AAV-vectored expression of monospecific or bispecific monoclonal antibodies protects mice from lethal Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa poses a significant threat to immunocompromised individuals and those with cystic fibrosis. Treatment relies on antibiotics, but persistent infections occur due to intrinsic and acquired resistance of P. aeruginosa towards multiple classes of antibiotics. To date, there are no licensed vaccines for this pathogen, prompting the urgent need for novel treatment approaches to combat P. aeruginosa infection and persistence. Here we validated AAV vectored immunoprophylaxis as a strategy to generate long-term plasma and mucosal expression of highly protective monoclonal antibodies (mAbs) targeting the exopolysaccharide Psl (Cam-003) and the PcrV (V2L2MD) component of the type-III secretion system injectosome either as single mAbs or together as a bispecific mAb (MEDI3902) in a mouse model. When administered intramuscularly, AAV-αPcrV, AAV-αPsl, and AAV-MEDI3902 significantly protected mice challenged intranasally with a lethal dose of P. aeruginosa strains PAO1 and PA14 and reduced bacterial burden and dissemination to other organs. While all AAV-mAbs provided protection, AAV-αPcrV and AAV-MEDI3902 provided 100% and 87.5% protection from a lethal challenge with 4.47 × 107 CFU PAO1 and 87.5% and 75% protection from a lethal challenge with 3 × 107 CFU PA14, respectively. Serum concentrations of MEDI3902 were ~10× lower than that of αPcrV, but mice treated with this vector showed a greater reduction in bacterial dissemination to the liver, lung, spleen, and blood compared to other AAV-mAbs. These results support further investigation into the use of AAV vectored immunoprophylaxis to prevent and treat P. aeruginosa infections and other bacterial pathogens of public health concern for which current treatment strategies are limited.

COPB2 loss of function causes a coatopathy with osteoporosis and developmental delay.

Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures, and developmental delay of variable severity. Electron microscopy of COPB2-deficient subjects' fibroblasts showed dilated endoplasmic reticulum (ER) with granular material, prominent rough ER, and vacuoles, consistent with an intracellular trafficking defect. We studied the effect of COPB2 deficiency on collagen trafficking because of the critical role of collagen secretion in bone biology. COPB2 siRNA-treated fibroblasts showed delayed collagen secretion with retention of type I collagen in the ER and Golgi and altered distribution of Golgi markers. copb2-null zebrafish embryos showed retention of type II collagen, disorganization of the ER and Golgi, and early larval lethality. Copb2+/- mice exhibited low bone mass, and consistent with the findings in human cells and zebrafish, studies in Copb2+/- mouse fibroblasts suggest ER stress and a Golgi defect. Interestingly, ascorbic acid treatment partially rescued the zebrafish developmental phenotype and the cellular phenotype in Copb2+/- mouse fibroblasts. This work identifies a form of coatopathy due to COPB2 haploinsufficiency, explores a potential therapeutic approach for this disorder, and highlights the role of the COPI complex as a regulator of skeletal homeostasis.

Female long-term sperm storage results in viable offspring in the Himalayan Mountain Pitviper, Ovophis monticola.

The ability of females to store sperm for extended periods in their reproductive tracts (termed long-term sperm storage, LTSS) has been reported across a diversity of vertebrate taxa. The evolutionary, ecological, and physiological significance of LTSS is wide-ranging and includes the ability to produce offspring when mates may be temporally scarce by way of decoupling copulation from ovulation, inbreeding avoidance, and the generation and maintenance of genetic diversity in progeny. Among vertebrate lineages, nonavian reptiles exhibit a remarkable capacity for LTSS, with the production of viable offspring reported after periods exceeding 6 years since prior contact with a potential mate. Given that female reptiles are able to store viable sperm for prolonged periods, it is important to disentangle LTSS from that of facultative parthenogenesis (FP), a reproductive trait which appears widespread among all reptile lineages. The implications of this distinction are particularly important in the context of the development and management of captive breeding programs. To accurately determine between the two reproductive strategies, genomic screening is highly recommended. Following a period of isolation for 13 months from a potential male mate, a female Himalayan Mountain Pitviper (Ovophis monticola) produced a clutch of three male offspring. Here, through genome-scale analyses of the female and her progeny, we document the first record of LTSS in this genus and exclude FP as the alternative hypothesis.

