Large-Scale Production of Anti-RNase A VHH Expressed in pyrG Auxotrophic Aspergillus oryzae.

Nanobodies, also referred to as VHH antibodies, are the smallest fragments of naturally produced camelid antibodies and are ideal affinity reagents due to their remarkable properties. They are considered an alternative to monoclonal antibodies (mAbs) with potential utility in imaging, diagnostic, and other biotechnological applications given the difficulties associated with mAb expression. Aspergillus oryzae (A. oryzae) is a potential system for the large-scale expression and production of functional VHH antibodies that can be used to meet the demand for affinity reagents. In this study, anti-RNase A VHH was expressed under the control of the glucoamylase promoter in pyrG auxotrophic A. oryzae grown in a fermenter. The feature of pyrG auxotrophy, selected for the construction of a stable and efficient platform, was established using homologous recombination. Pull-down assay, size exclusion chromatography, and surface plasmon resonance were used to confirm the binding specificity of anti-RNase A VHH to RNase A. The affinity of anti-RNase A VHH was nearly 18.3-fold higher (1.9 nM) when expressed in pyrG auxotrophic A. oryzae rather than in Escherichia coli. This demonstrates that pyrG auxotrophic A. oryzae is a practical, industrially scalable, and promising biotechnological platform for the large-scale production of functional VHH antibodies with high binding activity.

Expression and characterization of recombinant IL-1Ra in Aspergillus oryzae as a system.

BACKGROUND: The interleukin-1 receptor antagonist (IL-1Ra) is a crucial molecule that counteracts the effects of interleukin-1 (IL-1) by binding to its receptor. A high concentration of IL-1Ra is required for complete inhibition of IL-1 activity. However, the currently available Escherichia coli-expressed IL-1Ra (E. coli IL-1Ra, Anakinra) has a limited half-life. This study aims to produce a cost-effective, functional IL-1Ra on an industrial scale by expressing it in the pyrG auxotroph Aspergillus oryzae. RESULTS: We purified A. oryzae-expressed IL-1Ra (Asp. IL-1Ra) using ion exchange and size exclusion chromatography (53 mg/L). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that Asp. IL-1Ra is N-glycosylated and approximately 17 kDa in size. We conducted a comparative study of the bioactivity, binding kinetics, and half-life between Asp. IL-1Ra and E. coli IL-1Ra. Asp. IL-1Ra showed good bioactivity even at a low concentration of 0.5 nM. The in vitro half-life of Asp. IL-1Ra was determined for different time points (0, 24, 48, 72, and 96 h) and showed higher stability than E. coli IL-1Ra, despite exhibiting a 100-fold lower binding affinity (2 nM). CONCLUSION: This study reports the production of a functional Asp. IL-1Ra with advantageous stability, without extensive downstream processing. To our knowledge, this is the first report of a recombinant functional and stable IL-1Ra expressed in A. oryzae. Our results suggest that Asp. IL-1Ra has potential for industrial-scale production as a cost-effective alternative to E. coli IL-1Ra.

The influence of voxel size and artifact reduction on the detection of vertical root fracture in endodontically treated teeth.

OBJECTIVE: The aim of this study was to evaluate the effect of voxel size and artefact reduction (AR) on the identification of vertical root fractures (VRFs) in endodontically treated teeth. METHODS: A total of a hundred sound, extracted human mandibular single-rooted premolars were decoronated, after which root canal preparation was performed, canals were filled with gutta percha by single cone technique. Randomly selected fifty specimens were fractured, repositioned and glued together. The teeth were examined with cone beam computed tomography (CBCT) in five different voxel sizes (0.125, 0.200, 0.250, 0.300, and 0.400 voxels). Two scans were performed for each tooth, one with AR and one without AR. Two radiologists evaluated the CBCT scans. RESULTS: All voxel dimensions were successful in detecting VRFs in CBCT scans. But as the voxel size increased, the percentage of detecting VRFs decreased. High accuracy, sensitivity, specificity and predictive values were found for VRF detection on CBCT scans. Accuracy and sensitivity values decreased (from 100 to 82) while voxel dimensions increased (from 0.125 to 0.400). High-resolution images (0.125, 0.200, and 0.250 voxels) caused an increase in sensitivity for detection of VRFs. AR did not affect the accuracy, sensitivity, specificity and predictive values for VRF detection on CBCT scans. CONCLUSIONS: High-resolution CBCT images resulted in an increase in sensitivity and specificity for detection of VRFs compared with lower-resolution CBCT images. The use of AR did not further improve its diagnostic potential.

