Reticulon 2 deficiency results in an autosomal recessive distal motor neuropathy with lower limb spasticity.

Heterozygous RTN2 variants have been previously identified in a limited cohort of families affected by autosomal dominant spastic paraplegia (SPG12-OMIM:604805) with a variable age of onset. Nevertheless, the definitive validity of SPG12 remains to be confidently confirmed due to scarcity of supporting evidence. In our study, we identified and validated seven novel or ultra-rare homozygous loss-of-function RTN2 variants in 14 individuals from seven consanguineous families with distal hereditary motor neuropathy (dHMN) using exome, genome and Sanger sequencing coupled with deep-phenotyping. All affected individuals (seven males and seven females, aged 9-50 years) exhibited weakness in the distal upper and lower limbs, lower limb spasticity, hyperreflexia, with an onset in the first decade of life. Nerve conduction studies revealed axonal motor neuropathy with neurogenic changes in the electromyography. Despite a slowly progressive disease course, all patients remained ambulatory over a mean disease duration of 19.71 ± 13.70 years. Characterisation of C. elegans RTN2 homolog loss-of-function variants demonstrated morphological and behavioural differences compared to the parental strain. Treatment of the mutant with an endoplasmic/sarcoplasmic reticulum Ca2+ reuptake inhibitor (2,5-di-tert-butylhydroquinone) rescued key phenotypic differences, suggesting a potential therapeutic benefit for RTN2-disorder. Despite Reticulon-2 being an endoplasmic reticulum (ER)-resident membrane shaping protein, our analysis of patient fibroblast cells did not find significant alterations in ER structure or the response to ER stress. Our findings delineate a distinct form of autosomal recessive dHMN with pyramidal features associated with Reticulon-2 deficiency. This phenotype shares similarities with SIGMAR1-related dHMN, and Silver-like syndromes, providing valuable insights into the clinical spectrum and potential therapeutic strategies for RTN2-related dHMN.

Rectal and gastrointestinal temperature differ during passive heating and subsequent recovery.

We aimed to compare rectal temperature (Trec) and gastro-intestinal temperature (TGI) during passive heating and subsequent recovery with and without ice slurry ingestion. Twelve males (age: 25 ± 4 years, body mass index: 25.7 ± 2.5 kg m-2) were immersed in hot water on two occasions (Trec elevation: 1.82 ± 0.08°C). In the subsequent 60-min recovery in ambient conditions, participants ingested either 6.8 g kg-1 of ice slurry (-0.6°C, ICE) or control drink (37°C, CON). During passive heating, Trec was lower than TGI (P < 0.001), in the recovery, Trec was higher than TGI (P < 0.001). During passive heating, mean bias and 95%LoA (Limits of Agreement) were -0.10(±0.25)°C and -0.12(±0.36)°C for CON and ICE, respectively. In the recovery, mean bias and 95%LoA were 0.30(±0.60)°C and 0.42(±0.63)°C for CON and ICE, respectively. Trec and TGI differed during both heating and recovery, and less favourable agreement between Trec and TGI was found in the recovery from passive heating with or without ice slurry ingestion.

A new Caenorhabditis elegans model to study copper toxicity in Wilson disease.

Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a copper (Cu) transporting ATPase whose trafficking from the Golgi to endo-lysosomal compartments drives sequestration of excess Cu and its further excretion from hepatocytes into the bile. Loss of ATP7B function leads to toxic Cu overload in the liver and subsequently in the brain, causing fatal hepatic and neurological abnormalities. The limitations of existing WD therapies call for the development of new therapeutic approaches, which require an amenable animal model system for screening and validation of drugs and molecular targets. To achieve this objective, we generated a mutant Caenorhabditis elegans strain with a substitution of a conserved histidine (H828Q) in the ATP7B ortholog cua-1 corresponding to the most common ATP7B variant (H1069Q) that causes WD. cua-1 mutant animals exhibited very poor resistance to Cu compared to the wild-type strain. This manifested in a strong delay in larval development, a shorter lifespan, impaired motility, oxidative stress pathway activation, and mitochondrial damage. In addition, morphological analysis revealed several neuronal abnormalities in cua-1 mutant animals exposed to Cu. Further investigation suggested that mutant CUA-1 is retained and degraded in the endoplasmic reticulum, similarly to human ATP7B-H1069Q. As a consequence, the mutant protein does not allow animals to counteract Cu toxicity. Notably, pharmacological correctors of ATP7B-H1069Q reduced Cu toxicity in cua-1 mutants indicating that similar pathogenic molecular pathways might be activated by the H/Q substitution and, therefore, targeted for rescue of ATP7B/CUA-1 function. Taken together, our findings suggest that the newly generated cua-1 mutant strain represents an excellent model for Cu toxicity studies in WD.

