grained Austenitic Steel for Medical Applications.

In this paper, we study the corrosion-resistant austenitic steel Fe-0.02C-18Cr-8Ni for medical applications. The microstructure and mechanical properties (tensile mechanical properties, torsional strength, impact toughness, and static and cyclic crack resistance) under different types of loading of the steel are investigated. The results are compared for the two states of the steel: the initial (coarse-grained) state and the ultrafine-grained state produced by severe plastic deformation processing via equal-channel angular pressing. It is demonstrated that the ultrafine-grained steel 0.08C-18Cr-9Ni has essentially better properties and is very promising for the manufacture of medical products for various applications that experience various static and cyclic loads during operation.

Analysis of the results obtained by national research groups with the Russian state cancer program for the screening of diseases of the oral mucosa and the vermillion border.

AIMS AND OBJECTIVES: The purpose of the article is to analyze the results obtained by the national research groups with the Russian State Cancer Program for the screening of diseases of the oral mucosa and the vermillion border. This article analyzes the results obtained by national research groups in the implementation of the Russian State Cancer Program. BACKGROUND: Based on the Department of Therapeutic Dentistry of A.I. Yevdokimov Moscow State University of Medicine and Dentistry, national research groups were created to implement the state program draft for the screening of oncological and precancerous diseases of the oral mucosa and the vermillion border - a grant of the "A" format to support applied research on a competitive basis in the form of subsidies of the federal target program. MATERIALS AND METHODS: From 2015 to the first half of 2019, the national clinical groups studied the epidemiology (prevalence rate and structure) of oncological and precancerous diseases of the oral mucosa and the vermillion border in 486,059 persons aged 18-92 years living in Tula, the Tula region, Moscow and Noyabrsk as a result of active pathology detection during targeted examination of the population (screening) and appealability in the preclinical period, early, timely and late detection. RESULTS: the results of the research confirm low cancer alertness of practicing dentists. An individual patient registration card has been developed to systematize data and create a diagnostic register for the screening of cancer and precancerous diseases of the oral mucosa and the vermillion border. CONCLUSION: A project has been developed to implement the Russian State Cancer Program for oncological and precancerous diseases of the oral mucosa among the population of the Russian Federation.

Relaxin-expressing, fiber chimeric oncolytic adenovirus prolongs survival of tumor-bearing mice.

Selective replication of oncolytic viruses in tumor cells provides a promising approach for the treatment of human cancers. One of the limitations observed with oncolytic viruses currently used in the treatment of solid tumors is the inefficient spread of virus throughout the tumor mass following intratumoral injection. Data are presented showing that oncolytic adenoviruses expressing the relaxin gene and containing an Ad5/Ad35 chimeric fiber showed significantly enhanced transduction and increased virus spread throughout the tumor when compared with non-relaxin-expressing, Ad5-based viruses. The increased spread of such viruses throughout tumors correlated well with improved antitumor efficacy and overall survival in two highly metastatic tumor models. Furthermore, nonreplicating viruses expressing relaxin did not increase metastases, suggesting that high level expression of relaxin will not enhance metastatic spread of tumors. In summary, the data show that relaxin may play a role in rearranging matrix components within tumors, which helps recombinant oncolytic adenoviruses to spread effectively throughout the tumor mass and thereby increase the extent of viral replication within the tumor. Expressing relaxin from Ad5/Ad35 fiber chimeric adenoviruses may prove a potent and novel approach to treating patients with cancer.

Superior Strength and Multiple Strengthening Mechanisms in Nanocrystalline TWIP Steel.

The strengthening mechanism of the metallic material is related to the hindrance of the dislocation motion, and it is possible to achieve superior strength by maximizing these obstacles. In this study, the multiple strengthening mechanism-based nanostructured steel with high density of defects was fabricated using high-pressure torsion at room and elevated temperatures. By combining multiple strengthening mechanisms, we enhanced the strength of Fe-15 Mn-0.6C-1.5 Al steel to 2.6 GPa. We have found that solute segregation at grain boundaries achieves nanograined and nanotwinned structures with higher strength than the segregation-free counterparts. The importance of the use of multiple deformation mechanism suggests the development of a wide range of strong nanotwinned and nanostructured materials via severe plastic deformation process.

PEGylated IL-10 Activates Kupffer Cells to Control Hypercholesterolemia.