Facultative Parthenogenesis in a Zoo-Held Northern Water Snake, Nerodia sipedon.

Over the past several decades, facultative parthenogenesis (FP)-the ability of a sexually reproducing species to reproduce asexually-in vertebrates has been removed from the realm of obscurity and placed firmly in a position where it warrants focused scientific attention. Likely fueled by increased recognition of the trait, the availability of molecular tools capable of disentangling FP from long-term sperm storage, and the availability of potential cases originating from both zoological and private collections, a wealth of papers has been published revealing the diversity of vertebrate systems in which FP occurs. Specifically, cases have been reported in squamate reptiles (lizards and snakes), crocodiles, birds, and elasmobranch fishes (sharks, rays, and skates). Nonetheless, gaps remain in species documentation, and it is important to analyze and report on new cases. In this paper, we provide a DNA-based analysis confirming FP in a zoo-maintained northern water snake, Nerodia sipedon, a viviparous natricine species that is common and widely distributed in North America. Additionally, we provide information on the sexual development and health of the male parthenogen. Encouragingly, zoological institutions, aquaria, university laboratories, and private collections continue to be rich sources for the further study and documentation of FP in vertebrate species, advancing our understanding of this reproductive trait.

AAV-mediated delivery of actoxumab and bezlotoxumab results in serum and mucosal antibody concentrations that provide protection from C. difficile toxin challenge.

Clostridium difficile is the leading cause of antibiotic-associated nosocomial diarrhea in the developed world. When the host-associated colon microbiome is disrupted by the ingestion of antibiotics, C. difficile spores can germinate, resulting in infection. C. difficile secretes enterotoxin A (TcdA) and cytotoxin B (TcdB) that are responsible for disease pathology. Treatment options are limited as the bacterium demonstrates resistance to many antibiotics, and even with antibacterial therapies, recurrences of C. difficile are common. Actotoxumab and bezlotoxumab are human monoclonal antibodies that bind and neutralize TcdA and TcdB, respectively. In 2016, the US food and drug administration (FDA) approved bezlotoxumab for use in the prevention of C. difficile infection recurrence. To ensure the long-term expression of antibodies, gene therapy can be used. Here, adeno-associated virus (AAV)6.2FF, a novel triple mutant of AAV6, was engineered to express either actotoxumab or bezlotoxumab in mice and hamsters. Both antibodies expressed at greater than 90 µg/mL in the serum and were detected at mucosal surfaces in both models. Hundred percent of mice given AAV6.2FF-actoxumab survived a lethal dose of TcdA. This proof of concept study demonstrates that AAV-mediated expression of C. difficile toxin antibodies is a viable approach for the prevention of recurrent C. difficile infections.

Discovery of facultative parthenogenesis in a new world crocodile.

Over the past two decades, there has been an astounding growth in the documentation of vertebrate facultative parthenogenesis (FP). This unusual reproductive mode has been documented in birds, non-avian reptiles-specifically lizards and snakes-and elasmobranch fishes. Part of this growth among vertebrate taxa is attributable to awareness of the phenomenon itself and advances in molecular genetics/genomics and bioinformatics, and as such our understanding has developed considerably. Nonetheless, questions remain as to its occurrence outside of these vertebrate lineages, most notably in Chelonia (turtles) and Crocodylia (crocodiles, alligators and gharials). The latter group is particularly interesting because unlike all previously documented cases of FP in vertebrates, crocodilians lack sex chromosomes and sex determination is controlled by temperature. Here, using whole-genome sequencing data, we provide, to our knowledge, the first evidence of FP in a crocodilian, the American crocodile, Crocodylus acutus. The data support terminal fusion automixis as the reproductive mechanism; a finding which suggests a common evolutionary origin of FP across reptiles, crocodilians and birds. With FP now documented in the two main branches of extant archosaurs, this discovery offers tantalizing insights into the possible reproductive capabilities of the extinct archosaurian relatives of crocodilians and birds, notably members of Pterosauria and Dinosauria.

Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440.

Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic-catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.

Biological upgrading of pyrolysis-derived wastewater: Engineering Pseudomonas putida for alkylphenol, furfural, and acetone catabolism and (methyl)muconic acid production.

While biomass-derived carbohydrates have been predominant substrates for biological production of renewable fuels, chemicals, and materials, organic waste streams are growing in prominence as potential alternative feedstocks to improve the sustainability of manufacturing processes. Catalytic fast pyrolysis (CFP) is a promising approach to generate biofuels from lignocellulosic biomass, but it generates a complex, carbon-rich, and toxic wastewater stream that is challenging to process catalytically but could be biologically upgraded to valuable co-products. In this work, we implemented modular, heterologous catabolic pathways in the Pseudomonas putida KT2440-derived EM42 strain along with the overexpression of native toxicity tolerance machinery to enable utilization of 89% (w/w) of carbon in CFP wastewater. The dmp monooxygenase and meta-cleavage pathway from Pseudomonas putida CF600 were constitutively expressed to enable utilization of phenol, cresols, 2- and 3-ethyl phenol, and methyl catechols, and the native chaperones clpB, groES, and groEL were overexpressed to improve toxicity tolerance to diverse aromatic substrates. Next, heterologous furfural and acetone utilization pathways were incorporated, and a native alcohol dehydrogenase was overexpressed to improve methanol utilization, generating reducing equivalents. All pathways (encoded by genes totaling ~30 kilobases of DNA) were combined into a single strain that can catabolize a mock CFP wastewater stream as a sole carbon source. Further engineering enabled conversion of all aromatic compounds in the mock wastewater stream to (methyl)muconates with a ~90% (mol/mol) yield. Biological upgrading of CFP wastewater as outlined in this work provides a roadmap for future applications in valorizing other heterogeneous waste streams.

Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440.

Pseudomonas putida KT2440 has received increasing attention as an important biocatalyst for the conversion of diverse carbon sources to multiple products, including the olefinic diacid, cis,cis-muconic acid (muconate). P. putida has been previously engineered to produce muconate from glucose; however, periplasmic oxidation of glucose causes substantial 2-ketogluconate accumulation, reducing product yield and selectivity. Deletion of the glucose dehydrogenase gene (gcd) prevents 2-ketogluconate accumulation, but dramatically slows growth and muconate production. In this work, we employed adaptive laboratory evolution to improve muconate production in strains incapable of producing 2-ketogluconate. Growth-based selection improved growth, but reduced muconate titer. A new muconate-responsive biosensor was therefore developed to enable muconate-based screening using fluorescence activated cell sorting. Sorted clones demonstrated both improved growth and muconate production. Mutations identified by whole genome resequencing of these isolates indicated that glucose metabolism may be dysregulated in strains lacking gcd. Using this information, we used targeted engineering to recapitulate improvements achieved by evolution. Deletion of the transcriptional repressor gene hexR improved strain growth and increased the muconate production rate, and the impact of this deletion was investigated using transcriptomics. The genes gntZ and gacS were also disrupted in several evolved clones, and deletion of these genes further improved strain growth and muconate production. Together, these targets provide a suite of modifications that improve glucose conversion to muconate by P. putida in the context of gcd deletion. Prior to this work, our engineered strain lacking gcd generated 7.0 g/L muconate at a productivity of 0.07 g/L/h and a 38% yield (mol/mol) in a fed-batch bioreactor. Here, the resulting strain with the deletion of hexR, gntZ, and gacS achieved 22.0 g/L at 0.21 g/L/h and a 35.6% yield (mol/mol) from glucose in similar conditions. These strategies enabled enhanced muconic acid production and may also improve production of other target molecules from glucose in P. putida.

Temporal Artery Biopsy in the Workup of Giant Cell Arteritis: Diagnostic Considerations in a Veterans Administration Cohort.