Structure of active ß-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide.

The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of ß-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of ß-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate ß-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of ß-arrestin-1. The structure of the ß-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in ß-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of ß-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on ß-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.

Facilitators and barriers to referral compliance among dental patients with increased risk of obstructive sleep apnea.

OBJECTIVE: Our aims were to determine the adherence rate to dentist referrals for sleep apnea evaluation and the barriers and facilitators to referral compliance. MATERIAL AND METHODS: A sample of 1099 patients was screened with the STOP-Bang questionnaire. Those with elevated risk were referred for a sleep evaluation. An interview was conducted over the phone to determine compliance to referral and the barriers and facilitators to compliance. RESULTS: Of the 1099 patients (mean age: 45.1 ± 10 years) screened, 224 (20.4%) patients were determined to be at-risk for obstructive sleep apnea (OSA). Only 41 (18.3%) patients with increased risk adhered to referral recommendation. Demographic and health characteristics did not show significant differences between the compliant and non-compliant patients. The most common facilitators to compliance were increased awareness about OSA (N = 25, 65%) and dentist recommendation (N = 14, 34.1%), whereas the most common barriers to referral compliance were misconceptions about OSA (N = 69, 37.7%) and work responsibilities (N = 44, 24%). CONCLUSIONS: Only a small percentage of patients adhered to the recommendation of their dentist to see a sleep specialist. Increased awareness about OSA and dentist recommendation were the most common factors that facilitated compliance, whereas misconceptions about OSA and work responsibilities were the most common barriers to patient compliance.

Implant-Site Related and Patient-Based Factors With the Potential to Impact Patients' Satisfaction, Quality of Life Measures and Perceptions Toward Dental Implant Treatment.

OBJECTIVES: The present study aimed at evaluating both the implant site-related and patient-based factors with the potential to affect the extent of patients' satisfaction and also their perceptions regarding dental implant treatment. Potential differences between the esthetic evaluations of dental patients and dental specialists were also considered. MATERIALS AND METHODS: Implant-supported fixed prosthesis (n = 164) in 264 anterior esthetic implant sites were included. Patients' satisfaction, esthetic considerations, and perceptions toward dental implants, were evaluated by both Oral Health Impact Profile-14 (OHIP-14) questionnaire and visual analog scale. Pink Esthetic Score/White Esthetic Score were used for the professional esthetic evaluations of dental specialists. RESULTS: Overall patient satisfaction was high (87.42 ± 11.86). Compared with implant supported single-tooth restorations, patients with implant-supported bridges had lower OHIP scores (P = 0.001) and were relatively less satisfied with particular aspects of dental implant treatment (eg, cleanability, phonetics, surgical discomfort, and pretreatment information). Generally, type of prosthesis, history of soft/hard tissue augmentation, and reason for tooth loss had a clear impact on the extent of patients' satisfaction, esthetic considerations regarding treatment outcome, and their perceptions toward dental implant treatment (P < 0.05). CONCLUSIONS: Fixed implant-supported restorations generally provide with high levels of satisfaction and oral health-related quality of life.

Management of Limited Vertical Bone Height in the Posterior Mandible: Short Dental Implants Versus Nerve Lateralization With Standard Length Implants.