Interleukin-10 (IL-10) is a multifunctional cytokine that exerts potent context specific immunostimulatory and immunosuppressive effects. We have investigated the mechanism by which PEGylated rIL-10 regulates plasma cholesterol in mice and humans. In agreement with previous work on rIL-10, we report that PEGylated rIL-10 harnesses the myeloid immune system to control total plasma cholesterol levels. We have discovered that PEG-rMuIL-10's dramatic lowering of plasma cholesterol is dependent on phagocytotic cells. In particular, PEG-rHuIL-10 enhances cholesterol uptake by Kupffer cells. In addition, removal of phagocytotic cells dramatically increases plasma cholesterol levels, suggesting for the first time that immunological cells are implicitly involved in regulating total cholesterol levels. These data suggest that treatment with PEG-rIL-10 potentiates endogenous cholesterol regulating cell populations not currently targeted by standard of care therapeutics. Furthermore, we show that IL-10's increase of Kupffer cell cholesterol phagocytosis is concomitant with decreases in liver cholesterol and triglycerides. This leads to the reversal of early periportal liver fibrosis and facilitates the restoration of liver health. These data recommend PEG-rIL-10 for evaluation in the treatment of fatty liver disease and preventing its progression to non-alcoholic steatohepatitis. In direct confirmation of our in vivo findings in the treatment of hypercholesterolemic mice with PEG-rMuIL-10, we report that treatment of hypercholesterolemic cancer patients with PEG-rHuIL-10 lowers total plasma cholesterol by up to 50%. Taken together these data suggest that PEG-rIL-10's cholesterol regulating biology is consistent between mice and humans.

In vitro and in vivo pharmacology and pharmacokinetics of a human engineered monoclonal antibody to epithelial cell adhesion molecule.

ING-1(heMAb), a Human Engineered monoclonal antibody to epithelial cell adhesion molecule (Ep-CAM), was evaluated for its in vitro and in vivo activity. The dissociation constant of ING-1(heMAb) for binding to Ep-CAM on HT-29 human colon tumor cells was 2 to 5 nM, similar to chimeric ING-1. In antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays, ING-1(heMAb) caused a concentration-dependent lysis of BT-20 breast, MCF-7 breast, HT-29 colon, and CACO-2 colon tumor cells, with maximum cytolysis at approximately 1 microg/ml. After an intravenous injection in rats, plasma ING-1(heMAb) levels declined with an alpha half-life of 8 to 11 hours, and a beta half-life of 20 days, typical of an IgG in a species without the target for ING-1. In nude mice with human HT-29 colon tumors, plasma ING-1(heMAb) levels declined more rapidly than in non-tumor-bearing mice, suggesting an enhanced clearance via the tumor-associated human Ep-CAM. In nude mice, intravenous treatments with ING-1(heMAb) twice a week for 3 weeks significantly suppressed the growth of human HT-29 colon and PC-3 prostate tumors in a dose-dependent manner, with 1.0 mg/kg providing the greatest benefit. These results indicate that Human Engineered ING-1(heMAb) is a high-affinity antibody with potent in vitro activity that targets and suppresses the growth of human tumors in vivo.

Dynamical study of tyrosine hydroxylase expression and its correlation with vasopressin turnover in the magnocellular neurons of the supraoptico-posthypophysial system under long-term salt loading of adult rats.

Using immunocytochemistry, in situ hybridization and image analysis, we attempted to compare the dynamical expression of tyrosine hydroxylase (TH) and vasopressin (VP) mRNAs and proteins in the magnocellular neurons of the supraoptic nucleus in rats drinking 2% NaCl for 1, 2 and 3 weeks. Three stages in the reaction of VPergic neurons have been distinguished. The initial stage (first week) showed a synchronous activation of TH and VP mRNAs and protein expression as well as an increased number of TH-immunoreactive neurons. The next stage (second week) was characterized by a further increase in the number of TH-immunoreactive neurons. The number of VPergic neurons also increased significantly. Although the TH and VP mRNAs levels fell during the second week of osmotic stimulation, the TH content increased significantly, and the VP content remained at the same level. During the last stage (third week), TH-immunoreactive neurons increased in number and were as numerous as VP-immunoreactive neurons in intact rats. These data suggest that, finally, all the VPergic neurons begin to synthesize TH. The concentrations of VP and TH mRNAs did not change during the third week of osmotic stimulation, while the VP and TH contents increased. Thus, our study shows that there is a correlation between TH expression and VP expression and suggests similar mechanisms for the regulation of VP and TH gene expression and synthesis during long-term osmotic stimulation.

