Extracellular RNA Sensing Mediates Inflammation and Organ Injury in a Murine Model of Polytrauma.

Severe traumatic injury leads to marked systemic inflammation and multiorgan injury. Endogenous drivers such as extracellular nucleic acid may play a role in mediating innate immune response and the downstream pathogenesis. Here, we explored the role of plasma extracellular RNA (exRNA) and its sensing mechanism in inflammation and organ injury in a murine model of polytrauma. We found that severe polytrauma-bone fracture, muscle crush injury, and bowel ischemia-induced a marked increase in plasma exRNA, systemic inflammation, and multiorgan injury in mice. Plasma RNA profiling with RNA sequencing in mice and humans revealed a dominant presence of miRNAs and marked differential expression of numerous miRNAs after severe trauma. Plasma exRNA isolated from trauma mice induced a dose-dependent cytokine production in macrophages, which was almost abolished in TLR7-deficient cells but unchanged in TLR3-deficient cells. Moreover, RNase or specific miRNA inhibitors against the selected proinflammatory miRNAs (i.e., miR-7a-5p, miR-142, let-7j, miR-802, and miR-146a-5p) abolished or attenuated trauma plasma exRNA-induced cytokine production, respectively. Bioinformatic analyses of a group of miRNAs based on cytokine readouts revealed that high uridine abundance (>40%) is a reliable predictor in miRNA mimic-induced cytokine and complement production. Finally, compared with the wild-type, TLR7-knockout mice had attenuated plasma cytokine storm and reduced lung and hepatic injury after polytrauma. These data suggest that endogenous plasma exRNA of severely injured mice and ex-miRNAs with high uridine abundance prove to be highly proinflammatory. TLR7 sensing of plasma exRNA and ex-miRNAs activates innate immune responses and plays a role in inflammation and organ injury after trauma.

The O-glycan is essential for the induction of protective antibodies against lethal infection by flagella A-bearing Pseudomonas aeruginosa.

To address the problem of increased antimicrobial resistance, we developed a glycoconjugate vaccine comprised of O-polysaccharides (OPS) of the four most prevalent serotypes of Klebsiella pneumoniae (KP) linked to recombinant flagellin types A and B (rFlaA and rFlaB) of Pseudomonas aeruginosa (PA). Flagellin is the major subunit of the flagellar filament. Flagella A and B, essential virulence factors for PA, are glycosylated with different glycans. We previously reported that while both rFlaA and rFlaB were highly immunogenic, only the rFlaB antisera reduced PA motility and protected mice from lethal PA infection in a mouse model of thermal injury. Since recombinant flagellin is not glycosylated, we examined the possibility that the glycan on native FlaA (nFlaA) might be critical to functional immune responses. We compared the ability of nFlaA to that of native, deglycosylated FlaA (dnFlaA) to induce functionally active antisera. O glycan was removed from nFlaA with trifluoromethanesulfonic acid. Despite the similar high-titered anti-FlaA antibody levels elicited by nFlaA, rFlaA, and dnFlaA, only the nFlaA antisera inhibited PA motility and protected mice following lethal intraperitoneal bacterial challenge. Both the protective efficacy and carrier protein function of nFlaA were retained when conjugated to KP O1 OPS. We conclude that unlike the case with FlaB O glycan, the FlaA glycan is an important epitope for the induction of functionally active anti-FlaA antibodies.

Hybrid, transarticular external fixation with platelet-rich plasma injection as a treatment for partial calcaneal tendon disruption in dogs without primary tenorrhaphy.