Extracellular vesicles in the treatment and prevention of osteoarthritis: can horses help us translate this therapy to humans?

Osteoarthritis (OA) is a common joint disease affecting humans and horses, resulting in significant morbidity, financial expense, and loss of athletic use. While the pathogenesis is incompletely understood, inflammation is considered crucial in the development and progression of the disease. Mesenchymal stromal cells (MSCs) have received increasing scientific attention for their anti-inflammatory, immunomodulatory, and pro-regenerative effects. However, there are concerns about their ability to become a commercially available therapeutic. Extracellular vesicles (EVs) are now recognized to play a crucial role in the therapeutic efficacy observed with MSCs and offer a potentially novel cell-free therapeutic that may negate many of the concerns with MSCs. There is evidence that EVs have profound anti-inflammatory, immunomodulatory, and pro-regenerative effects equal to or greater than the MSCs they are derived from in the treatment of OA. Most of these studies are in small animal models, limiting the translation of these results to humans. However, highly translational animal models are crucial for further understanding the efficacy of potential therapeutics and for close comparisons with humans. For this reason, the horse, which experiences the same gravitational impacts on joints similar to people, is a highly relevant large animal species for testing. The equine species has well-designed and validated OA models, and additionally, therapies can be further tested in naturally occurring OA to validate preclinical model testing. Therefore, the horse is a highly suitable model to increase our knowledge of the therapeutic potential of EVs.

Wheelchair rugby players maintain sprint performance but alter propulsion biomechanics after simulated match play.

The study aimed to explore the influence of a sports-specific intermittent sprint protocol (ISP) on wheelchair sprint performance and the kinetics and kinematics of sprinting in elite wheelchair rugby (WR) players with and without spinal cord injury (SCI). Fifteen international WR players (age 30.3 ± 5.5 years) performed two 10-s sprints on a dual roller wheelchair ergometer before and immediately after an ISP consisting of four 16-min quarters. Physiological measurements (heart rate, blood lactate concentration, and rating of perceived exertion) were collected. Three-dimensional thorax and bilateral glenohumeral kinematics were quantified. Following the ISP, all physiological parameters significantly increased (p ≤ 0.027), but neither sprinting peak velocity nor distance traveled changed. Players propelled with significantly reduced thorax flexion and peak glenohumeral abduction during both the acceleration (both -5°) and maximal velocity phases (-6° and 8°, respectively) of sprinting post-ISP. Moreover, players exhibited significantly larger mean contact angles (+24°), contact angle asymmetries (+4%), and glenohumeral flexion asymmetries (+10%) during the acceleration phase of sprinting post-ISP. Players displayed greater glenohumeral abduction range of motion (+17°) and asymmetries (+20%) during the maximal velocity phase of sprinting post-ISP. Players with SCI (SCI, n = 7) significantly increased asymmetries in peak power (+6%) and glenohumeral abduction (+15%) during the acceleration phase post-ISP. Our data indicates that despite inducing physiological fatigue resulting from WR match play, players can maintain sprint performance by modifying how they propel their wheelchair. Increased asymmetry post-ISP was notable, which may be specific to impairment type and warrants further investigation.

Successful surgical removal of a pheochromocytoma in a mare via trans-costal approach.