BACKGROUND: Giant cell arteritis (GCA) is the most common systemic vasculitis in the American population older than 50 years and is a sight-threatening and life-threatening disease. It is definitively diagnosed with a temporal artery biopsy. Although there are many studies focusing on the clinical presentation and laboratory values in diagnosing GCA in the general population, studies focusing on the veteran population are lacking. This is the first study describing the diagnostic features of GCA in the US military veterans. METHODS: We performed a retrospective chart review in the Veterans Information Systems and Technology Architecture Computerized Patient Record System (CPRS 1.0, Department of Veterans Affairs Health Data Systems). Anatomic pathology reports from temporal artery biopsies (TABs) were collected, as well as the clinical presentation and laboratory values for each case. Frequency, sensitivity, and specificity were calculated for clinical variables, such as new-onset headache and vision changes, including diplopia, ischemic vision loss/optic disc disease, and amaurosis fugax. A logistic regression (LR) prediction model was then developed to compare veteran risk factors with those of the general population. RESULTS: Of 292 patients, 40 had positive TABs (13.7%). The average age of subjects with positive TABs was 73 ± 8.8 years (mean ± SD). The average erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in patients with positive TABs (69.1 mm/hr and 56.6 mg/L, respectively) were significantly higher than ESR and CRP in patients with negative TABs (50.5 mm/hr, P = 0.0016 and 32.2 mg/L, P = 0.0394, respectively). Mean platelet levels were much higher (317.6 × 10/L) in patients with positive TABs than platelet levels in those with negative TABs (260.6 × 10/L, P = 0.0005). CRP was the most sensitive variable at 83.3%, followed by ESR with a sensitivity of 80% and new-onset headache with a sensitivity of 62.5%. Jaw claudication and polymyalgia rheumatica (PMR) were most specific (81.3% and 89.3%, respectively). Headache was the most common presenting symptom overall (71.6%), followed by vision changes (50.3%), scalp tenderness (25.7%), jaw claudication (20.9%), and PMR-related symptoms (12.7%). The LR prediction model included scalp tenderness, log (CRP), log (platelets), vision changes, and age, with 50% sensitivity and 88.36% specificity. Platelets (odds ratio [OR] = 4.309, P = 0.049), CRP (OR = 1.504, P = 0.022) and scalp tenderness (OR = 3.860, P = 0.016) were statistically significant predictors of a positive TAB in this population. CONCLUSIONS: Veterans Administration (VA) patients present with symptomatology similar to that of the general population. A positive biopsy was found in female veterans more frequently than in their male counterparts. Platelet count and scalp tenderness were most predictive. Our LR model provided a highly specific method for detecting GCA in the veteran population at this institution, but further studies are needed to determine the generalizability of the model. This retrospective study serves as a basis for future multicenter VA-wide studies to characterize the unique features in this population.

GBA Variants are associated with a distinct pattern of cognitive deficits in Parkinson's disease.

BACKGROUND: Loss-of-function mutations in the GBA gene are associated with more severe cognitive impairment in PD, but the nature of these deficits is not well understood and whether common GBA polymorphisms influence cognitive performance in PD is not yet known. METHODS: We screened the GBA coding region for mutations and the E326K polymorphism in 1,369 PD patients enrolled at eight sites from the PD Cognitive Genetics Consortium. Participants underwent assessments of learning and memory (Hopkins Verbal Learning Test-Revised), working memory/executive function (Letter-Number Sequencing Test and Trail Making Test A and B), language processing (semantic and phonemic verbal fluency), visuospatial abilities (Benton Judgment of Line Orientation), and global cognitive function (MoCA). We used linear regression to test for association between genotype and cognitive performance with adjustment for important covariates and accounted for multiple testing using Bonferroni's corrections. RESULTS: Mutation carriers (n = 60; 4.4%) and E326K carriers (n = 65; 4.7%) had a higher prevalence of dementia (mutations, odds ratio = 5.1; P = 9.7 × 10(-6) ; E326K, odds ratio = 6.4; P = 5.7 × 10(-7) ) and lower performance on Letter-Number Sequencing (mutations, corrected P[Pc ] = 9.0 × 10(-4) ; E326K, Pc = 0.036), Trail Making B-A (mutations, Pc = 0.018; E326K, Pc = 0.018), and Benton Judgment of Line Orientation (mutations, Pc = 0.0045; E326K, Pc = 0.0013). CONCLUSIONS: Both GBA mutations and E326K are associated with a distinct cognitive profile characterized by greater impairment in working memory/executive function and visuospatial abilities in PD patients. The discovery that E326K negatively impacts cognitive performance approximately doubles the proportion of PD patients we now recognize are at risk for more severe GBA-related cognitive deficits.