OBJECTIVE: To report the use of a hybrid, transarticular external skeletal fixator and platelet-rich plasma injection without primary tenorrhaphy for management of partial calcanean tendon disruption in dogs. STUDY DESIGN: Multi-institutional retrospective study. ANIMALS: A total of 11 client-owned dogs. METHODS: Medical records from two referral centers from 2019 to 2022 were reviewed for inclusion in the study. Dogs diagnosed with partial, non-traumatic calcanean tendon disruption treated with hybrid, transarticular external skeletal fixation and platelet-rich plasma injection were included in the study. Cases that did not include complete postoperative follow-up over 8-12 weeks and owner-reported outcomes at >6 months were excluded from the study. Dogs with traumatic laceration or those in which a primary tenorrhaphy was performed were also excluded. RESULTS: A total of 11 (n = 11) dogs met the inclusion criteria. Median follow-up time was 18.5 months (6 months-34 months). Mean time of fixation was 9 weeks (6 weeks-12 weeks). Five dogs returned to full, pain-free function (5/11 = 45%). Five dogs had an acceptable return to function (5/11 = 45%). One dog had an unacceptable outcome (1/11 = 9%). Pin tract complications occurred in five dogs (5/11 = 45%) and resolved with medical intervention. CONCLUSION: The results of this study suggest that temporary tibiotarsal immobilization with a hybrid, transarticular external skeletal fixator and platelet-rich plasma injection without primary tenorrhaphy can lead to a successful outcome in dogs with partial calcanean tendon disruption. CLINICAL SIGNIFICANCE: Temporary hybrid, transarticular external skeletal fixation and platelet-rich plasma injection without primary tenorrhaphy may play a role in the management of partial calcanean tendon disruption in dogs.

Effect of mGluR2 positive allosteric modulation on frontostriatal working memory activation in schizophrenia.

Negative symptoms and cognitive deficits contribute strongly to disability in schizophrenia, and are resistant to existing medications. Recent drug development has targeted enhanced NMDA function by increasing mGluR2/3 signaling. However, the clinical utility of such agents remains uncertain, and markers of brain circuit function are critical for clarifying mechanisms and understanding individual differences in efficacy. We conducted a double-blind, placebo-controlled, randomized cross-over (14 day washout) pilot study evaluating adjunctive use of the mGluR2 positive allosteric modulator AZD8529 (80 mg daily for 3 days), in chronic stable patients with schizophrenia (n = 26 analyzed). We focused on 3 T fMRI response in frontostriatal regions during an n-back working memory task, testing the hypothesis that AZD8529 produces fMRI changes that correlate with improvement in negative symptoms and cognition. We found that AZD8529 did not produce significant group-average effects on symptoms or cognitive accuracy. However, AZD8529 did increase n-back fMRI activation in striatum (p < 0.0001) and anterior cingulate/paracingulate (p = 0.002). Greater drug-versus-placebo effects on caudate activation significantly correlated with greater reductions in PANSS negative symptom scores (r = -0.42, p = 0.031), and exploratory correlations suggested broader effects across multiple symptom domains and regions of interest. These findings demonstrate that fMRI responses to mGluR2 positive modulation relate to individual differences in symptom reduction, and could be pursued for future biomarker development. Negative clinical results at the group level should not lead to premature termination of investigation of this mechanism, which may benefit an important subset of individuals with schizophrenia. Imaging biomarkers may reveal therapeutic mechanisms, and help guide treatment toward specific populations.

The effect of grip location on Kirschner wire bend characteristics: An in vitro study.

OBJECTIVE: To determine the effect of Kirschner wire (K-wire) grip location on bend angle, bend radius, and torque when performing a Z-bend technique. STUDY DESIGN: Experimental study. SAMPLE POPULATION: Ten samples at each of five grip locations for each of three K-wire diameters. METHODS: K-wires of three diameters (0.9, 1.1, 1.6 mm) were drilled into PVC pipe, and a Jacob's chuck was used to bend the wires at five periodic grip locations (distance from the bone model). Torque, bend angle, and bend radius were determined for each sample. Outcome variables were statistically analyzed by grip location to determine significant relationships. RESULTS: A grip location of 2.0 cm in the 0.9 mm K-wire group minimized bend angle (mean ± SD: 75.92° ± 0.81) and bend radius (2.89 mm ± 0.08). A grip location of 3.0 cm in the 1.1 mm K-wire group minimized bend angle (72.88° ± 0.98) and bend radius (2.47 mm ± 0.20). A grip location of 3.0 cm minimized bend angle (74.38° ± 1.93) and bend radius (2.71 mm ± 0.27) in the 1.6 mm K-wire group. Torque at these grip locations for the 0.9, 1.1, and 1.6 mm K-wires was 6.50 N-m ± 0.0, 11.00 N-m ± 0.0, and 19.05 N-m ± 0.16, respectively. CONCLUSION: Bend angle and bend radius can be minimized by bending K-wires at specific grip locations, though torque is not minimized at these locations. Clinical significance These findings provide an evidence-based recommendation of where surgeons should grip K-wires when bending them.

