Agonists or positive allosteric modulators of α7 nicotinic acetylcholine receptor prevent interaction of SARS-Cov-2 receptor-binding domain with astrocytoma cells.

SARS-Cov-2, the virus causing COVID-19, penetrates host target cells via the receptor of angiotensin-converting enzyme 2 (ACE2). Disrupting the virus interaction with ACE2 affords a plausible mechanism for prevention of cell penetration and inhibiting dissemination of the virus. Our studies demonstrate that ACE2 interaction with the receptor binding domain of SARS-Cov-2 spike protein (RBD) can be impaired by modulating the α7 nicotinic acetylcholine receptor (α7 nAChR) contiguous with ACE2. U373 cells of human astrocytoma origin were shown to bind both ACE2-specific antibody and recombinant RBD in Cell-ELISA. ACE2 was found to interact with α7 nAChR in U373 cell lysates studied by Sandwich ELISA. Our studies demonstrate that inhibition of RBD binding to ACE2-expressing U373 cells were defined with α7 nAChR agonists choline and PNU282987, but not a competitive antagonist methyllicaconitine (MLA). Additionally, the type 2 positive allosteric modulator (PAM2) PNU120596 and hydroxyurea (HU) also inhibited the binding. Our studies demonstrate that activation of α7 AChRs has efficacy in inhibiting the SARS-Cov-2 interaction with the ACE2 receptor and in such a way can prevent virus target cell penetration. These studies also help to clarify the consistent efficacy and positive outcomes for utilizing HU in treating COVID-19.

Hydroxyurea interaction with α7 nicotinic acetylcholine receptor can underlie its therapeutic efficacy upon COVID-19.

In this paper the authors provide evidence that hydroxyurea (hydroxycarbamide) interacts with α7 nicotinic acetylcholine receptor, exerts anti-inflammatory and pro-survival effect, prevents α7 nicotinic receptor interaction with angiotensin-converting enzyme-2 and stimulates IgM to IgG class switch upon immunization with SARS spike protein fragment 674-685. Hydroxyurea shifts immunoglobulin glycosylation profile to anti-inflammatory phenotype and prevents the appearance of anti-idiotypic α7(179-190)-specific antibodies, as well as memory impairment. According to these results, interaction with α7 nicotinic acetylcholine receptor may underlie positive therapeutic effects of hydroxyurea upon SARS-Cov-2 infection by interfering with virus penetration into the cell and providing anti-inflammatory and immunomodulatory effects.

A ribosomal operon database and MegaBLAST settings for strain-level resolution of microbiomes.

Current methods to characterize microbial communities generally employ sequencing of the 16S rRNA gene (<500 bp) with high accuracy (∼99%) but limited phylogenetic resolution. However, long-read sequencing now allows for the profiling of near-full-length ribosomal operons (16S-ITS-23S rRNA genes) on platforms such as the Oxford Nanopore MinION. Here, we describe an rRNA operon database with >300 ,000 entries, representing >10 ,000 prokaryotic species and ∼ 150, 000 strains. Additionally, BLAST parameters were identified for strain-level resolution using in silico mutated, mock rRNA operon sequences (70-95% identity) from four bacterial phyla and two members of the Euryarchaeota, mimicking MinION reads. MegaBLAST settings were determined that required <3 s per read on a Mac Mini with strain-level resolution for sequences with >84% identity. These settings were tested on rRNA operon libraries from the human respiratory tract, farm/forest soils and marine sponges ( n = 1, 322, 818 reads for all sample sets). Most rRNA operon reads in this data set yielded best BLAST hits (95 ± 8%). However, only 38-82% of library reads were compatible with strain-level resolution, reflecting the dominance of human/biomedical-associated prokaryotic entries in the database. Since the MinION and the Mac Mini are both portable, this study demonstrates the possibility of rapid strain-level microbiome analysis in the field.

Offline Next Generation Metagenomics Sequence Analysis Using MinION Detection Software (MINDS).