BACKGROUND: Pheochromocytomas have been previously reported in horses, but successful antemortem diagnosis and surgical removal without recurrence of clinical signs have not been described. OBJECTIVE: To report the clinical presentation, diagnostic evaluation, surgical technique, anaesthetic management and post-operative care of a mare diagnosed with pheochromocytoma. STUDY DESIGN: Clinical case report. METHODS: An 18-year-old Quarter Horse mare presented for recurrent episodes of colic, profuse sweating, muscle fasciculations and agitation over a 2-month period. Clinical, clinicopathologic and ultrasonographic (transcutaneous, transrectal) abnormalities were consistent with a unilateral left-sided adrenal mass. Surgical removal of the mass was performed via a trans-costal approach with removal of the 18th rib and retraction of the left kidney to improve exposure. Associated vasculature was ligated, and the adrenal mass was removed and submitted for histopathology and immunohistochemistry. RESULTS: A trans-costal surgical approach provided excellent visualisation of the adrenal mass and allowed for identification and ligation of associated vessels. Total surgical and anaesthesia time were 86 and 114 min, respectively. Several intraoperative (hypertension, tachycardia) and post-operative (colic with tachycardia, tachypnea, large colon pelvic flexure impaction and nasogastric reflux) complications were encountered and managed successfully. Immunohistochemistry demonstrated positive labelling for synaptophysin and chromogranin A, confirming diagnosis of pheochromocytoma. The mare had recovered well at 6-week recheck post-operatively and returned to training at 6 months post-operatively. No further clinical signs consistent with pheochromocytoma have been observed following removal. CONCLUSIONS: The trans-costal approach allowed for surgical removal of a pheochromocytoma in a mare. Surgical removal of adrenal masses in horses may be associated with complications yet was successfully performed without subsequent recurrence of clinical signs associated with tumour presence and return to athletic use in this mare.

Brain monoamine vesicular transport disease caused by homozygous SLC18A2 variants: A study in 42 affected individuals.

PURPOSE: Brain monoamine vesicular transport disease is an infantile-onset movement disorder that mimics cerebral palsy. In 2013, the homozygous SLC18A2 variant, p.Pro387Leu, was first reported as a cause of this rare disorder, and dopamine agonists were efficient for treating affected individuals from a single large family. To date, only 6 variants have been reported. In this study, we evaluated genotype-phenotype correlations in individuals with biallelic SLC18A2 variants. METHODS: A total of 42 affected individuals with homozygous SLC18A2 variant alleles were identified. We evaluated genotype-phenotype correlations and the missense variants in the affected individuals based on the structural modeling of rat VMAT2 encoded by Slc18a2, with cytoplasm- and lumen-facing conformations. A Caenorhabditis elegans model was created for functional studies. RESULTS: A total of 19 homozygous SLC18A2 variants, including 3 recurrent variants, were identified using exome sequencing. The affected individuals typically showed global developmental delay, hypotonia, dystonia, oculogyric crisis, and autonomic nervous system involvement (temperature dysregulation/sweating, hypersalivation, and gastrointestinal dysmotility). Among the 58 affected individuals described to date, 16 (28%) died before the age of 13 years. Of the 17 patients with p.Pro237His, 9 died, whereas all 14 patients with p.Pro387Leu survived. Although a dopamine agonist mildly improved the disease symptoms in 18 of 21 patients (86%), some affected individuals with p.Ile43Phe and p.Pro387Leu showed milder phenotypes and presented prolonged survival even without treatment. The C. elegans model showed behavioral abnormalities. CONCLUSION: These data expand the phenotypic and genotypic spectra of SLC18A2-related disorders.

Ice Slurry Ingestion Lowers Thermoregulatory Strain in Wheelchair Tennis Players During Repeated Sprint Intervals in the Heat.