The effect of two platelet-rich plasma aspiration techniques on plasma cellular concentrations using a double syringe gravitational centrifugation system.

OBJECTIVE: To identify which aspiration technique increased plasma platelet concentration and which technique minimized plasma leukocyte and erythrocyte concentrations using a gravitational double-syringe platelet rich plasma (PRP) system. STUDY DESIGN: Controlled laboratory study. ANIMALS: Thirty adult dogs. METHODS: Whole blood was collected into two autologous conditioned plasma (ACP) syringes and an ethylenediaminetetraacetic acid (EDTA tube) (control samples). The ACP syringes were centrifuged for 5 min at 1500 rpm. The proximal 2 mL of plasma from one ACP syringe was deposited in an EDTA tube (preflash samples). Plasma from the second ACP syringe was withdrawn until the buffy coat was pierced, producing a "flash" of red blood cells, agitated and deposited into an EDTA tube (flash samples). Complete blood counts were performed. RESULTS: Mean plasma platelet concentrations of the control, preflash, and flash samples were 2.4 × 105 /dL, 3.3 × 105 /dL and 4.1 × 105 /dL, respectively. The mean platelet concentration of the flash samples was 7.9 × 104 /dL higher than the preflash samples (p = .005). The mean platelet concentration was lower in the control samples than the preflash (p = .002) and flash (p < .0001) samples. The median plasma leukocyte concentration of the preflash samples (0/dL) was lower than in the flash samples (2.4 × 103 /dL) (p = .001). The median plasma hematocrit value of the preflash samples (0%) was lower than in the flash samples (1.0%) (p = .002). CONCLUSION: The flash method is not necessary to produce a PRP sample. CLINICAL SIGNIFICANCE: Both methods produced PRP. However, clinicians should avoid aspirating the buffy coat when processing PRP for therapies where leukocytes and erythrocytes are contraindicated.

Effect of screw insertion angle and speed on the incidence of transcortical fracture development in a canine tibial diaphyseal model.

OBJECTIVE: To assess the incidence of transcortical fracture (TCF) development based on screw insertion angle and screw insertion speed. STUDY DESIGN: Cadaveric experimental study. SAMPLE POPULATION: Sixty-six canine tibiae. METHODS: Sixty-six cadaveric tibiae were randomly assigned to one of six groups that varied based on screw insertion angle relative to the pilot hole (0, 5, or 10°) and screw insertion speed (650 or 1350 revolutions per minute [rpm]). Each tibia was mounted in a custom jig. Locking self-tapping screws (3.5 mm) were inserted at varying speeds and insertion angles, based on group assignment. Orthogonal radiographs were evaluated for TCFs. Fisher's exact tests with a Bonferroni correction were performed to evaluate differences in the frequency of TCF between groups. RESULTS: In Group A (0°/650 rpm: control), a 0% TCF rate was observed (n = 0/80). Group B (5°/650 rpm) had a 3.75% TCF rate (n = 3/80). Group C (10°/650 rpm) had a 12.5% TCF rate (n = 10/80). Group D (10°/hand insertion) had a 3.75% TCF rate (n = 3/80). Group E (10°/1350 rpm) had a 17.5% TCF rate (n = 14/80). Group F (0°/1350 rpm) had a 0% TCF rate (n = 0/80). Groups C and E had the highest TCF rates with a difference in TCF rates observed between the control group and Group C (p = .001) and between the control group and Group E (p < .001). CONCLUSION: Increased screw insertion angle and insertion speed appear to be predisposing factors for TCF development in cadaveric bone. CLINICAL SIGNIFICANCE: Ensuring screw insertion is coaxial with the pilot hole and using slower screw insertion speeds may help reduce the risk of TCF development.

