Ether Bond Cleavage of a Phenylcoumaran ß-5 Lignin Model Compound and Polymeric Lignin Catalysed by a LigE-type Etherase from Agrobacterium sp.

A LigE-type beta-etherase enzyme from lignin-degrading Agrobacterium sp. has been identified, which assists degradation of polymeric lignins. Testing against lignin dimer model compounds revealed that it does not catalyse the previously reported reaction of Sphingobium SYK-6 LigE, but instead shows activity for a ß-5 phenylcoumaran lignin dimer. The reaction products did not contain glutathione, indicating a catalytic role for reduced glutathione in this enzyme. Three reaction products were identified: the major product was a cis-stilbene arising from C-C fragmentation involving loss of formaldehyde; two minor products were an alkene arising from elimination of glutathione, and an oxidised ketone, proposed to arise from reaction of an intermediate with molecular oxygen. Testing of the recombinant enzyme against a soda lignin revealed the formation of new signals by two-dimensional NMR analysis, whose chemical shifts are consistent with the formation of a stilbene unit in polymeric lignin.

Depletion of SAM leading to loss of heterochromatin drives muscle stem cell ageing.

The global loss of heterochromatin during ageing has been observed in eukaryotes from yeast to humans, and this has been proposed as one of the causes of ageing. However, the cause of this age-associated loss of heterochromatin has remained enigmatic. Here we show that heterochromatin markers, including histone H3K9 di/tri-methylation and HP1, decrease with age in muscle stem cells (MuSCs) as a consequence of the depletion of the methyl donor S-adenosylmethionine (SAM). We find that restoration of intracellular SAM in aged MuSCs restores heterochromatin content to youthful levels and rejuvenates age-associated features, including DNA damage accumulation, increased cell death, and defective muscle regeneration. SAM is not only a methyl group donor for transmethylation, but it is also an aminopropyl donor for polyamine synthesis. Excessive consumption of SAM in polyamine synthesis may reduce its availability for transmethylation. Consistent with this premise, we observe that perturbation of increased polyamine synthesis by inhibiting spermidine synthase restores intracellular SAM content and heterochromatin formation, leading to improvements in aged MuSC function and regenerative capacity in male and female mice. Together, our studies demonstrate a direct causal link between polyamine metabolism and epigenetic dysregulation during murine MuSC ageing.

Structure-Based Design and Synthesis of Potent and Selective KRAS G12D Inhibitors.

KRAS G12D mutation has been found in approximately 45% of pancreatic ductal adenocarcinoma (PDAC) cases, making it an attractive therapeutic target. Through structure-based drug design, a series of potent and selective KRAS G12D inhibitors were designed. The lead compound, ERAS-5024, inhibited ERK1/2 phosphorylation and cell proliferation in three-dimensional Cell-Titer Glo assays in AsPC-1 PDAC cells with single-digit nanomolar potency and caused tumor regression in the in vivo efficacy studies. We describe here the details of the design and synthesis program that led to the discovery of ERAS-5024.

The pharmacologic and toxicologic characterization of the potent and selective KRAS G12D inhibitors ERAS-4693 and ERAS-5024.

Two potent and selective KRASG12D inhibitors, ERAS-4693 and ERAS-5024, were generated as possible clinical candidates to treat patients harboring G12D mutations in solid tumors. Both molecules exhibited strong anti-tumor activity in the KRASG12D mutant PDAC xenograft mouse models while ERAS-5024 also showed tumor growth inhibition when administered on an intermittent dosing regimen. Acute dose-limiting toxicity consistent with an allergic reaction was observed for both molecules shortly after administration at doses just above those which demonstrated anti-tumor activity, indicative of a narrow therapeutic index. A series of studies were subsequently conducted to identify a common underlying mechanism for the observed toxicity, including CETSA® (Cellular Thermal Shift Assay) as well as several functional off-target screens. Both ERAS-4693 and ERAS-5024 were identified to agonize MRGPRX2 which has been linked to pseudo-allergic reactions. In vivo toxicologic characterization of both molecules included repeat-dose studies in the rat and dog. Dose-limiting toxicities were observed in both species with ERAS-4693 and ERAS-5024 and plasma exposure levels at the maximum tolerated doses were generally below that which caused strong anti-tumor activity, supporting the initial observation of a narrow therapeutic index. Additional overlapping toxicities included a reduction in reticulocytes and clinical pathological changes suggestive of an inflammatory response. Furthermore, increases in plasma histamine were observed in dogs administered ERAS-5024, supporting the hypothesis that MRGPRX2 agonism may be the cause of the pseudo-allergic reaction. This work highlights the importance of balancing both the safety and efficacy of KRASG12D inhibitors as this class of molecules begins to enter clinical development.