PURPOSE: To examine the efficacy of per-cooling via ice slurry ingestion (ICE) in wheelchair tennis players exercising in the heat. METHOD: Eight wheelchair tennis players undertook sprints (4 sets of 10 × 5 s over 40 min) in a hot environment (∼32 °C), interspersed by 3 boluses of 2.67 g·kg (6.8 g·kg total) ICE or drinking temperate water (control condition). Athletes performed an on-court test of repeated sprint ability (20 × 20 m) in temperate conditions immediately before and 20 minutes after the heat exposure, and time to complete each sprint as well as intermediate times were recorded. Gastrointestinal and weighted mean skin and forehead temperatures were collected throughout the heat exposure, as were thermal sensation, heart rate, and blood lactate concentration. Sweat rate was calculated from body mass changes and fluid/ice intakes. RESULTS: Compared with the control condition, ICE resulted in a significantly lower gastrointestinal temperature (95% CI, 0.11-0.17 °C; P < .001), forehead temperature (0.58-1.06 °C; P < .001), thermal sensation (0.07-0.50 units; P = .017), and sweat rate (0.06-0.46 L·h-1; P = .017). Skin temperature, heart rate, and blood lactate concentration were not significantly different between conditions (P ≥ .598). There was no overall change preheating to postheating (P ≥ .114) or an effect of condition (P ≥ .251) on repeated sprint times. CONCLUSIONS: ICE is effective at lowering objective and subjective thermal strain when consumed between sets of repeated wheelchair sprints in the heat. However, ICE has no effect on on-court repeated 20-m sprint performance.

Bi-allelic loss-of-function variants in PPFIBP1 cause a neurodevelopmental disorder with microcephaly, epilepsy, and periventricular calcifications.

PPFIBP1 encodes for the liprin-ß1 protein, which has been shown to play a role in neuronal outgrowth and synapse formation in Drosophila melanogaster. By exome and genome sequencing, we detected nine ultra-rare homozygous loss-of-function variants in 16 individuals from 12 unrelated families. The individuals presented with moderate to profound developmental delay, often refractory early-onset epilepsy, and progressive microcephaly. Further common clinical findings included muscular hyper- and hypotonia, spasticity, failure to thrive and short stature, feeding difficulties, impaired vision, and congenital heart defects. Neuroimaging revealed abnormalities of brain morphology with leukoencephalopathy, ventriculomegaly, cortical abnormalities, and intracranial periventricular calcifications as major features. In a fetus with intracranial calcifications, we identified a rare homozygous missense variant that by structural analysis was predicted to disturb the topology of the SAM domain region that is essential for protein-protein interaction. For further insight into the effects of PPFIBP1 loss of function, we performed automated behavioral phenotyping of a Caenorhabditis elegans PPFIBP1/hlb-1 knockout model, which revealed defects in spontaneous and light-induced behavior and confirmed resistance to the acetylcholinesterase inhibitor aldicarb, suggesting a defect in the neuronal presynaptic zone. In conclusion, we establish bi-allelic loss-of-function variants in PPFIBP1 as a cause of an autosomal recessive severe neurodevelopmental disorder with early-onset epilepsy, microcephaly, and periventricular calcifications.

The effect of pre-cooling or per-cooling in athletes with a spinal cord injury: A systematic review and meta-analysis.

OBJECTIVES: For individuals with a spinal cord injury, thermoregulatory challenges presented by the environment are amplified, increasing the risk of exertional heat illness. Thus, this systematic review and meta-analysis aims to quantify the effects of pre- and per-cooling on core temperature (Tc), skin temperature (Tsk) and thermal sensation in participants with spinal cord injury and assess the influence of lesion level on the effects of cooling. DESIGN: Systematic review with meta-analysis. METHODS: Out of 2107 potential studies, 17 were identified via the inclusion criteria for a total of 145 research participants. A total of 12 studies were included in the primary analysis of Tc; 9 included in the analysis of Tsk; and 9 included in the analysis of thermal perceptions. 15 experimental conditions were included in the secondary analysis of lesion level on the effects of cooling. RESULTS: Cooling reduced Tc (Hedges' g = 0.44; 95% confidence intervals 0.16, 0.72; p < 0.001), Tsk (Hedges' g = 1.11; 95% confidence intervals 0.56, 1.66; p < 0.002) and thermal sensation (Hedges' g = 0.60; 95% confidence intervals 0.27, 0.93; p < 0.001). Subgroup analysis revealed pre-cooling (Hedges' g = 0.92), reduced Tc to a greater extent than per-cooling (Hedges' g = 0.25; p = 0.020). The effect of lesion level on the effectiveness of cooling on Tc had a moderate, positive association (r = 0.518, p = 0.048). CONCLUSION: Pre-cooling may reduce Tc to a greater extent than per-cooling during subsequent exercise. Pre- and per-cooling can attenuate the increase in thermal strain in athletes with a spinal cord injury. The beneficial effects of cooling are greater in tetraplegic individuals.