Distribution of serotypes and antibiotic resistance of invasive Pseudomonas aeruginosa in a multi-country collection.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections and is frequently associated with healthcare-associated infections. Because of its ability to rapidly acquire resistance to antibiotics, P. aeruginosa infections are difficult to treat. Alternative strategies, such as a vaccine, are needed to prevent infections. We collected a total of 413 P. aeruginosa isolates from the blood and cerebrospinal fluid of patients from 10 countries located on 4 continents during 2005-2017 and characterized these isolates to inform vaccine development efforts. We determined the diversity and distribution of O antigen and flagellin types and antibiotic susceptibility of the invasive P. aeruginosa. We used an antibody-based agglutination assay and PCR for O antigen typing and PCR for flagellin typing. We determined antibiotic susceptibility using the Kirby-Bauer disk diffusion method. RESULTS: Of the 413 isolates, 314 (95%) were typed by an antibody-based agglutination assay or PCR (n = 99). Among the 20 serotypes of P. aeruginosa, the most common serotypes were O1, O2, O3, O4, O5, O6, O8, O9, O10 and O11; a vaccine that targets these 10 serotypes would confer protection against more than 80% of invasive P. aeruginosa infections. The most common flagellin type among 386 isolates was FlaB (41%). Resistance to aztreonam (56%) was most common, followed by levofloxacin (42%). We also found that 22% of strains were non-susceptible to meropenem and piperacillin-tazobactam. Ninety-nine (27%) of our collected isolates were resistant to multiple antibiotics. Isolates with FlaA2 flagellin were more commonly multidrug resistant (p = 0.04). CONCLUSIONS: Vaccines targeting common O antigens and two flagellin antigens, FlaB and FlaA2, would offer an excellent strategy to prevent P. aeruginosa invasive infections.

A Nonlethal Murine Flame Burn Model Leads to a Transient Reduction in Host Defenses and Enhanced Susceptibility to Lethal Pseudomonas aeruginosa Infection.

Of the 486,000 burn injuries that required medical treatment in the United States in 2016, 40,000 people were hospitalized, with >3,000 fatalities. After burn injury, humans are at increased risk of sepsis and mortality from infections caused by Pseudomonas aeruginosa, an opportunistic pathogen. We hypothesize that systemic events were initiated from the burn that increased the host's susceptibility to P. aeruginosa. A nonlethal 10% total body surface area (TBSA), full-thickness flame burn was performed in CD-1 mice without and with subsequent P. aeruginosa (strain M2) infection. The 50% lethal dose for subcutaneous infection with P. aeruginosa M2 at the burn site immediately after the burn decreased by 6 log, with mortality occurring between 18 and 26 h, compared with P. aeruginosa-infected mice without burn injury. Bacteria in distal organs were detected by 18 h, concurrent with the onset of clinical symptoms. Serum proinflammatory cytokines (interleukin-6 [IL-6], IL-1ß, gamma interferon, and tumor necrosis factor alpha) and the anti-inflammatory cytokine IL-10 were first detected at 12 h postburn with infection and continued to increase until death. Directly after burn alone, serum levels of HMGB1, a danger-associated molecular pattern and TLR4 agonist, transiently increased to 50 ng/ml before returning to 20 ng/ml. Burn with P. aeruginosa infection increased serum HMGB1 concentrations >10-fold (250 ng/ml) at the time of death. This HMGB1-rich serum stimulated TLR4-mediated NF-κB activation in a TLR4 reporter cell line. Treatment of infected burned mice with P5779, a peptide inhibitor of HMGB1, increased the mean survival from 23 to 42 h (P < 0.0001). We conclude that the high level of serum HMGB1, which preceded the increase in proinflammatory cytokines, is associated with postburn mortality.

Schizophrenia risk variants influence multiple classes of transcripts of sorting nexin 19 (SNX19).