Evaluation of the Effects of Synovial Multipotent Cells on Deep Digital Flexor Tendon Repair in a Large Animal Model of Intra-Synovial Tendinopathy.

Intra-synovial tendon injuries are a common orthopedic problem with limited treatment options. The synovium is a specialized connective tissue forming the inner encapsulating lining of diarthrodial joints and intra-synovial tendons. It contains multipotent mesenchymal stromal cells that render it a viable source of progenitors for tendon repair. This study evaluated the effects of autologous implantation of cells derived from normal synovium (synovial membrane cells [SMCs]) in augmenting repair in an ovine model of intra-synovial tendon injury. For this purpose, synovial biopsies were taken from the right digital flexor tendon sheath following creation of a defect to the lateral deep digital flexor tendon. Mononuclear cells were isolated by partial enzymatic digestion and assessed for MSC characteristics. Cell tracking and tendon repair were assessed by implanting 5 × 106 cells into the digital flexor tendon sheath under ultrasound guidance with the effects evaluated using magnetic resonance imaging and histopathology. Synovial biopsies yielded an average 4.0 × 105 ± 2.7 × 105 SMCs that exhibited a fibroblastic morphology, variable osteogenic, and adipogenic responses but were ubiquitously strongly chondrogenic. SMCs displayed high expression of CD29 with CD271NEGATIVE and MHC-IILOW cell-surface marker profiles, and variable expression of CD73, CD90, CD105, CD166, and MHC-I. Implanted SMCs demonstrated engraftment within the synovium, though a lack of repair of the tendon lesion over 24 weeks was observed. We conclude healthy synovium is a viable source of multipotent cells, but that the heterogeneity of synovium underlies the variability between different SMC populations, which while capable of engraftment and persistence within the synovium exhibit limited capacity of influencing tendon repair. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 38:128-138, 2020.

Virtual reality for management of pain in hospitalized patients: A randomized comparative effectiveness trial.

OBJECTIVES: Therapeutic virtual reality (VR) has emerged as an effective, drug-free tool for pain management, but there is a lack of randomized, controlled data evaluating its effectiveness in hospitalized patients. We sought to measure the impact of on-demand VR versus "health and wellness" television programming for pain in hospitalized patients. METHODS: We performed a prospective, randomized, comparative effectiveness trial in hospitalized patients with an average pain score of ≥3 out of 10 points. Patients in the experimental group received a library of 21 VR experiences administered using the Samsung Gear Oculus headset; control patients viewed specialized television programming to promote health and wellness. Clinical staff followed usual care; study interventions were not protocolized. The primary outcome was patient-reported pain using a numeric rating scale, as recorded by nursing staff during usual care. Pre- and post-intervention pain scores were compared immediately after initial treatment and after 48- and 72-hours. RESULTS: There were 120 subjects (61 VR; 59 control). The mean within-subject difference in immediate pre- and post-intervention pain scores was larger in the VR group (-1.72 points; SD 3.56) than in the control group (-0.46 points; SD 3.01); this difference was significant in favor of VR (P < .04). When limited to the subgroup of patients with severe baseline pain (≥7 points), the effect of VR was more pronounced vs. control (-3.04, SD 3.75 vs. -0.93, SD 2.16 points; P = .02). In regression analyses adjusting for pre-intervention pain, time, age, gender, and type of pain, VR yielded a .59 (P = .03) and .56 (P = .04) point incremental reduction in pain versus control during the 48- and 72-hour post-intervention periods, respectively. CONCLUSIONS: VR significantly reduces pain versus an active control condition in hospitalized patients. VR is most effective for severe pain. Future trials should evaluate standardized order sets that interpose VR as an early non-drug option for analgesia.