A High-Intensity Warm-Up Increases Thermal Strain But Does Not Affect Repeated Sprint Performance in Athletes With a Cervical Spinal Cord Injury.

PURPOSE: To compare the effects of typical competition versus high-intensity intermittent warm-up (WU) on thermoregulatory responses and repeated sprint performance during wheelchair rugby game play. METHODS: An intermittent sprint protocol (ISP) simulating the demands of wheelchair rugby was performed by male wheelchair rugby players (7 with cervical spinal cord injury [SCI] and 8 without SCI) following 2 WU protocols. These included a typical competition WU (control) and a WU consisting of high-intensity efforts (INT). Core temperature (Tcore), thermal sensation, and thermal comfort were recorded. Wheelchair performance variables associated to power, speed, and fatigue were also calculated. RESULTS: During the WU, Tcore was similar between conditions for both groups. During the ISP, a higher Tcore was found for SCI compared to NON-SCI (38.1 [0.3] vs 37.7 [0.3] °C: P = .036, d = 0.75), and the SCI group experienced a higher peak Tcore for INT compared with control (39.0 [0.4] vs 38.6 [0.6] °C; P = .004). Peak Tcore occurred later in the ISP for players with SCI (96 [5.8] vs 48 [2.7] min; P < .001). All players reported a higher thermal sensation and thermal comfort following INT (P < .001), with no differences between conditions throughout the ISP. No significant differences were found in wheelchair performance variables during the ISP between conditions (P ≥ .143). CONCLUSIONS: The high-INT WU increased thermal strain in the SCI group during the ISP, potentially due to increased metabolic heat production and impaired thermoregulation, while not impacting on repeated sprint performance. It may be advisable to limit high-INT bouts during a WU in players with SCI to mitigate issues related to hyperthermia in subsequent performance.

Virulence and antimicrobial resistance genes are enriched in the plasmidome of clinical Escherichia coli isolates compared with wastewater isolates from western Kenya.

Many low-middle income countries in Africa have poorly-developed infectious disease monitoring systems. Here, we employed whole genome sequencing (WGS) to investigate the presence/absence of antimicrobial resistance (AMR) and virulence-associated (VA) genes in a collection of clinical and municipal wastewater Escherichia coli isolates from Kakamega, west Kenya. We were particularly interested to see whether, given the association between infection and water quality, the isolates from these geographically-linked environments might display similar genomic signatures. Phylogenetic analysis based on the core genes common to all of the isolates revealed two broad divisions, corresponding to the commensal/enterotoxigenic E. coli on the one hand, and uropathogenic E. coli on the other. Although the clinical and wastewater isolates each contained a very similar mean number of antibiotic resistance-encoding genes, the clinical isolates were enriched in genes required for in-host survival. Furthermore, and although the chromosomally encoded repertoire of these genes was similar in all sequenced isolates, the genetic composition of the plasmids from clinical and wastewater E. coli was more habitat-specific, with the clinical isolate plasmidome enriched in AMR and VA genes. Intriguingly, the plasmid-borne VA genes were often duplicates of genes already present on the chromosome, whereas the plasmid-borne AMR determinants were more specific. This reinforces the notion that plasmids are a primary means by which infection-related AMR and VA-associated genes are acquired and disseminated among these strains.

Shoulder Tendon Adaptations Following a Graded Exercise Test to Exhaustion in Highly Trained Wheelchair Rugby Athletes With Different Impairments.