Inferior alveolar nerve lateralization (IANL) and short dental implants (SDI) are 2 viable implant-based treatment approaches in the presence of atrophied posterior mandible. Despite the risks of dysfunction, infection, and pathologic fractures in IANL, it becomes possible to place standard implants. The purpose of this study was to compare SDI and IANL approaches from clinical and radiographic aspects. Fifteen subjects having unilateral atrophic mandibles were allocated to SDI and IANL treatment groups. Following surgical procedures, early postoperative complications, implant survival, and periimplant clinical and radiographic parameters including probing pocket depth, attachment level, keratinized tissue amount, vertical tissue recession, and marginal bone loss were recorded at baseline and 1-year after prosthetic rehabilitation. In both groups, no implant was lost. Except usual postoperative complications, 2 patients had transient paraesthesia after IANL. According to time-dependent evaluation, both groups showed significant increase in probing pocket depth and attachment level at 1-year follow-up compared with baseline (P < 0.05). Except a slight but significant increase in mesial surface of SDI group (P < 0.05), no remarkable time-dependent change was identified in vertical tissue recession. Keratinized tissue amount did not exhibit any inter- or intragroup difference during whole study period. Marginal bone loss did not show any difference between IANL and SDI groups at follow-up. SDI placement or standard length implant placement with IANL can be considered promising alternatives in the treatment of atrophic mandibular posterior regions. However, SDI may be preferred in terms of lower complication risk.

Radiographic Fractal and Clinical Resonance Frequency Analyses of Posterior Mandibular Dental Implants: Their Possible Association With Mandibular Cortical Index With 12-Month Follow-up.

BACKGROUND: The aim of the present study was to investigate whether the mandibular cortical index (MCI) has a relationship with fractal dimension of bone and/or implant stability, and to justify the possible association between MCI, fractal dimension, and stability. MATERIALS AND METHODS: Eighty-two subjects who received dental implants to replace missing mandibular premolar/molar sites were selected. Three months after surgical placement, implants were restored with fixed ceramic fused metal crowns. MCI was evaluated at baseline; fractal dimensions were measured with fractal analysis (FA) and implant stability quotient (ISQ) with resonance frequency analysis immediately after surgery and 12-month follow-up. RESULTS: FA at mesial and distal regions for Class 1, Class 2, and Class 3 MCI resulted with significant increases at 12-month follow-up compared to baseline. The ISQ in patients with Class 2 and Class 3 MCI resulted with a significant decrease compared to Class 1 MCI at baseline and at 12 months. All MCI classes evaluated with ISQ and FA at baseline values resulted with significant increases at 12 months. Significant correlations were considered for all mandibular posterior implants between baseline and 12-month measurements for ISQ and FA evaluations. CONCLUSIONS: Fractal analysis may be a useful method for understanding the healing process around implants and implant stability quotient values. Mandibular cortical index evaluations should be considered before implant procedures, which may provide a presurgical treatment plan and may provide information about the mandibular bone quality.

Evaluation of the fungiform papillae number in Behçet's disease.

OBJECTIVES: The aim of this study was to evaluate the number, area, circumference, and circularity of lingual fungiform papillae and taste sensitivity in Behçet's disease (BD) and the correlation with the disease. MATERIALS AND METHODS: Eighty-six Behçet's patients and 78 healthy control subjects were examined. After taking digital photographs from the tongue, the fungiform papillae evaluations were performed for the right and left tongue tip sites separately. Taste sense was evaluated with an electrogustometer (EG) from the same sites. RESULTS: The ANOVA test was used to evaluate the fungiform papillae number difference between BD and control groups, and the difference was statistically significant. The fungiform papillae number was significantly reduced in BD, 37.93% at the left site and 40.13% at the right site compared to controls. There was no significant difference in papillae area, circumference, circularity, and EG measurements between BD and control group. CONCLUSION: According to these findings, it is conceivable that the reduction of the fungiform papillae number could be a sign of BD. CLINICAL RELEVANCE: BD has different clinical features that are used for the diagnosis. This study provided a new possible clinical feature. Further studies should be carried out to investigate the usability of this new clinical feature for the diagnosis of BD.

Generating conformation-specific synthetic antibodies to trap proteins in selected functional states.

A set of phage display sorting strategies and validation methodologies are presented that are capable of producing high performance synthetic antibodies (sABs) with customized properties. Exquisite control of antigen and conditions during the phage display selection process can yield sABs that: (1) recognize conformational states, (2) target specific regions of the surface of a protein, (3) induce conformational changes, and (4) capture and stabilize multi-protein complexes. These unique capabilities open myriad opportunities to study complex macromolecular processes inaccessible to traditional affinity reagent technology. We present detailed protocols for de novo isolation of binders, as well as examples of downstream biophysical characterization. The methods described are generalizable and can be adapted to other in vitro direct evolution approaches based on yeast or mRNA display.