Field laboratories interested in using the MinION often need the internet to perform sample analysis. Thus, the lack of internet connectivity in resource-limited or remote locations renders downstream analysis problematic, resulting in a lack of sample identification in the field. Due to this dependency, field samples are generally transported back to the lab for analysis where internet availability for downstream analysis is available. These logistics problems and the time lost in sample characterization and identification, pose a significant problem for field scientists. To address this limitation, we have developed a stand-alone data analysis packet using open source tools developed by the Nanopore community that does not depend on internet availability. Like Oxford Nanopore Technologies' (ONT) cloud-based What's In My Pot (WIMP) software, we developed the offline MinION Detection Software (MINDS) based on the Centrifuge classification engine for rapid species identification. Several online bioinformatics applications have been developed surrounding ONT's framework for analysis of long reads. We have developed and evaluated an offline real time classification application pipeline using open source tools developed by the Nanopore community that does not depend on internet availability. Our application has been tested on ATCC's 20 strain even mix whole cell (ATCC MSA-2002) sample. Using the Rapid Sequencing Kit (SQK-RAD004), we were able to identify all 20 organisms at species level. The analysis was performed in 15 min using a Dell Precision 7720 laptop. Our offline downstream bioinformatics application provides a cost-effective option as well as quick turn-around time when analyzing samples in the field, thus enabling researchers to fully utilize ONT's MinION portability, ease-of-use, and identification capability in remote locations.

Occurrence of Bacillus amyloliquefaciens as a systemic endophyte of vanilla orchids.

We report the occurrence of Bacillus amyloliquefaciens in vanilla orchids (Vanilla phaeantha) and cultivated hybrid vanilla (V. planifolia × V. pompona) as a systemic bacterial endophyte. We determined with light microscopy and isolations that tissues of V. phaeantha and the cultivated hybrid were infected by a bacterial endophyte and that shoot meristems and stomatal areas of stems and leaves were densely colonized. We identified the endophyte as B. amyloliquefaciens using DNA sequence data. Since additional endophyte-free plants and seed of this orchid were not available, additional studies were performed on surrogate hosts Amaranthus caudatus, Ipomoea tricolor, and I. purpurea. Plants of A. caudatus inoculated with B. amyloliquefaciens demonstrated intracellular colonization of guard cells and other epidermal cells, confirming the pattern observed in the orchids. Isolations and histological studies suggest that the bacterium may penetrate deeply into developing plant tissues in shoot meristems, forming endospores in maturing tissues. B. amyloliquefaciens produced fungal inhibitors in culture. In controlled experiments using morning glory seedlings we showed that the bacterium promoted seedling growth and reduced seedling necrosis due to pathogens. We detected the gene for phosphopantetheinyl transferase (sfp), an enzyme in the pathway for production of antifungal lipopeptides, and purified the lipopeptide "surfactin" from cultures of the bacterium. We hypothesize that B. amyloliquefaciens is a robust endophyte and defensive mutualist of vanilla orchids. Whether the symbiosis between this bacterium and its hosts can be managed to protect vanilla crops from diseases is a question that should be evaluated in future research.

Cyclic loading of Achilles tendon repairs: a comparison of polyester and polyblend suture.

BACKGROUND: Early functional rehabilitation is widely used after open suture repair of the Achilles tendon. To our knowledge, no previous studies have assessed gap formation from cyclic loading and subsequent failure loads of simulated Achilles tendon repairs. A synthetic (polyblend) suture has been introduced for tendon repairs with reportedly greater strength than polyester suture. This stronger, stiffer suture material may provide stronger repairs with less elongation of the tendon repair. METHODS: Simulated Achilles tendon ruptures in bovine Achilles tendon were repaired with a four-strand Krackow suture technique using No. 2 polyester suture. Specimens were loaded for 3,000 cycles at maximal loads of 50, 75, 100, or 125 N, and gap formation at the repair site was continuously measured. After cyclic loading, each specimen was loaded to failure. Identical repairs were performed with number 2 polyblend suture and cyclically loaded to 75 N for 3,000 cycles. All specimens were loaded to failure. RESULTS: Cyclically loading polyester suture repairs to 50, 75, 100, or 125 N for 3,000 cycles resulted in mean gapping at the repair site of 3.0 +/- 0.8, 4.9 +/- 1.0, 7.2 +/- 0.9, and 7.9 +/- 0.8 mm, respectively. Cyclically loading the polyblend suture repairs for 3,000 cycles at 75 N, resulted in 3.3 +/- 0.3 mm of gap formation at the repair site, significantly less than polyester suture repairs (p < 0.001). The mean load to failure for polyester suture repair was 222 +/- 19 N and for polyblend suture repair was 582 +/- 49 N, a statistically significant difference (p < 0.001). Gap formation at 100, 1,000, and 2,000 cycles, as a percentage of total gap formation at 3,000 cycles, was 64.3%, 87.5%, and 95.4% for polyester suture and 45.8%, 78.5%, and 90.1% for polyblend repairs. All specimens in all groups failed at the knots during load-to-failure testing. CONCLUSIONS: Cyclic loading of simulated Achilles tendon repairs using a Krackow, four-core polyester suture technique showed progressive gap formation with increasing load. All repairs failed at the knot, and suture pull-out from tendon was not observed. Polyblend suture repair, when compared to identical repairs with braided polyester suture, resulted in a 260% higher load to failure and 33% less gap formation at the repair site after 3,000 cycles. CLINICAL RELEVANCE: The use of polyblend suture in a four-stranded Krackow configuration provides stronger repairs with less gap formation, which may provide increased security during early functional rehabilitation.