Congenital block vertebrae and intervertebral disc protrusion in a young cat.

CASE SUMMARY: A 6-month-old domestic shorthair cat was evaluated for proprioceptive ataxia of the pelvic limbs. Over 2 months, the cat became poorly ambulatory, paraparetic with proprioceptive ataxia and developed a firm, distended bladder with intermittent overflow urinary incontinence. Block vertebrae (T1-3, T4-5 and T10-11) and lordosis were identified on radiographs of the vertebral column. MRI revealed T3/4 intervertebral disc protrusion with severe extradural compression, secondary syringohydromyelia caudal to the protrusion and generalised intervertebral disc disease throughout the cervical and thoracic vertebrae. Dorsal laminectomy at T3/4 resulted in resolution of paraparesis and marked improvement in coordination and strength. Block vertebrae are usually considered an incidental finding. In this patient, angular deformation (lordosis) and adjacent segment disease probably contributed to clinically significant intervertebral disc degeneration and protrusion. RELEVANCE AND NOVEL INFORMATION: There are few case reports in the literature of multiple congenital vertebral malformations causing neurological deficits in cats. This is the first reported case of successful surgical management of intervertebral disc protrusion, possibly secondary to block vertebrae and lordosis in a cat.

A rare case of a gabapentin-induced cardiomyopathy.

WHAT IS KNOWN AND OBJECTIVE: Gabapentin, a γ-aminobutyric acid derivative, is used for the treatment of partial onset seizures, postherpetic neuralgia, diabetic neuropathy and a host of other neurological disorders. CASE DESCRIPTION: A 44-year-old woman with spinal stenosis was prescribed gabapentin for pain. Two months after initiating therapy, she was diagnosed with a new-onset non-ischaemic cardiomyopathy with an ejection fraction of 36% measured on a transthoracic echocardiogram. WHAT IS NEW AND CONCLUSION: A patient with suspected gabapentin-induced cardiomyopathy is reported. However, to date, gabapentin therapy has not been associated with risk of the developing a cardiomyopathy.

Abdominal Wall Schwannoma.

This rare form of subcutaneous nodule can be identified through the combination of imaging and biopsy, but the definitive diagnosis is made on complete excision of the mass.

Clinical signs, imaging findings, and outcome in twelve cats with internal ophthalmoparesis/ophthalmoplegia.

OBJECTIVE: To retrospectively evaluate the clinical signs, imaging findings, and outcome of feline internal ophthalmoparesis/ophthalmoplegia. PROCEDURE: Medical records were reviewed from 2008 to 2015. Inclusion criteria included cats that presented with internal ophthalmoparesis/ophthalmoplegia, underwent diagnostic imaging, and had follow-up information available. RESULTS: Twelve cases of feline internal ophthalmoparesis/ophthalmoplegia were identified. Nine cats were unilaterally affected, and three cats were bilaterally affected. Affected cats had a median age of 10.54 years (range 5.75 to 13.17), and both sexes of varying breeds were affected (nine males; three females). Clinical signs including abnormal mental status (n = 9; 75%) and additional neurologic abnormalities (n = 10; 83%) were observed. Magnetic resonance imaging and/or computed tomography (MRI/CT) of the head were performed in ten cats, revealing a mass lesion in all cases with varying locations. Multicentric lymphoma was diagnosed in two cats via abdominal ultrasound and cytology. All twelve cats were euthanized due to deterioration of clinical signs and/or quality-of-life concerns. Median time from diagnosis to euthanasia was 3.5 days (range 0 to 80 days). CONCLUSIONS: Feline internal ophthalmoparesis/ophthalmoplegia rarely presents as the sole clinical sign in a referral hospital. Advanced imaging (MRI/CT) may be necessary to reach a definitive diagnosis in these cases. However, abdominal ultrasound would be advocated in cats with systemic clinical signs as a less expensive and less invasive diagnostic test to further investigate the possible etiology of internal ophthalmoparesis/ophthalmoplegia prior to advanced imaging. Feline cases with internal ophthalmoparesis/ophthalmoplegia associated with other intracranial signs and/or systemic clinical signs have a poor prognosis.

Rabbits with naturally occurring cataracts referred for phacoemulsification and intraocular lens implantation: a preliminary study of 12 cases.