Mechanism of activation gating in the full-length KcsA K+ channel.

Using a constitutively active channel mutant, we solved the structure of full-length KcsA in the open conformation at 3.9 Å. The structure reveals that the activation gate expands about 20 Å, exerting a strain on the bulge helices in the C-terminal domain and generating side windows large enough to accommodate hydrated K(+) ions. Functional and spectroscopic analysis of the gating transition provides direct insight into the allosteric coupling between the activation gate and the selectivity filter. We show that the movement of the inner gate helix is transmitted to the C-terminus as a straightforward expansion, leading to an upward movement and the insertion of the top third of the bulge helix into the membrane. We suggest that by limiting the extent to which the inner gate can open, the cytoplasmic domain also modulates the level of inactivation occurring at the selectivity filter.

Transient receptor potential cation channel, subfamily C, member 5 (TRPC5) is a cold-transducer in the peripheral nervous system.

Detection and adaptation to cold temperature is crucial to survival. Cold sensing in the innocuous range of cold (>10-15 °C) in the mammalian peripheral nervous system is thought to rely primarily on transient receptor potential (TRP) ion channels, most notably the menthol receptor, TRPM8. Here we report that TRP cation channel, subfamily C member 5 (TRPC5), but not TRPC1/TRPC5 heteromeric channels, are highly cold sensitive in the temperature range 37-25 °C. We found that TRPC5 is present in mouse and human sensory neurons of dorsal root ganglia, a substantial number of peripheral nerves including intraepithelial endings, and in the dorsal lamina of the spinal cord that receives sensory input from the skin, consistent with a potential TRPC5 function as an innocuous cold transducer in nociceptive and thermosensory nerve endings. Although deletion of TRPC5 in 129S1/SvImJ mice resulted in no temperature-sensitive behavioral changes, TRPM8 and/or other menthol-sensitive channels appear to underpin a much larger component of noxious cold sensing after TRPC5 deletion and a shift in mechanosensitive C-fiber subtypes. These findings demonstrate that highly cold-sensitive TRPC5 channels are a molecular component for detection and regional adaptation to cold temperatures in the peripheral nervous system that is distinct from noxious cold sensing.

Investigation of the variability in the tool marks produced by modified blank firing pistols on fired 7.65 mm projectiles.

Firearms identification has an important place in forensic ballistic investigations since the weapons are widely used in criminal offences. Firearm examiners resolve many case files, through the use of automatic systems or comparison microscopes. Advanced forensic technologies like BALISTIKA helps to record and analyze non standard ballistic evidence. In today's world, with the ease of access to materials and production technique information, pistols modified from blank firers are frequently encountered as crime tools. In this study, the characteristics of 7.65 mm fired cartridge cases obtained by controlled shots from blank firing modified pistols were examined, and their detection performances were compared by means of the Balistika system. Although distinctive differences are not expected after successive test shootings, balistically important changes were seen after the use of blank firing modified pistols and the 3D imaging system proved to be useful in observing such differences. The analyses showed that the modifications in weapons lead to variation in the ballistic characteristics and reduce the accuracy of the detection performance, which may result in flawed forensic decisions. It was also found that the deviations in ballistic impressions of modified blank firing pistols were greater than that of standard fabricated and hand-made pistols. This unique study contributed to the forensic sciences literature by focusing on the impact of modified weapons on ballistic characteristics.

Evaluation of the temporomandibular joint morphology and condylar position with cone-beam computerized tomography in patients with internal derangement.

OBJECTIVES: To evaluate the relationship between internal derangement and morphological features of the temporomandibular joint by comparing joint morphology between patients with ID and control patients. METHODS: The current study prospectively is evaluated the cone-beam computed images of 106 ID patients and 122 patients with asymptomatic joints; recorded angular and linear measurements. The relationships between these measurements and ID are investigated; the differences between the two groups using paired t tests. RESULTS: There were significant differences in the values of articular slope and the condyle height/glenoid fossa depth (CH/GFD) ratio between patients in the ID and control groups (p < 0.05). There was also a relationship between these parameters and the gender of patients with ID. The condylar position was posterior in the ID group. CONCLUSION: Posteriorly and superiorly located condyles with an increased CH/GFD ratio, condyles located eccentrically, and a steeper posterior slope may be risk factors for ID.