An in vitro study comparing the use of suture anchors and drill hole fixation for flexor digitorum longus transfer to the navicular.

BACKGROUND: The surgical management of posterior tibial tendon dysfunction often includes transfer of the flexor digitorum longus (FDL) tendon through a tunnel in the navicular. Fixation often is obtained by sewing the tendon back onto itself. The purpose of this study was to compare this standard method of fixation with suture anchor fixation, a technique that may be associated with less surgical morbidity, because it requires the harvesting of less tendon length. METHODS: FDL tendon transfer to the navicular was done in 13 fresh-frozen cadaver specimens. In six feet comprising the standard group, the FDL tendon was transected distal to the master knot of Henry, placed through a drill hole into the navicular, and sutured back onto itself. In seven feet the FDL tendon was transected proximal to the master knot of Henry, placed into a drill hole into the navicular, and fixed with a suture anchor. Load was applied to the proximal FDL muscle and tendon using a materials testing system (MTS) machine and peak load to failure was measured. RESULTS: The mean load to failure was 142.48 N +/- 38.06 N for the standard group and 142.12 N +/- 59.26 N for the suture anchor group (p = 0.305 for the Student-t test and p = 0.945 for the Mann-Whitney test). CONCLUSION: Transfer of the FDL tendon to the navicular using suture anchor fixation requires less tendon length yet provides similar fixation strength as compared to sewing the tendon back onto itself. However, suture anchors are considerably more expensive than sutures. CLINICAL IMPLICATIONS: Suture anchors allow comparable fixation of FDL tendon transfer into a navicular without the need to disrupt the master knot of Henry. This technique may be associated with less morbidity including a shorter incision, decreased risk of medial plantar nerve injury, and decreased loss of lesser toe plantarflexion strength secondary to maintenance of the normal interconnections between the flexor hallucis longus (FHL) and FDL tendons.

Mechanisms of c-reactive protein up-regulation in arterialized vein grafts.

BACKGROUND: C-reactive protein (CRP), an acute phase reactant, is an independent predictor of coronary artery syndromes and a mediator of the vascular response to injury. CRP has been found in arterialized vein grafts and has been linked to atherogenesis; however, its involvement in vein graft early failure or intimal hyperplasia has not been assessed. This study was designed to investigate the mechanism(s) of CRP up-regulation in arterialized vein grafts. METHODS: Carotid artery bypass with arterialized jugular vein grafts (AVG) was performed in 18 dogs. AVG were harvested at 3, 8, and 24 hours and 4, 14, and 28 days, using the femoral vein obtained at the time of AVG harvest as a control. Serum CRP levels were characterized by enzyme-linked immunosorbent assay; AVG expression of CRP was studied by immunofluorescence, Western blotting, in situ hybridization, Northern blotting, and quantitative RT-PCR. RESULTS: CRP levels peaked at 24 hours in serum and AVG but remained at baseline in control veins. By double immunofluorescence, CRP was associated with the media and adventitia of AVG. However, Northern blotting analysis showed no CRP mRNA expression in AVG. Reverse transcriptase polymerase chain reaction analysis confirmed the lack of up-regulation of CRP in AVG. CONCLUSION: CRP levels are increased in AVG, peaking 24 hours after arterialization. However, no significant production of CRP was detected in AVG. Therefore, increased CRP levels within AVG appear to originate mostly from CRP diffusion from the systemic circulation. These results have significant implications for the development of strategies aimed at blocking CRP up-regulation in bypass grafts.

The effect of triceps surae contracture force on plantar foot pressure distribution.

BACKGROUND: Triceps surae contractures have been associated with foot and ankle pathology. Achilles tendon contractures have been shown to shift plantar foot pressure from the heel to the forefoot. The purpose of this study was to determine whether isolated gastrocnemius contractures had similar effects and to assess the effects of gastrocnemius or soleus contracture on midfoot plantar pressure. METHODS: Ten fresh frozen cadaver below-knee specimens were loaded to 79 pounds (350 N) plantar force with the foot unconstrained on a 10-degree dorsiflexed plate. Combinations of static gastrocnemius or soleus forces were applied in 3-lb increments and plantar pressure recordings were obtained for the hindfoot, midfoot, and forefoot regions. RESULTS: The percentage of plantar force borne by the forefoot and midfoot increased with triceps surae force, while that borne by the hindfoot decreased (p

Ligamentous Lisfranc joint injuries: a biomechanical comparison of dorsal plate and transarticular screw fixation.