OBJECTIVE: To describe the presentation of 15 rabbits with naturally occurring cataracts referred for phacoemulsification surgery, the procedure in 13 cases and the follow-up in 12. ANIMALS INCLUDED: Fifteen rabbits (30 eyes), nine of which stopped following visual cues in association with cataract progression. MATERIALS AND METHODS: Rabbits underwent preoperative ophthalmic and ocular ultrasound examination. RESULTS: Thirteen rabbits (22 eyes) had mature cataracts. Ten were bilateral and three unilateral. Two rabbits had an anterior chamber abscess. The cataract in one of these was incipient. One rabbit had bilateral immature cataracts. One rabbit had a subluxated lens, and one had a retinal detachment. Thirteen rabbits (22 eyes) underwent phacoemulsification. Eighteen, 13.5-mm capsular tension rings (CTRs) and seventeen, 13-mm IOLs (Acrivet® , Berlin, Germany) were fitted including one 41D 60V-model, and three 49D and thirteen 58D 20S-models. Intraoperative complications included one unilateral posterior-capsular tear, one lens subluxation, and one expulsive choroidal hemorrhage. One rabbit died during anesthetic recovery. Nine cases were PCR-tested for Encephalitozoon cuniculi, and only three were positive. The median follow-up time was 12 months (4-24 months). Rabbits that were not following visual cues preoperatively did so postoperatively, and surgery resulted in a clear visual axis for the follow-up period in every case except in two, due to reasons other than the surgery. CONCLUSIONS: Phacoemulsification with CTR and IOL implantation offers good long-term results and can improve the quality of life of pet rabbits. Retinal detachment, lens luxation, expulsive choroidal hemorrhage, and anesthetic death are potential complications.

Rapid postoperative recurrence of a cranial multilobular tumor of bone in a young dog.

Although resection of multilobular tumors of bone can be associated with a good prognosis and long disease-free intervals in dogs, osteosarcomatous transformation should be considered a cause for rapid recurrence of clinical signs.

Computed tomographic morphometry of tympanic bulla shape and position in brachycephalic and mesaticephalic dog breeds.

Anatomic variations in skull morphology have been previously described for brachycephalic dogs; however there is little published information on interbreed variations in tympanic bulla morphology. This retrospective observational study aimed to (1) provide detailed descriptions of the computed tomographic (CT) morphology of tympanic bullae in a sample of dogs representing four brachycephalic breeds (Pugs, French Bulldogs, English Bulldog, and Cavalier King Charles Spaniels) versus two mesaticephalic breeds (Labrador retrievers and Jack Russell Terriers); and (2) test associations between tympanic bulla morphology and presence of middle ear effusion. Archived head CT scans for the above dog breeds were retrieved and a single observer measured tympanic bulla shape (width:height ratio), wall thickness, position relative to the temporomandibular joint, and relative volume (volume:body weight ratio). A total of 127 dogs were sampled. Cavalier King Charles Spaniels had significantly flatter tympanic bullae (greater width:height ratios) versus Pugs, English Bulldogs, Labrador retrievers, and Jack Russell terriers. French Bulldogs and Pugs had significantly more overlap between tympanic bullae and temporomandibular joints versus other breeds. All brachycephalic breeds had significantly lower tympanic bulla volume:weight ratios versus Labrador retrievers. Soft tissue attenuating material (middle ear effusion) was present in the middle ear of 48/100 (48%) of brachycephalic breeds, but no significant association was found between tympanic bulla CT measurements and presence of this material. Findings indicated that there are significant interbreed variations in tympanic bulla morphology, however no significant relationship between tympanic bulla morphology and presence of middle ear effusion could be identified.

Surgical treatment of a paraspinal abscess with osteomyelitis and spinal cord compression in a rabbit.