Objective: This study aimed to identify acute changes in biceps and supraspinatus tendon characteristics before and after a graded exercise test to exhaustion (GXT) in highly trained wheelchair rugby (WR) athletes. A secondary aspect was to define chronic tendon adaptations related to the impairment of the athlete and the occupation of the tendon within the subacromial space (occupation ratio). Methods: Twelve WR athletes with different impairments (age = 32 ± 6 years; body mass = 67.2 ± 11.2 kg; 9.0 ± 3.6 years competing) volunteered for this study. Performance Corrected Wheelchair Users Shoulder Pain Index was used to quantify shoulder pain. Quantitative Ultrasound Protocols (QUS) were used to define supraspinatus and biceps tendon thickness, echogenicity, and echogenicity ratio of both dominant and non-dominant shoulder before and after the GXT including 22 ± 3.1 min submaximal propulsion and 10.2 ± 1.7 min maximal propulsion on a treadmill. Furthermore, the acromio-humeral distance (AHD) defined from ultrasound (US) images was used to calculate the occupation ratios. Results: A mixed-effect multilevel analysis that included shoulder as grouping variable, demonstrated a significant reduction in the echogenicity of the biceps following GXT whilst controlling for impairment [spinal cord injury (SCI) and non-SCI] and the occupation ratio (ß = -9.01, SEß = 2.72, p = 0.001, 95% CI = [-14.34; -3.68]). This points toward fluid inflow into the tendon that may be related to overload and acute inflammation. In addition, persons with a SCI (n = 8) had a thicker supraspinatus tendon in comparison to persons with non-SCI (n = 3) which may be related to chronic tendon adaptations (ß = -0.53 mm, SEß = 0.26, p = 0.038, 95% CI = [-1.04; -0.03]). Finally, a greater occupation ratio was associated with signs of tendinopathy (i.e., greater biceps and supraspinatus tendon thickness, and lower supraspinatus echogenicity and echogenicity ratio). Conclusion: Acute biceps tendon adaptations in response to the GXT in highly trained WR athletes were evident with chronic adaptations in the supraspinatus tendon being related to the impairment of the athlete. Ultrasound can be used to monitor tendon adaptations in WR athletes for medical diagnosis to assist the scheduling and type of training.

Factors associated with survival and racing performance of 114 Thoroughbred foals with septic arthritis compared with maternal siblings (2009-2015).

BACKGROUND: There is little consensus on factors associated with survival in foals with septic arthritis and limited data on long-term racing performance of Thoroughbred foals treated for septic arthritis. A more thorough understanding of short- and long-term outcome is necessary to help inform owners, and subsequently guide treatment. OBJECTIVES: To investigate factors associated with survival, and to analyse racing performance of foals with septic arthritis compared with their maternal siblings. STUDY DESIGN: Retrospective cohort and a case-control study. METHODS: Veterinary clinical records of Thoroughbred foals ≤180 days old that underwent arthroscopic, cannulae or through-and-through needle lavage for the treatment of septic arthritis between 2009 and 2015 were reviewed. Data included signalment, and clinicopathological information. The dam's foaling records were reviewed and the lifetime racing records were obtained for affected foals and two of their maternal siblings. Logistic regression analysis was used to determine factors associated with survival to discharge or racing. Comparisons between treated foals and their maternal siblings were made. RESULTS: Ninety (78%) of 115 foals diagnosed with septic arthritis were discharged alive. Foals <26 days old at the time of admission were five times less likely (P = .003) and foals with concurrent multisystemic disease were six times less likely (P = .02) to be discharged alive. Sixty (67%) foals discharged alive started in ≥1 race, and there was no difference in the proportion of foals that started in a race or racing performance between foals treated for septic arthritis and their maternal siblings. MAIN LIMITATIONS: Retrospective study design, limited number of foals with multiple joint involvement and failure to accurately record duration of clinical signs. CONCLUSIONS: Foals treated for septic arthritis at the Scone Equine Hospital, New South Wales, Australia had a good prognosis for survival, and for this cohort, foals that survived to discharge had a similar ability to race as their maternal siblings.

Recapitulation of polymicrobial communities associated with cystic fibrosis airway infections: a perspective.

The airways of persons with cystic fibrosis are prone to infection by a diverse and dynamic polymicrobial consortium. Currently, no models exist that permit recapitulation of this consortium within the laboratory. Such microbial ecosystems likely have a network of interspecies interactions, serving to modulate metabolic pathways and impact upon disease severity. The contribution of less abundant/fastidious microbial species on this cross-talk has often been neglected due to lack of experimental tractability. Here, we critically assess the existing models for studying polymicrobial infections. Particular attention is paid to 3Rs-compliant in vitro and in silico infection models, offering significant advantages over mammalian infection models. We outline why these models will likely become the 'go to' approaches when recapitulating polymicrobial cystic fibrosis infection.

Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis.

An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using (13)C-labeled sugars and [(15)N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. The results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals.

Early screw fixation versus casting in the treatment of acute Jones fractures.

BACKGROUND: There is considerable variability in the literature concerning the optimal treatment of acute Jones fractures. HYPOTHESIS: Early surgical fixation of acute Jones fractures will result in shorter times to union and return to athletics compared with cast treatment. STUDY DESIGN: Randomized controlled clinical trial; Level of evidence, 1. METHODS: Eighteen patients were randomized to cast treatment, and 19 patients were randomized to screw fixation. Success of treatment and the times to union and return to sports were calculated for each patient. RESULTS: Mean follow-up was 25.3 months (range, 15-42 months). Eight of 18 (44%) in the cast group were considered treatment failures: 5 nonunions, 1 delayed union, and 2 refractures. One of 19 patients in the surgery group was considered a treatment failure. For the surgery group, the median times to union and return to sports were 7.5 and 8.0 weeks, respectively. For the cast group, the median times were 14.5 and 15.0 weeks, respectively. The Mann-Whitney test showed a statistically significant difference between the groups in both parameters, with P < 001. CONCLUSION: There is a high incidence (44%) of failure after cast treatment of acute Jones fractures. Early screw fixation results in quicker times to union and return to sports compared with cast treatment.

Radiation-induced strain differences in mouse alveolar inflammatory cell apoptosis.

Whole-thorax irradiation results in the development of the diffuse inflammatory response alveolitis in C3H/HeJ (C3H) mice and a milder alveolitis with fibrosis in C57BL/6J (B6) mice. In this study, we investigate if this mouse strain difference in response to radiation is due to differences in lung inflammatory cell apoptosis. Mice of the C3H and B6 strains were given a radiation dose of 18 Gy to the thorax and the animals were sacrificed at 11 or 18 weeks following exposure or when they were moribund. Active caspase-3 staining was used to identify apoptotic cells in the alveolar space of histological lung sections from the mice. The apoptotic index of B6 mice was greater than that of C3H mice at 11 weeks postirradiation (17.8% of airspace cells vs. 7.8%, p = 0.028) and in mice sacrificed because of illness (27.3% vs. 14.4%, p = 0.036). No C3H mice survived to the later time point. The inflammatory cells undergoing apoptosis in the mouse lungs were morphologically consistent with alveolar macrophages. We conclude that a difference in inflammatory cell apoptosis may contribute to the disparate pulmonary radiation response of these mouse strains.

Side-chain dynamics of an alpha-helical polypeptide monitored by fluorescence.

The "blob" model, developed to analyze the fluorescence decays of polymers randomly labeled with pyrene, has been applied to a series of pyrene-labeled poly(glutamic acid)s (PyPGA) in DMF and carbonated buffer solutions at pH 9. Poly(glutamic acid) (PGA) exists in the ionized form in the buffer solutions as poly(sodium glutamate) (PGNa). PGA adopts an alpha-helical conformation in DMF, whereas in aqueous solution PGNa is a random coil. Fluorescence, UV-vis absorption, and circular dichroism measurements indicate that in our studies pyrene pendants attached themselves along PGA in a clustered manner. Simulations were carried out to establish that the geometry of the PGA alpha-helix induces the high level of pyrene clustering. Since the level of pyrene clustering decreased with lower pyrene content, information about naked PGA was retrieved by extrapolating the trends obtained by fluorescence to zero pyrene content. Analysis of the fluorescence decays demonstrated that during its lifetime an excited pyrene probes a 32 amino acid section of the PGA alpha-helix. This result was supported by molecular mechanics optimizations. This study establishes that the blob model, originally used to monitor the encounters between pyrenes attached randomly onto a polymer adopting a random coil conformation, can also be applied to study the dynamics of the side chains of structured proteins. Since the blob model helps in monitoring the encounters between amino acids in the initial state (i.e., random coil) and in the final state (i.e., structured protein) of the folding pathway of a protein, it could be applicable to the study of protein folding.