Genome-wide association studies (GWAS) have identified many genomic loci associated with risk for schizophrenia, but unambiguous identification of the relationship between disease-associated variants and specific genes, and in particular their effect on risk conferring transcripts, has proven difficult. To better understand the specific molecular mechanism(s) at the schizophrenia locus in 11q25, we undertook cis expression quantitative trait loci (cis-eQTL) mapping for this 2 megabase genomic region using postmortem human brain samples. To comprehensively assess the effects of genetic risk upon local expression, we evaluated multiple transcript features: genes, exons, and exon-exon junctions in multiple brain regions-dorsolateral prefrontal cortex (DLPFC), hippocampus, and caudate. Genetic risk variants strongly associated with expression of SNX19 transcript features that tag multiple rare classes of SNX19 transcripts, whereas they only weakly affected expression of an exon-exon junction that tags the majority of abundant transcripts. The most prominent class of SNX19 risk-associated transcripts is predicted to be overexpressed, defined by an exon-exon splice junction between exons 8 and 10 (junc8.10) and that is predicted to encode proteins that lack the characteristic nexin C terminal domain. Risk alleles were also associated with either increased or decreased expression of multiple additional classes of transcripts. With RACE, molecular cloning, and long read sequencing, we found a number of novel SNX19 transcripts that further define the set of potential etiological transcripts. We explored epigenetic regulation of SNX19 expression and found that DNA methylation at CpG sites near the primary transcription start site and within exon 2 partially mediate the effects of risk variants on risk-associated expression. ATAC sequencing revealed that some of the most strongly risk-associated SNPs are located within a region of open chromatin, suggesting a nearby regulatory element is involved. These findings indicate a potentially complex molecular etiology, in which risk alleles for schizophrenia generate epigenetic alterations and dysregulation of multiple classes of SNX19 transcripts.

Evaluation of the risk of patella fracture as the result of decreasing tibial plateau angle following tibial plateau leveling osteotomy.

OBJECTIVE: To evaluate the effect of postoperative tibial plateau angle (TPA) following tibial plateau leveling osteotomy (TPLO) on the risk of patella fracture during the convalescent period. STUDY DESIGN: Retrospective study. SAMPLE POPULATION: Fracture group: 20 stifles; reference group: 65 stifles. METHODS: Medical records were reviewed for stifles with patellar fractures after a TPLO procedure (fracture group) and stifles with >180 days radiographic examination with no complications following TPLO (reference group). Stifle radiographs were masked to group and final TPA (fTPA) was measured, at the time of fracture diagnosis (fracture group) and at last follow-up (reference group), using PACS software. TPAs in the fracture and reference groups were compared using the Wilcoxon rank-sum test. Statistical significance was set at .05. RESULTS: Patellar fractures were diagnosed at a median of 69 days (range 31-189) after surgery. The median duration of follow-up time for the reference group was 471 days (range 180-1868). Median fTPA for the fracture group was 1.4° (range [-10.3]-7.1). Median fTPA for the reference group was 4.1° (range [-3.9]-14.1). The odds of patellar fractures increased by 21.7% (95% CI: 8.6%-35.6%) for every 1° decrease in fTPA. CONCLUSION: The risk of patella fractures increased as TPAs after TPLOs decreased. CLINICAL SIGNIFICANCE: Care should be taken to avoid excessive rotation during TPLO to decrease the likelihood of postoperative patellar fractures.

Evaluation of the clinical value of routine radiographic examination during convalescence for tibial plateau-leveling osteotomy.

OBJECTIVE: To determine how frequently routine follow-up radiographic findings would result in a change to the postoperative plan following tibial plateau-leveling osteotomy (TPLO) in dogs. STUDY DESIGN: Retrospective study SAMPLE POPULATION: Short-term group: 100 cases; intermediate-term group: 50 cases. METHODS: Medical records of 100 consecutive cases meeting the inclusion criteria were reviewed (the short-term group). The cases had no owner-perceived issues and underwent routinely prescribed radiographic follow up between 40 and 60 postoperative days after TPLO performed by one experienced surgeon. Complications identified on physical examination (PE) and radiographic examination (RE) were recorded, along with any changes to the postoperative plan. Medical records of 50 consecutive cases that had short-term and intermediate-term (≥180 days) REs and PEs were reviewed similarly (intermediate-term group). RESULTS: Fifty-one cases in the short-term group had no complications on PE or RE. Forty-nine dogs were diagnosed with minor complications (patellar ligament desmitis, patella or fibula fracture, gait abnormalities): 42 on RE only; 6 on PE and RE; 1 on PE only. Exercise restriction was extended for 2 weeks in 2 cases with radiographic patellar ligament desmitis. Two cases in the intermediate-term group had minor complications at intermediate-term RE. No new PE or RE complications developed between short-term and intermediate-term evaluations. CONCLUSION: At routine rechecks of dogs with no owner-perceived issues after TPLO, 49% had minor complications but only 2% were deemed significant enough to alter patient management. The likelihood of new radiographic complications developing after short-term evaluation is low. CLINICAL SIGNIFICANCE: Routine radiographic recheck examinations rarely altered the postoperative plan in TPLO cases with unremarkable clinical recoveries.