CASE DESCRIPTION A 16-month-old neutered male Continental Giant rabbit (Lepus curpaeums) was referred for evaluation of a 7-day history of acute-onset, progressive, symmetric paraparesis. CLINICAL FINDINGS On initial examination, the rabbit was nonambulatory, and results of neurologic examination were consistent with a lesion affecting the T3-L3 spinal cord segments. Thoracic radiography showed irregular widening of the left T11-12 articular process joint. Marked dorsolateral and lateral extradural spinal cord compression with contrast enhancement of the adjacent epaxial muscles was evident on MRI images of the spine. TREATMENT AND OUTCOME A left-sided T11-T12 hemilaminectomy was performed, which revealed an abnormal and hypertrophic T11-12 articular process joint and an osteolytic lesion communicating with the vertebral canal. Copious purulent material causing marked spinal cord compression was evident, and the surgical site was lavaged extensively with sterile (0.9% NaCl) saline solution. Results of aerobic, anaerobic, and enriched bacteriologic cultures of swab specimens obtained from the surgical site were negative. Histologic analysis of biopsy samples revealed chronic purulent osteomyelitis, myositis, and fasciitis with necrosis, fibrosis, and dystrophic mineralization. The rabbit was discharged 48 hours after surgery. Ten weeks after surgery, the rabbit was ambulatory with mild paraparesis. On telephone follow-up 21 months after surgery, the owners indicated that the rabbit was healthy and expressed satisfaction with the treatment and outcome. CLINICAL RELEVANCE Paraspinal abscess with vertebral canal involvement should be considered as a differential diagnosis for rabbits with clinical signs of progressive T3-L3 myelopathy. Outcome for the patient of the present report suggested that surgical treatment including decompression and debridement can result in a favorable long-term outcome.

Phase I clinical trial of sipuleucel-T combined with escalating doses of ipilimumab in progressive metastatic castrate-resistant prostate cancer.

BACKGROUND: Sipuleucel-T (SIP-T), which functions by stimulating cancer-specific dendritic cells, prolongs survival in men with prostate cancer. Ipilimumab (IPI) achieved a borderline survival advantage in a large randomized trial. SIP-T and IPI are potentially synergistic. PATIENTS AND METHODS: Nine men with progressive metastatic castrate-resistant prostate cancer (mCRPC) were treated prospectively with SIP-T followed immediately by IPI with one of the following doses of IPI: 1 mg/kg at 1 week after SIP-T; 1 mg/kg at 1 and 4 weeks after SIP-T; or 1 mg/kg at 1, 4, and 7 weeks after SIP-T. Three patients were evaluated at each level. Cancer-specific immunoglobulins directed at granulocyte-macrophage-colony-stimulating factor/prostatic acid phosphatase (PAP) fusion protein (PA2024) and PAP were measured prior to SIP-T, after SIP-T, 1 week after IPI, every other month for 5 months, then every 3 months for an additional 12 months. RESULTS: Adverse events of SIP-T were consistent with previous reports. IPI only caused a transient grade 1 rash in one patient. Median age, Gleason score, and number of previous hormonal interventions were 77 years, 8, and 3, respectively. Eight men had bone metastases and one had lymph node metastasis. Statistically significant increases in serum immunoglobulin G (IgG) and IgG-IgM specific for PA2024 and PAP occurred after SIP-T. An additional statistically significant increase in the aforementioned immunoglobulins - above the levels achieved by SIP-T - occurred after IPI. Median clinical follow-up was 36 months (range: 26-40). Three patients died from progressive disease after 9, 18, and 20 months. Out of the remaining six patients, five of them needed further treatment that included abiraterone acetate, enzalutamide, radium-223 dichloride, and spot radiation. One patient had an undetectable PSA, who did not receive any other treatment except spot radiation. Median PSA at last follow-up for the surviving patients was 3.8 (range: 0.6-7.47). CONCLUSION: In this small trial, the addition of IPI to SIP-T was well tolerated. IPI increased immunoglobulins specific for the PA2024 protein and PAP above the level achieved with SIP-T alone.

Performance characteristics and relationship of PSA value/kinetics on carbon-11 acetate PET/CT imaging in biochemical relapse of prostate cancer.