Periotestometry and tissue hydration. How are these indicators related?

The mobility of teeth in terms of periotestometry and hydration of periodontal tissues in terms of bioimpedance spectrometry were studied in healthy volunteers without dental and concomitant somatic diseases. It was found that teeth of these subjects have both low and pathological mobility. It was also revealed as increased hydration (edema), indicating the presence of inflammation, and reduced hydration, indicating atrophic processes in periodontal tissues. A comparison of the data of periotestometry and bioimpedance spectrometry showed that the indicators of mobility of the teeth and hydration of the periodontal tissues reflect different characteristics of the functional state of dentoalveolar system, and can be used as complementary in the diagnostic studies.

Neonatal CSF oxytocin levels are associated with parent report of infant soothability and sociability.

Oxytocin (OT) has been linked to social behavior in rodents, non-human primates, and adult humans, but almost nothing is known about brain OT activity in human newborns or its impact on social development. To better understand the role of OT biology in human social functioning, a multi-disciplinary, longitudinal study was conducted. Cerebral spinal fluid (CSF) OT levels from 18 human neonates were evaluated and examined in relationship to social-seeking behavior at term, at 3 months, and at 6 months of age. Higher neonatal CSF OT levels were consistently associated with solicitation of parental soothing and interest in social engagement with others. This is the first study to link CSF OT levels to normative human social functioning. Research is now required to test whether early OT levels serve as a biomarker for subsequent social abnormalities.

Percutaneous peripheral nerve stimulation.

Since its inception in the 1970s, peripheral neuromodulation has become an increasingly common procedure to treat chronic neuropathic disorders. Historically, peripheral nerve stimulation (PNS) originated with the placement of large surface cuff electrodes, which was refined by the introduction of functional nerve mapping with circumferential electrical stimulation. This substantially improved the targeting of sensory fascicles. Surgical placement of spinal cord stimulation (SCS) 'button type' paddle electrodes was replaced when the introduction of percutaneous cylindrical SCS electrodes expanded the spectrum of PNS applications and improved the ability to target afferent sensory fibers as well as reducing the complication rate. To further refine functional mapping for the placement of these percutaneous electrodes, radiofrequency needle probes have more recently been employed to elicit paresthesias in awake patients to map the pain generators and guide treatment. In this chapter, we provide a description of the development and basic mechanisms of peripheral nerve stimulation, as well as a more detailed description of the two most commonly employed forms of peripheral nerve stimulation: occipital nerve stimulation for occipital neuralgia, and subcutaneous peripheral nerve field stimulation to stimulate free nerve endings within the subcutaneous tissue when the pain is limited to a small, well-localized area. The closely related ideas of internal and external targeted subcutaneous stimulation are also discussed.

Feasibility of anterior cervical discectomy and fusion as an outpatient procedure.

BACKGROUND: Anterior cervical discectomy and fusion (ACDF) procedures are increasingly being managed on an outpatient basis. Currently there are no definitive guidelines within the literature that delineate which patient population can safely be managed as such. The purpose of this study is to demonstrate that ACDF procedures, within a selective patient population at our institution, can be safely performed on an outpatient basis. METHODS: This is a retrospective chart review within one physician's practice of patients undergoing instrumented ACDF procedures using allograft. This sample included 117 patients who underwent one- and two-level ACDF procedures from November 2005 to April 2009. Hospital length of stay and hospital readmissions were noted. Complication rates in the outpatient population were assessed to determine the feasibility of outpatient management for selective patients undergoing ACDF procedures. RESULTS: A total of 59 patients (50%) were treated on an outpatient basis. Sixty-eight patients underwent single level ACDF procedures, 38 patients (56%) of which were discharged on the same day. Forty-nine patients underwent two-level ACDF procedures, 21 patients (43%) of which were discharged on the same day. There was one complication (1.4%) in patients who were discharged on the same day. That patient required readmission for 23-hour observation secondary to neck swelling. CONCLUSIONS: ACDF procedures involving single and two-level fusions can safely be performed on an outpatient basis. Complication rates associated with this procedure are low, with critical postoperative complications involving respiratory compromise occurring very infrequently and in the immediate postoperative period.