Neuraminidase 1-mediated desialylation of the mucin 1 ectodomain releases a decoy receptor that protects against Pseudomonas aeruginosa lung infection.

Pseudomonas aeruginosa (Pa) expresses an adhesin, flagellin, that engages the mucin 1 (MUC1) ectodomain (ED) expressed on airway epithelia, increasing association of MUC1-ED with neuraminidase 1 (NEU1) and MUC1-ED desialylation. The MUC1-ED desialylation unmasks both cryptic binding sites for Pa and a protease recognition site, permitting its proteolytic release as a hyperadhesive decoy receptor for Pa. We found here that intranasal administration of Pa strain K (PAK) to BALB/c mice increases MUC1-ED shedding into the bronchoalveolar compartment. MUC1-ED levels increased as early as 12 h, peaked at 24-48 h with a 7.8-fold increase, and decreased by 72 h. The a-type flagellin-expressing PAK strain and the b-type flagellin-expressing PAO1 strain stimulated comparable levels of MUC1-ED shedding. A flagellin-deficient PAK mutant provoked dramatically reduced MUC1-ED shedding compared with the WT strain, and purified flagellin recapitulated the WT effect. In lung tissues, Pa increased association of NEU1 and protective protein/cathepsin A with MUC1-ED in reciprocal co-immunoprecipitation assays and stimulated MUC1-ED desialylation. NEU1-selective sialidase inhibition protected against Pa-induced MUC1-ED desialylation and shedding. In Pa-challenged mice, MUC1-ED-enriched bronchoalveolar lavage fluid (BALF) inhibited flagellin binding and Pa adhesion to human airway epithelia by up to 44% and flagellin-driven motility by >30%. Finally, Pa co-administration with recombinant human MUC1-ED dramatically diminished lung and BALF bacterial burden, proinflammatory cytokine levels, and pulmonary leukostasis and increased 5-day survival from 0% to 75%. We conclude that Pa flagellin provokes NEU1-mediated airway shedding of MUC1-ED, which functions as a decoy receptor protecting against lethal Pa lung infection.

The effect of extracorporeal shockwave on liposomal bupivacaine in a tibial plateau leveling osteotomy model.

OBJECTIVE: To determine the effect of extracorporeal shock wave (ESWT) on liposomal bupivacaine in a tibial-plateau-leveling osteotomy model. STUDY DESIGN: In vitro study. SAMPLE POPULATION: Ten samples per group. METHODS: In addition to a control group (sham treatment), five treatment groups were defined as A, energy (E) 3 (0.22 mJ/mm2 ), 360 pulses per minute (p/m); B, E6 (0.29 mJ/mm2 ), 360 p/m; C, E8 (0.39 mJ/mm2 ), 360 p/m; D, E6, 480 p/m; E, E8 480 p/m. Two-milliliter aliquots of liposomal bupivacaine were placed in a gelatin chamber and treated with 1000 pulses according to group. All samples remained in the chamber for 170 seconds to reflect the longest treatment group. Free bupivacaine concentrations were determined after treatment with high-performance liquid chromatography. RESULTS: The median free bupivacaine concentration was reported as control, 1.90 mg/mL; A, 2.10 mg/mL; B, 2.03 mg/mL; C, 2.94 mg/mL; D, 2.71 mg/mL; E, 4.35 mg/mL. Groups C (P = .027), D (P = .034), and E (P = .002) were different from the control group. Groups C (P = .0025) and D (P = .0025) were different from group E. Additional intertreatment group differences were found. CONCLUSION: Extracorporeal shock wave therapy caused a dose-dependent release of bupivacaine; however, there was no significant release of bupivacaine from liposomes when ESWT was applied at currently recommended therapeutic settings in this model. CLINICAL SIGNIFICANCE: This in vitro study provides evidence that concurrent electrohydraulic ESWT and liposomal bupivacaine is likely safe at currently recommended settings, however, higher energy and pulse frequency settings should be avoided.