An elevated serum prostate-specific antigen (PSA) level alone cannot distinguish between local-regional recurrences and distant metastases after treatment with curative intent. With available salvage treatments, it has become important to localize the site of recurrence. 11C-Acetate PET/CT was performed in patients with rising PSA, with statistical analysis of detection rates, sites/location of detection, PSA kinetics and comparison with other tracers (FDG and Choline). Correlation to biopsy, subsequent imaging and PSA response to focal treatment was also performed. 88% (637) of 721 11C-Acetate PET/CT scans performed were positive. There was a statistically significant difference in PSA values between the positive and negative scans (P < 0.001 for mean difference) with the percentage of positive scans and PSA having a positive correlation. A PSA of 1.09 ng/mL was found to be an optimal cutoff. PSAdT was significantly correlated with a positive scan only when the PSA was < 1.0 ng/mL. For this subgroup, a PSAdT of < 3.8 months appeared significant (P < 0.05) as an optimal cutoff point. 11C-Acetate PET/CT demonstrates a high detection rate for the site of recurrence/metastasis in biochemical relapsed prostate cancer (88% overall detection rate, PPV 90.8%). This analysis suggests an optimal PSA threshold of > 1.09 ng/mL or a PSAdT of < 3.8 months when the PSA is below 1.0 ng/mL as independent predictors of positive findings.

Successful medical management of a domestic longhair cat with subdural intracranial empyema and multifocal pneumonia.

OBJECTIVE: To describe a case of successful medical management of subdural intracranial empyema and multifocal pneumonia in a domestic longhaired cat. CASE SUMMARY: A 7-year-and-8-month-old male neutered domestic longhair cat presented with tachypnea, respiratory compromise, vestibular ataxia, obtundation, left-sided head tilt, and multiple cranial nerve deficits. Neuroanatomical localization was multifocal with central vestibular involvement. Magnetic resonance imaging of the head indicated diffuse subdural empyema, mainly affecting the middle cranial fossa and the right cerebrum. Analysis of cerebrospinal fluid revealed degenerate neutrophils with a mixed population of intracellular bacilli. Computed tomography (CT) of the thorax was suggestive for multifocal pneumonia. Aggressive medical management with IV fluids, oxygen supplementation, mannitol boluses, dexamethasone, and broad-spectrum antimicrobials was initiated. The cat demonstrated gradual improvement within 24 hours following initiation of treatment. General physical and neurological examinations, 9 weeks after initiating treatment, did not reveal any abnormalities. A CT examination performed at this time revealed resolution of the cat's pulmonary lesions. The cat was still free of clinical signs, 9 months after treatment was started. NEW OR UNIQUE INFORMATION PROVIDED: Subdural empyema is infrequently reported in cats and has high mortality rates even following surgical treatment. To the authors' knowledge, this is the first reported case of successful medical management of a cat with subdural empyema and suggests that aggressive medical management should be attempted in cats that are not considered surgical candidates.

IMPROVING CONSPICUITY OF THE CANINE GASTROINTESTINAL WALL USING DUAL PHASE CONTRAST-ENHANCED COMPUTED TOMOGRAPHY: A RETROSPECTIVE CROSS-SECTIONAL STUDY.

Gastrointestinal (GI) disease is a common clinical complaint in small animal patients; computed tomography (CT) examinations enable a global overview of the GI tract and associated structures. Previously, the GI wall has been reportedly identified from serosa to mucosa in 77% of standard postcontrast CT studies and wall layers seen in ultrasound have not been distinguished. Inconsistent strong contrast enhancement of the inner layer of the GI mucosal surface was noted on dual phase CT studies acquired in our institution, which increased the visibility of the GI tract and disease processes. The aim of this retrospective, observational, cross-sectional study was to determine the optimal portal vein attenuation for maximizing GI wall conspicuity using dual phase contrast-enhanced CT. Patients with abdominal CT for a non-GI related disease were included. In a pilot study, 175 GI segments from 35 CT studies were graded for presence of mucosal surface enhancement (MSE). The strongest mucosal surface enhancement grade correlated with portal vein attenuation of 43-150 HU; this value was used as inclusion criterion in the main study. A total of 441 GI segments were evaluated in 42 CT studies postcontrast for GI wall conspicuity. The GI wall was conspicuous in 56.7% precontrast, 84.5% at 30s, and 77.3% late postcontrast; 4.7% of segments were removed due to motion blur. At 30 s distinct mucosal surface enhancement was seen in the small intestine and gastric mucosal surface enhancement was poor. Findings supported the use of dual phase contrast-enhanced CT for improving conspicuity of the GI wall.