Development of etching paste for serial number restoration on aluminum engine block.

Engine numbers, which involve information regarding the engine type, production number, and year and place of manufacture, are used for identification purposes. Comprising of unique alphanumeric characters, the engine numbers are fully or partially obliterated especially in auto theft and smuggling cases to conceal the origin, identity, and owner of vehicles. The limitations of the current restoration techniques such as the difficulty of using chemical liquid etching in vertical sites, the restrictions of magnetic and optical methods, and the applicability of several techniques like electron backscatter diffraction only in the laboratory environment prompt the development of new techniques. In view of these limitations and the importance of restoring engine numbers in criminal investigations, this unique study aimed to develop an etching paste that would restore the effaced characters on a real aluminum alloy engine block. The characters which were cold-stamped on the engine block were milled at varying depths and restoration attempts were conducted using etching pastes formed with different chemicals and materials. The analyses indicate that the etching paste formed with 200 mg of perlite, 400 mg of iron powder, and 450 µL of 20 M NaOH provided restoration to a good extent. The prevention of over-etching through the controlling of the chemical reaction and the cost-effectiveness appears to be the advantages of this technique. The success of recovery on the real engine block, the facilitation of restoration on curved surfaces, and the chance of on-site usage will likely make the etching paste a widely used tool in serial number restoration.

The effects of four different solvent vapours on the restoration of obliterated stamp markings from five different wooden surfaces.

The stamp markings on wooden surfaces, which are placed on trees and products including antiques, indicate the status of trees and involve identifying data regarding the products. Such markings are obliterated either to facilitate illegal logging or to conceal product information. Despite the wide literature on the restoration of obliterated characters on metal and polymer surfaces, the recovery of defaced characters on wooden surfaces appears to be understudied. Several reference texts in the forensic marks' examination literature suggest that water, water vapor, and alkaline solutions are useful in restoring the abraded markings on the wood. Since there does not seem to be any experimental study proving such success, this study aimed to fill this gap. This study conducted experimental research by using water, ethanol, ammonia, and chloroform to recover the scraped characters on samples obtained from walnut, beech, spruce, oak, and cedar trees. The cold-stamped characters, which were defaced at varying depths, were restored using vapor and liquid phases of four solvents. While the vapor phases of water, ethanol, and ammonia yielded good outcomes on all types of wooden surfaces, the liquid phases did not seem to be useful in the revisualization process. The response of the vapors, which varied between 62 and 220 s, depended on the type of wood. The restoration technique developed in this research offers the possibility of on-site usage, easy application, utilization of low-cost solvents, rapid recovery, and effectiveness on various wooden surfaces. Overall, the restoration methodology used in this research appears to be fruitful in retrieving identifying information on wooden samples.

A recombinant Aspergillus oryzae fungus transmitted from larvae to adults of Anopheles stephensi mosquitoes inhibits malaria parasite oocyst development.

The control of malaria parasite transmission from mosquitoes to humans is hampered by decreasing efficacies of insecticides, development of drug resistance against the last-resort antimalarials, and the absence of effective vaccines. Herein, the anti-plasmodial transmission blocking activity of a recombinant Aspergillus oryzae (A. oryzae-R) fungus strain, which is used in human food industry, was investigated in laboratory-reared Anopheles stephensi mosquitoes. The recombinant fungus strain was genetically modified to secrete two anti-plasmodial effector peptides, MP2 (midgut peptide 2) and EPIP (enolase-plasminogen interaction peptide) peptides. The transstadial transmission of the fungus from larvae to adult mosquitoes was confirmed following inoculation of A. oryzae-R in the water trays used for larval rearing. Secretion of the anti-plasmodial effector peptides inside the mosquito midguts inhibited oocyst formation of P. berghei parasites. These results indicate that A. oryzae can be used as a paratransgenesis model carrying effector proteins to inhibit malaria parasite development in An. stephensi. Further studies are needed to determine if this recombinant fungus can be adapted under natural conditions, with a minimal or no impact on the environment, to target mosquito-borne infectious disease agents inside their vectors.