Comprehensive assessment of multiple biases in small RNA sequencing reveals significant differences in the performance of widely used methods.

BACKGROUND: RNA sequencing offers advantages over other quantification methods for microRNA (miRNA), yet numerous biases make reliable quantification challenging. Previous evaluations of these biases have focused on adapter ligation bias with limited evaluation of reverse transcription bias or amplification bias. Furthermore, evaluations of the quantification of isomiRs (miRNA isoforms) or the influence of starting amount on performance have been very limited. No study had yet evaluated the quantification of isomiRs of altered length or compared the consistency of results derived from multiple moderate starting inputs. We therefore evaluated quantifications of miRNA and isomiRs using four library preparation kits, with various starting amounts, as well as quantifications following removal of duplicate reads using unique molecular identifiers (UMIs) to mitigate reverse transcription and amplification biases. RESULTS: All methods resulted in false isomiR detection; however, the adapter-free method tested was especially prone to false isomiR detection. We demonstrate that using UMIs improves accuracy and we provide a guide for input amounts to improve consistency. CONCLUSIONS: Our data show differences and limitations of current methods, thus raising concerns about the validity of quantification of miRNA and isomiRs across studies. We advocate for the use of UMIs to improve accuracy and reliability of miRNA quantifications.

The Fyn kinase inhibitor, AZD0530, suppresses mouse alcohol self-administration and seeking.

Fyn is a member of the Src family of protein tyrosine kinases (PTKs) that plays an important role not only in normal synaptic functions but also in brain pathologies including alcohol use disorder. We previously reported that repeated cycles of binge drinking and withdrawal activate Fyn in the dorsomedial striatum (DMS) of rodents, and that Fyn signaling in the DMS contributes to rat alcohol intake and relapse. Here, we used AZD0530, a CNS penetrable inhibitor of Src PTKs developed for the treatment of Alzheimer disease and cancer and tested its efficacy to suppress alcohol-dependent molecular and behavioral effects. We show that systemic administration of AZD0530 prevents alcohol-induced Fyn activation and GluN2B phosphorylation in the DMS of mice. We further report that a single dose of AZD0530 reduces alcohol operant self-administration and promotes extinction of alcohol self-administration without altering basal and dopamine D1 receptor-dependent locomotion. Together, our findings suggest that AZD0530, through its inhibitory actions on Fyn kinase, dampens alcohol seeking and drinking.

Use of an amikacin-infused collagen sponge concurrent with implant removal for treatment of tibial plateau leveling osteotomy surgical site infection in 31 cases.

OBJECTIVE: To report the use and long-term outcome of dogs with surgical site infection (SSI) after tibial plateau leveling osteotomy (TPO), treated with an amikacin-infused collagen sponge and implant removal. STUDY DESIGN: Retrospective study. ANIMALS: Thirty-one client-owned dogs. METHODS: Medical records were reviewed for dogs with SSI after a TPLO that were treated with surgical implant removal and concurrent implantation of an amikacin-infused collagen sponge. Relevant clinical and surgical data were recorded. The TPLO implants were routinely removed; the surgical site was swabbed for culture. The sponge was aseptically infused with amikacin prior to implantation. Postprocedure examinations consisted of visual inspection of the incision by the surgeon and lameness scoring. RESULTS: Thirty-one dogs met all inclusion criteria. Median follow-up time was 687 days. Short-term examination after implant removal and sponge implantation revealed uneventful incisional healing in 24 dogs. Six (19.4%) dogs exhibited inflamed incision sites a median of 4 days (range, 3-9) postoperatively that resolved without additional treatment. One (3.2%) dog required empirical antibiotic treatment 7 days postoperatively but was lost to long-term follow-up. Long-term follow-up examination revealed no clinical evidence of SSI recurrence and no lameness in the remaining 30 cases. CONCLUSION: Surgical implant removal and implantation of an absorbable collagen sponge infused with amikacin alone was an effective treatment for postoperative TPLO SSI. CLINICAL SIGNIFICANCE: This procedure had a 96.8% long-term resolution of SSI. It should be considered as a treatment option for TPLO SSI.