BACKGROUND: The current treatment of displaced ligamentous injuries of the tarsometatarsal (TMT) joints is open reduction and rigid fixation using transarticular screws. This technique causes further articular surface damage that theoretically may increase the risk of arthritis. Should the screws break, hardware removal is difficult. An alternative method that avoids these potential complications is rigid fixation using dorsal plates. METHODS: The displacement between the first metatarsal and medial cuneiform, the second metatarsal and intermediate cuneiform, the first and second metatarsal bases, and the medial cuneiform and second metatarsal base were measured in 10 matched pairs of fresh-frozen cadaver lower extremities in the unloaded and loaded condition. After sectioning the Lisfranc and TMT joint ligaments, measurements were repeated in the loaded condition. The first and second TMT joints of the right feet were fixed with transarticular 3.5-mm cortical screws while those of the left feet with were fixed with dorsal 2.7-mm 1/4 tubular plates. Measurements were then repeated in the unloaded and loaded condition. RESULTS: After ligament sectioning, significantly increased first and second TMT joint subluxation with loading was seen. No significant difference was noted with direct comparison between plates and screws with respect to ability to realign the first and second TMT joints and to maintain TMT joint alignment during loading. The amount of articular surface destruction caused by one 3.5-mm screw was 2.0 +/- 0.7% for the medial cuneiform, 2.6 +/- 0.5% for the first metatarsal, 3.6 +/- 1.2% for the intermediate cuneiform, and 3.6 +/- 1.0% for the second metatarsal. CONCLUSIONS: The model reliably produced displacement of the first and second TMT joints consistent with a ligamentous Lisfranc injury. Transarticular screws and dorsal plates showed similar ability to reduce the first and second TMT joints after TMT and Lisfranc ligament transection and to resist TMT joint displacement with weightbearing load. CLINICAL RELEVANCE: Dorsal plating may be an alternative to transarticular screws in the treatment of displaced Lisfranc injuries.

Arthroscopic treatment of soft-tissue impingement lesions of the ankle in adolescents.

BACKGROUND: Ankle sprains are common injuries in adolescents. Most are treated conservatively with rest, ice, antiinflammatory medication, and rehabilitation. A small percentage of patients develop chronic pain and functional instability secondary to intraarticular soft-tissue impingement. This study evaluated the effectiveness of ankle arthroscopy for the treatment of functional instability of the ankle with pain specifically in adolescents between the ages of 13 and 19 years. METHODS: We evaluated 11 patients at an average of 25 (range 15 to 38) months after arthroscopic debridement. The American Orthopaedic Foot and Ankle Society (AOFAS) ankle and hindfoot score was used to evaluate outcomes. In addition, four questions were asked: 1) Are you satisfied with the outcome of your surgery? 2) Would you have the procedure again? 3) Would you recommend it to a friend? 4) Have you returned to your preinjury level of activity. RESULTS: The average postoperative AOFAS score was 95 (range 75 to 100). We found that 10 of 11 patients had good to excellent results with arthroscopic debridement of soft-tissue, intraarticular impingement lesions. All but one patient was satisfied with the procedure and all stated that they would have the procedure again. Two patients had repeat injuries and one subsequently developed reflex sympathetic dystrophy. These two patients had not yet returned to their preinjury level of activity at last followup. CONCLUSIONS: We found ankle arthroscopy to be a safe and effective means of treatment in adolescents with functional instability from soft-tissue ankle impingement.

Correction of severe residual clubfoot deformity in adolescents with the Ilizarov technique.

Correction of severe residual clubfoot deformities in adolescents is a difficult undertaking and historically has had mediocre success without an accompanying arthrodesis procedure. Soft tissue release alone has yielded extremely high recurrence rates. Additional osteotomies have been used for correction of single deformities in one plane. The use of Ilizarov external fixation techniques has allowed for improvement of correction, stabilization, and decrease in recurrence rates. The technique, however, is difficult and should be performed by surgeons who are familiar with correction of pediatric foot and ankle deformities and are versed fully in Ilizarov fixation techniques.

Flexor hallucis longus transfer for repair of chronic achilles tendinopathy.