The discovery of LRRK2 p.R1441S, a novel mutation for Parkinson's disease, adds to the complexity of a mutational hotspot.

Mutations in the LRRK2 gene result in autosomal dominant, late onset Parkinson's disease (PD). Three such mutations (p.R1441C, p.R1441G, and p.R1441H) are known to occur within codon 1441, and haplotype analyses indicate that each one has arisen independently on multiple occasions. We sequenced the entire coding region of 18 casual genes for PD or other parkinsonian neurodegenerative disorders in the proband of a family with autosomal dominant PD. We discovered a new missense mutation in the LRRK2 gene, c.4321C>A (p.R1441S). The mutation was predicted to be highly deleterious in silico (Combined Annotation Dependent Depletion score of 25.5) and segregated with disease in the pedigree. The clinical characteristics of affected family members were similar to those described in PD families with other mutations in LRRK2 codon 1441 and included resting tremor, rigidity, bradykinesia, unilateral onset, and a good response to levodopa. Age at onset ranged from 41 to 76. Two of the affected members of the pedigree underwent detailed, longitudinal neuropsychological testing, and both displayed evidence of mild cognitive deficits at or slightly preceding the onset of motor symptoms. LRRK2 p.R1441S represents the fourth pathogenic mutation observed within codon 1441 and its discovery adds to the remarkable complexity of a mutational hotspot within the ROC domain of the LRRK2 protein. © 2016 Wiley Periodicals, Inc.

Small Ne of the Isolated and Unmanaged Horse Population on Sable Island.

For small, isolated populations 2 common conservation concerns relate to genetic threats: inbreeding and negative consequences associated with loss of genetic diversity due to drift. Mitigating these threats often involves conservation actions that can be controversial, such as translocations or captive breeding programs. Although such actions have been successful in some situations, in others they have had undesirable outcomes. Here, we estimated the effective population size (N e ) of the Sable Island horses to assess the risk to this population of these genetic threats. We found surprising consistency of N e estimates across the 5 different methods used, with a mean of 48 effective individuals. This estimate falls below the 50 criterion of the "50/500 rule," below which inbreeding depression is a concern for population viability. However, simulations and knowledge of population history indicate that this population is still in its early stages of approaching equilibrium between mutation, drift, and genetic diversity; and no negative consequences have been identified that could be associated with inbreeding depression. Therefore, we do not recommend taking management action (such as translocations) at this stage. Rather, we propose continued monitoring of genetic diversity and fitness over time so that trends and any substantial changes can be detected. This represents one of the few unmanaged horse populations in the world, and therefore these data will not only alert us to serious concerns regarding their conservation status, but will also provide a wealth of information about how natural processes drive patterns of reproduction, mortality, and population growth over time.

Evaluation of mild cognitive impairment subtypes in Parkinson's disease.

Mild cognitive impairment in Parkinson's disease (PD-MCI) is common and increases the risk for dementia. Establishing distinct PD-MCI cognitive subtypes could be valuable for eventually predicting those most likely to convert to dementia. However, the study of PD-MCI subtypes has not yielded consistent results among cohorts. To determine whether there are distinct cognitive subtypes among participants diagnosed with PD-MCI in the Pacific Northwest Udall Center Clinical Consortium, we cognitively subtyped 95 patients with PD-MCI, using the Movement Disorders Society Task Force diagnostic guidelines. Psychometric test scores were then subjected to principle components factor analysis to determine whether similar cognitive subgroups could be identified using statistical methodology. Multiple-domain PD-MCI was diagnosed in 95% of the sample, and a range of cognitive impairments were noted. Factor analysis yielded seven factors and demonstrated overlap of phonemic verbal fluency on two factors, as well as the loading of verbal fluency on the same factor as a visuospatial measure; however, these factors did not partition the sample into distinct cognitive subtypes. Separation of cognitive subtypes based on the current PD-MCI criteria, or via statistical methods, may not provide sufficient information to describe distinct PD groups. Future efforts to validate the PD-MCI criteria and identify combinations of genetic or other risk factors for cognitive impairment are warranted.