Human ACE2 orthologous peptide sequences show better binding affinity to SARS-CoV-2 RBD domain: Implications for drug design.

Computational methods coupled with experimental validation play a critical role in the identification of novel inhibitory peptides that interact with viral antigenic determinants. The interaction between the receptor binding domain (RBD) of SARS-CoV-2 spike protein and the helical peptide of human angiotensin-converting enzyme-2 (ACE2) is a necessity for the initiation of viral infection. Herein, natural orthologs of human ACE2 helical peptide were evaluated for competitive inhibitory binding to the viral RBD by use of a computational approach, which was experimentally validated. A total of 624 natural ACE2 orthologous 32-amino acid long peptides were identified through a similarity search. Molecular docking was used to virtually screen and rank the peptides based on binding affinity metrics, benchmarked against human ACE2 peptide docked to the RBD. Molecular dynamics (MD) simulations were done for the human reference and the Nipponia nippon peptide as it exhibited the highest binding affinity (Gibbs free energy; -14 kcal/mol) predicted from the docking results. The MD simulation confirmed the stability of the assessed peptide in the complex (-12.3 kcal/mol). The top three docked-peptides (from Chitinophaga sancti, Nipponia nippon, and Mus musculus) and the human reference were experimentally validated by use of surface plasmon resonance technology. The human reference exhibited the weakest binding affinity (Kd of 318-441 pM) among the peptides tested, in agreement with the docking prediction, while the peptide from Nipponia nippon was the best, with 267-538-fold higher affinity than the reference. The validated peptides merit further investigation. This work showcases that the approach herein can aid in the identification of inhibitory biosimilar peptides for other viruses.

Are marginal bone levels and implant stability/mobility affected by single-stage platform switched dental implants? A comparative clinical study.

OBJECTIVES: The aim of this study was to evaluate short-term bone level and stability/mobility measurement alterations at platform switched (PS) and standard platform (SP) implants placed in mandibular premolar/molar regions using a single-stage protocol. MATERIALS AND METHODS: Sixteen PS and 16 SP implants restorated with fixed prosthesis were included. Standard implant dimensions were used for both implant systems. After 3 months of osseointegration, implants were connected to abutments and final restorations were performed. Marginal bone loss was measured by standardized periapical radiographs. Implant stability/mobility was determined by resonance frequency analysis (RFA) and mobility measuring (MM) device values. Peri-implant parameters were evaluated with clinical periodontal indices and all parameters were assessed at baseline, 1, 3, and 6 months after the surgery. RESULTS: After 6 months, all implants showed uneventful healing. Radiographic evaluation showed a mean bone loss of 0.72 mm for PS and 0.56 mm for SP implants, and there were no significant differences between implant types. At 6 months, mean implant stability quotient (ISQ) values were 73.38 and 77 for PS and SP implants, respectively. Mean MM values were -4.75 for PS and -6.38 for SP implants. Mean MM values were lower for SP implants compared to PS implants at all time points. No significant differences were detected between implant types according to clinical peri-implant parameters. CONCLUSIONS: The micro-gap at crestal level which immediately exposed to the oral cavity in non-submerged two part implants seems to have adverse influence on the marginal bone level.

An engineered substance P variant for receptor-mediated delivery of synthetic antibodies into tumor cells.

We have developed and tested a robust delivery method for the transport of proteins to the cytoplasm of mammalian cells without compromising the integrity of the cell membrane. This receptor-mediated delivery (RMD) technology utilizes a variant of substance P (SP), a neuropeptide that is rapidly internalized upon interaction with the neurokinin-1 receptor (NK1R). Cargos in the form of synthetic antibody fragments (sABs) were conjugated to the engineered SP variant (SPv) and efficiently internalized by NK1R-expressing cells. The sABs used here were generated to bind specific conformational forms of actin. The internalized proteins appear to escape the endosome and retain their binding activity within the cells as demonstrated by co-localization with the actin cytoskeleton. Further, since the NK1R is over-expressed in many cancers, SPv-mediated delivery provides a highly specific method for therapeutic utilization of affinity reagents targeting intracellular processes in diseased tissue.