BACKGROUND: The flexor hallucis longus (FHL) tendon has been used to augment the repairs for chronic Achilles tendinopathy. Two common methods of FHL harvesting include a single incision (posterior) technique and a double incision (posterior and medial utility) technique. This cadaver study was designed to measure and compare the lengths of FHL tendon obtainable for reconstruction with each technique. METHODS: Fourteen fresh-frozen cadaver lower limbs were utilized for FHL harvest. The tendon was first exposed through the single posterior-medial incision approach adjacent to the Achilles. A second medial utility midfoot incision was then made and the FHL was marked at the level of Henry's knot with a suture, to approximate the level of potential harvest via a two-incision technique. The FHL was then harvested and delivered into the posterior wound. Single incision technique graft length was then measured from the tip of the calcaneal tuber to the level of transection. The remaining in situ tendon was then also measured between its level of transection and the more distal suture placed at Henry's knot. These two lengths were then combined to determine the total potential tendon graft length obtainable using a double incision technique. RESULTS: The average length of the FHL tendon harvested through the single posterior incision technique measured 5.16 cm (range, 3.4-6.9 cm, SD = 1.29). The average total tendon graft length available using the double incision technique measured 8.09 cm (range, 5.1-11.1 cm, SD = 1.63). The difference between the lengths obtained from these two techniques was significant (p < .001). CONCLUSIONS: These results demonstrate approximate FHL graft lengths obtainable by using either a single or double incision harvest technique and show that a significantly longer graft can be obtained using a double incision technique. Further data need to be obtained, however, to support whether the extra surgery and graft length obtained from a double incision technique are of any benefit in improving the ultimate functional outcome of these repairs.

Molecular characterization and expression in Escherichia coli of three beta-1,3-glucanase genes from Lysobacter enzymogenes strain N4-7.

Lysobacter enzymogenes strain N4-7 produces multiple biochemically distinct extracellular beta-1,3-glucanase activities. The gluA, gluB, and gluC genes, encoding enzymes with beta-1,3-glucanase activity, were identified by a reverse-genetics approach following internal amino acid sequence determination of beta-1,3-glucanase-active proteins partially purified from culture filtrates of strain N4-7. Analysis of gluA and gluC gene products indicates that they are members of family 16 glycoside hydrolases that have significant sequence identity to each other throughout the catalytic domain but that differ structurally by the presence of a family 6 carbohydrate-binding domain within the gluC product. Analysis of the gluB gene product indicates that it is a member of family 64 glycoside hydrolases. Expression of each gene in Escherichia coli resulted in the production of proteins with beta-1,3-glucanase activity. Biochemical analyses of the recombinant enzymes indicate that GluA and GluC exhibit maximal activity at pH 4.5 and 45 degrees C and that GluB is most active between pH 4.5 and 5.0 at 41 degrees C. Activity of recombinant proteins against various beta-1,3 glucan substrates indicates that GluA and GluC are most active against linear beta-1,3 glucans, while GluB is most active against the insoluble beta-1,3 glucan substrate zymosan A. These data suggest that the contribution of beta-1,3-glucanases to the biocontrol activity of L. enzymogenes may be due to complementary activities of these enzymes in the hydrolysis of beta-1,3 glucans from fungal cell walls.

Different faces of the triple arthrodesis.

Patients with severe pes planovalgus or cavovarus foot deformities who fail conservative treatment may require a triple arthrodesis. Modifying the triple arthrodesis to include extended bone wedge resections allows for improved correction. The goal of each procedure is to obtain a less painful, plantigrade foot, and to improve function. Additional hindfoot or midfoot osteotomies may be needed in the modified triple arthrodesis. Midfoot or forefoot cavus can be addressed with either the Japas, Cole, or Jahss osteotomies, as described above. Residual hindfoot valgus can be adequately corrected with a medial displacement osteotomy of the calcaneus. Residual hindfoot varus is preferably corrected through a lateral closing wedge calcaneal osteotomy. This allows for adequate correction without the need for bone graft or an extended medial incision in the area of the tibial neurovascular bundle. Good results have been obtained with these types of complicated reconstructive procedures.

Adolescent osteochondral lesion of the talus. Ankle arthroscopy in pediatric patients.

Osteochondral lesions of the talus can be difficult to diagnose and can result in a significant functional limitation in young, active patients. New imaging modalities have improved the diagnosis and staging of these lesions. In general, nonoperative treatment results in poorer outcomes compared with operative treatment, and arthroscopic treatment has results similar to open treatment. Although the literature is limited, the treatment of adolescents results in outcomes similar to the adult population.