Characterization of α-galacto-oligosaccharides formed via heterologous expression of α-galactosidases from Lactobacillus reuteri in Lactococcus lactis.

α-Galacto-oligosaccharides (α-GOS) are produced by transgalactosylation reactions of α-galactosidase (α-Gal) or by conversion of raffinose family oligosaccharides by levansucrase. Similarly to ß-GOS, α-GOS have the potential to mimic glycan receptors on eukaryotic cells and act as molecular decoys to prevent bacterial infection; however, data on transgalactosylation reactions of α-Gal remain scarce. The α-Gal gene sequence from Lactobacillus reuteri was cloned into an α-Gal negative strain of Lactococcus lactis. Transgalactosylation reactions were achieved using crude cell extracts with melibiose or raffinose as galactosyl donor and fucose, N-acetylglucosamine or lactose as galactosyl acceptor. The composition, sequence and most linkage types of α-GOS formed with acceptors saccharides were determined by liquid chromatography-tandem mass spectrometry. α-Gal of Lactobacillus reuteri formed (1 → 3)-, (1 → 4)- or (1 → 6)-linked α-GOS but exhibited a preference for formation of (1 → 6)-linkages. Fucose, N-acetylglucosamine and lactose were suitable galactosyl acceptors for α-Gal of L. reuteri, resulting in formation of (1 → 3)-, (1 → 4)- or (1 → 6)-linked hetero-oligosaccharides. By determining the structural specificity of α-Gal and increasing the variation of oligosaccharides produced by introducing alternative acceptor sugars, this work supports further studies to assess α-GOS pathogen adhesion prevention in mammalian hosts.

Radiation-induced changes in energy metabolism result in mitochondrial dysfunction in salivary glands.

Salivary glands are indirectly damaged during radiotherapy for head and neck cancer, resulting in acute and chronic hyposalivation. Current treatments for radiation-induced hyposalivation do not permanently restore function to the gland; therefore, more mechanistic understanding of the damage response is needed to identify therapeutic targets for lasting restoration. Energy metabolism reprogramming has been observed in cancer and wound healing models to provide necessary fuel for cell proliferation; however, there is limited understanding of alterations in energy metabolism reprogramming in tissues that fail to heal. We measured extracellular acidification and oxygen consumption rates, assessed mitochondrial DNA copy number, and tested fuel dependency of irradiated primary salivary acinar cells. Radiation treatment leads to increases in glycolytic flux, oxidative phosphorylation, and ATP production rate at acute and intermediate time points. In contrast, at chronic radiation time points there is a significant decrease in glycolytic flux, oxidative phosphorylation, and ATP production rate. Irradiated salivary glands exhibit significant decreases in spare respiratory capacity and increases in mitochondrial DNA copy number at days 5 and 30 post-treatment, suggesting a mitochondrial dysfunction phenotype. These results elucidate kinetic changes in energy metabolism reprogramming of irradiated salivary glands that may underscore the chronic loss of function phenotype.

Associations between baseline cognitive status and motor outcomes after treadmill training in people with Parkinson's disease: a pilot study.