Brucella canis sacroiliitis and discospondylitis in a dog.

A 1.5-year-old neutered male Labrador retriever dog was presented with a 1-month history of pelvic limb lameness. Physical examination revealed lumbosacral and pelvic pain. Diagnostic imaging findings were consistent with discospondylitis and bilateral sacroiliitis. Brucellosis was definitively diagnosed with rapid slide agglutination (RSAT) and agar gel immunodiffusion (AGID) tests. Brucella canis has not been associated with sacroiliitis and should be included as a differential diagnosis for dogs presented with clinical or radiographic signs of sacroiliitis.

Sacro-iliite et discospondylite à Brucella canis chez un chien. Un chien mâle castré de race Labrador âgé de 1,5 ans fut présenté avec une histoire de boiterie du membre pelvien ayant débuté 1 mois plus tôt. L'examen physique révéla une douleur lombo-sacrée et pelvienne. Les trouvailles d'imagerie étaient cohérentes avec une discospondylite et une sacro-iliite bilatérale. La brucellose fut diagnostiquée de manière définitive par un test d'agglutination rapide sur lame (RSAT) et un test d'immunodiffusion en gel (AGID). Brucella canis n'a pas été associé avec une sacro-iliite et devrait être inclus dans le diagnostic différentiel de chiens présentant des signes cliniques ou radiographiques de sacro-iliite.(Traduit par Dr Serge Messier).

Differential Pharmacology and Binding of mGlu2 Receptor Allosteric Modulators.

Allosteric modulation of metabotropic glutamate receptor 2 (mGlu2) has demonstrated efficacy in preclinical rodent models of several brain disorders, leading to industry and academic drug discovery efforts. Although the pharmacology and binding sites of some mGlu2 allosteric modulators have been characterized previously, questions remain about the nature of the allosteric mechanism of cooperativity with glutamate and whether structurally diverse allosteric modulators bind in an identical manner to specific allosteric sites. To further investigate the in vitro pharmacology of mGlu2 allosteric modulators, we developed and characterized a novel mGlu2 positive allosteric modulator (PAM) radioligand in parallel with functional studies of a structurally diverse set of mGlu2 PAMs and negative allosteric modulators (NAMs). Using an operational model of allosterism to analyze the functional data, we found that PAMs affect both the affinity and efficacy of glutamate at mGlu2, whereas NAMs predominantly affect the efficacy of glutamate in our assay system. More importantly, we found that binding of a novel mGlu2 PAM radioligand was inhibited by multiple structurally diverse PAMs and NAMs, indicating that they may bind to the mGlu2 allosteric site labeled with the novel mGlu2 PAM radioligand; however, further studies suggested that these allosteric modulators do not all interact with the radioligand in an identical manner. Together, these findings provide new insights into the binding sites and modes of efficacy of different structurally and functionally distinct mGlu2 allosteric modulators and suggest that different ligands either interact with distinct sites or adapt different binding poses to shared allosteric site(s).

Novel inhibitors of As(III) S-adenosylmethionine methyltransferase (AS3MT) identified by virtual screening.

Due to increased interest in As(III) S-adenosylmethionine methyltransferase (AS3MT), a search for chemical probes that can help elucidate function was initiated. A homology model was built based on related enzymes, and virtual screening produced 426 potential hits. Evaluation of these compounds in a functional enzymatic assay revealed several modest inhibitors including an O-substituted 2-amino-3-cyano indole scaffold. Two iterations of near neighbor searches revealed compound 5 as a potent inhibitor of AS3MT with good selectivity over representative methyltransferases DOT1L and NSD2 as well as a representative set of diverse receptors. Compound 5 should prove to be a useful tool to investigate the role of AS3MT and a potential starting point for further optimization.