PURPOSE: To determine the effect of baseline cognition on gait outcomes after a treadmill training program for people with Parkinson's disease (PD). METHODS: This pilot clinical trial involved people with PD who were classified as having no cognitive impairment (PD-NCI) or mild cognitive impairment (PD-MCI). Baseline executive function and memory were assessed. The intervention was a 10-week gait training program (twice-weekly treadmill sessions), with structured speed and distance progression and verbal cues for gait quality. Response to intervention was assessed by gait speed measured after week 2 (short-term) and week 10 (long-term). RESULTS: Participants (n = 19; 12 PD-NCI, 7 PD-MCI) had a mean (standard deviation) age of 66.5 (6.3) years, disease duration of 8.8 (6.3) years, and MDS-UPDRS III score of 21.3 (10.7). Gait speed increased at short-term and long-term assessments. The response did not differ between PD-NCI and PD-MCI groups; however, better baseline memory performance and milder PD motor severity were independently associated with greater improvements in gait speed in unadjusted and adjusted models. CONCLUSIONS: These findings suggest that memory impairments and more severe motor involvement can influence the response to gait rehabilitation in PD and highlight the need for treatments optimized for people with greater cognitive and motor impairment.IMPLICATIONS FOR REHABILITATIONCognitive deficits in Parkinson's disease (PD) could impact motor learning and gait rehabilitation, yet little is known about the effects of cognitive impairments on the response to rehabilitation in people with PD.This study demonstrates that the response to gait rehabilitation did not differ between people with PD who had no cognitive impairment and those with mild cognitive impairment.Across all participants, better baseline memory was associated with greater improvements in gait speed.Rehabilitation professionals should be mindful of PD severity, as those with more substantial memory and motor impairments may require additional dosing or support to maximize gait training benefits.

Identification of conjugated linoleic acid (CLA) isomers by silver ion-liquid chromatography/in-line ozonolysis/mass spectrometry (Ag+-LC/O3-MS).

A novel method for the identification of conjugated linoleic acid (CLA) isomers has been developed in which silver ion liquid chromatography is coupled to in-line ozonolysis/mass spectrometry (Ag(+)-LC/O3-MS). The mobile phase containing CLA isomers eluting from the Ag(+)-LC column flows through a length of gas-permeable tubing within an ozone rich environment. Ozone penetrating the tubing wall reacts with the conjugated double bonds forming ozonolysis product aldehydes. These, and their corresponding methanol loss fragment ions formed within the atmospheric pressure photoionization (APPI) source, were detected by in-line MS and used for the direct assignment of double bond positions. Assignment of positional isomers is based entirely on the two pairs of diagnostic ions seen in the in-line O3-MS mass spectra. In this way, de novo identification of CLA positional isomers, i.e. without requiring comparison to CLA standards, was achieved. The Ag(+)-LC/O3-MS method was applied to the analysis of CLA isomers in a commercial CLA supplement, milk fat, and the lipid extract from a Lactobacillus plantarum TMW1460 culture. The results demonstrate how Ag(+)-LC/O3-MS can be used for the direct and fast determination of CLA isomers at low concentrations and in complex lipid mixtures.

Antifungal hydroxy fatty acids produced during sourdough fermentation: microbial and enzymatic pathways, and antifungal activity in bread.

Lactobacilli convert linoleic acid to hydroxy fatty acids; however, this conversion has not been demonstrated in food fermentations and it remains unknown whether hydroxy fatty acids produced by lactobacilli have antifungal activity. This study aimed to determine whether lactobacilli convert linoleic acid to metabolites with antifungal activity and to assess whether this conversion can be employed to delay fungal growth on bread. Aqueous and organic extracts from seven strains of lactobacilli grown in modified De Man Rogosa Sharpe medium or sourdough were assayed for antifungal activity. Lactobacillus hammesii exhibited increased antifungal activity upon the addition of linoleic acid as a substrate. Bioassay-guided fractionation attributed the antifungal activity of L. hammesii to a monohydroxy C(18:1) fatty acid. Comparison of its antifungal activity to those of other hydroxy fatty acids revealed that the monohydroxy fraction from L. hammesii and coriolic (13-hydroxy-9,11-octadecadienoic) acid were the most active, with MICs of 0.1 to 0.7 g liter(-1). Ricinoleic (12-hydroxy-9-octadecenoic) acid was active at a MIC of 2.4 g liter(-1). L. hammesii accumulated the monohydroxy C(18:1) fatty acid in sourdough to a concentration of 0.73 ± 0.03 g liter(-1) (mean ± standard deviation). Generation of hydroxy fatty acids in sourdough also occurred through enzymatic oxidation of linoleic acid to coriolic acid. The use of 20% sourdough fermented with L. hammesii or the use of 0.15% coriolic acid in bread making increased the mold-free shelf life by 2 to 3 days or from 2 to more than 6 days, respectively. In conclusion, L. hammesii converts linoleic acid in sourdough and the resulting monohydroxy octadecenoic acid exerts antifungal activity in bread.