Abdominal ultrasound image quality is comparable among veterinary sonographers with varying levels of expertise for healthy canine and feline patients.

Abdominal ultrasonography is increasingly used as a standard diagnostic test in veterinary practices, however, there is little published information regarding the effects of operator experience on image quality. In this prospective observer agreement study, image quality was assessed for abdominal ultrasound examinations performed by nine sonographers (three general practitioners, three credentialed veterinary technicians, and three board-certified specialists). Each sonographer independently performed abdominal ultrasound examinations on the same group of 4 sedated clinically healthy animals (3 dogs, 1 cat) using the same model machine and standardized presets. Twenty-five organs and anatomical landmarks per exam (26 for male dog) were evaluated. Still images and cine loops were recorded for each one of the organs. The final scoring of image quality for each examination was performed by two board-certified veterinary radiologists in a randomized and blinded fashion. Semiquantitative scoring system was used for each reading: 0 - not seen, 1- seen but poor quality/partial seen, 2 - average/good quality, and 3 - excellent quality. The average score for each animal and sonographer was tallied and sonographer groups and individual sonographers were compared. Scores were assessed for normality and data were ranked transformed prior to statistical analysis. No significant differences were found regarding the completeness and quality scores of sonographers of different experience levels and disciplines when measuring specific standard components of a full abdominal scan. There were no statistical differences between individual sonographers or groups of sonographers. Although not statistically significant, the general practitioner's group showed the greatest variability of their individual scores.

Geranylgeranyl generosity: a new prenyl-transferase gives a fat to a SNARE protein.

Protein prenylation, a well-defined protein consensus motifs direct modification by one of three prenyl-transferases, has been an area of fairly settled science for 20 or 30 years. Protein prenylation, the specific prenyl modification (farnesyl or geranylgeranyl), as well as the prenyl-transferases involved can be inferred by protein sequence. Two new papers now upset this settled wisdom with the discovery of a fourth prenyl-transferase, namely geranylgeranyl-transferase-III (GGTase-III) (Kuchay et al, 2019; Shirakawa et al, 2020).

A mixed-method evaluation of the New York State Eat Well Play Hard Community Projects: Building local capacity for sustainable childhood obesity prevention.

This study used a mixed-method, comparative case study approach to assess the level of capacity built for childhood obesity prevention among seven New York State Eat Well Play Hard-Community Projects (EWPH-CP). Data were collected through a self-reported survey in 2007, semi-structured interviews in 2009, and EWPH-CP program documentation throughout the 2006-2010 funding cycle. Quantitative and qualitative analyses were used along with an integrative framework for assessing local capacity building to characterize the capacity built by the study coalitions. Four coalitions rated membership characteristics as a challenge at the beginning of the funding cycle. Towards the end of the funding cycle, all seven coalitions reported activities that were initially focused on building their membership (i.e., member capacity) or positive working relationships (i.e. relational capacity), before eventually pursuing support and resources (i.e., organizational capacity) for implementing their chosen community-oriented programmatic goals (i.e., programmatic capacity). Five coalitions reported environmental changes aimed at increasing physical activity or fruit and vegetable intake. Technical assistance provided to coalitions was credited with contributing to the achievement of programmatic goals. These results suggest that the coalitions succeeded in building local capacity for increasing age-appropriate physical activity or fruit and vegetables intake in the target communities.

Neuronal ceroid lipofuscinosis with DNAJC5/CSPα mutation has PPT1 pathology and exhibit aberrant protein palmitoylation.

Neuronal ceroid lipofuscinoses (NCL) are a group of inherited neurodegenerative disorders with lysosomal pathology (CLN1-14). Recently, mutations in the DNAJC5/CLN4 gene, which encodes the presynaptic co-chaperone CSPα were shown to cause autosomal-dominant NCL. Although 14 NCL genes have been identified, it is unknown if they act in common disease pathways. Here we show that two disease-associated proteins, CSPα and the depalmitoylating enzyme palmitoyl-protein thioesterase 1 (PPT1/CLN1) are biochemically linked. We find that in DNAJC5/CLN4 patient brains, PPT1 is massively increased and mis-localized. Surprisingly, the specific enzymatic activity of PPT1 is dramatically reduced. Notably, we demonstrate that CSPα is depalmitoylated by PPT1 and hence its substrate. To determine the consequences of PPT1 accumulation, we compared the palmitomes from control and DNAJC5/CLN4 patient brains by quantitative proteomics. We discovered global changes in protein palmitoylation, mainly involving lysosomal and synaptic proteins. Our findings establish a functional link between two forms of NCL and serve as a springboard for investigations of NCL disease pathways.

Tracking brain palmitoylation change: predominance of glial change in a mouse model of Huntington's disease.

Protein palmitoylation, a reversible lipid modification of proteins, is widely used in the nervous system, with dysregulated palmitoylation being implicated in a variety of neurological disorders. Described below is ABE/SILAM, a proteomic strategy that couples acyl-biotinyl exchange (ABE) purification of palmitoyl-proteins to whole animal stable isotope labeling (SILAM) to provide an accurate tracking of palmitoylation change within rodent disease models. As a first application, we have used ABE/SILAM to look at Huntington's disease (HD), profiling palmitoylation change in two HD-relevant mouse mutants: the transgenic HD model mouse YAC128 and the hypomorphic Hip14-gt mouse, which has sharply reduced expression for HIP14 (Zdhhc17), a palmitoyl-transferase implicated in the HD disease process. Rather than mapping to the degenerating neurons themselves, the biggest disease changes instead map to astrocytes and oligodendrocytes (i.e., the supporting glial cells).

The yeast kinase Yck2 has a tripartite palmitoylation signal.

The yeast kinase Yck2 tethers to the cytoplasmic surface of the plasma membrane through dual palmitoylation of its C-terminal Cys-Cys dipeptide, mediated by the Golgi-localized palmitoyl-transferase Akr1. Here, the Yck2 palmitoylation signal is found to consist of three parts: 1) a 10-residue-long, conserved C-terminal peptide (CCTP) that includes the C-terminal Cys-Cys dipeptide; 2) the kinase catalytic domain (KD); and mapping between these two elements; and 3) a 176-residue-long, poorly conserved, glutamine-rich sequence. The CCTP, which contains the C-terminal cysteines as well as an important Phe-Phe dipeptide, likely serves as an Akr1 recognition element, because CCTP mutations disrupt palmitoylation within a purified in vitro palmitoylation system. The KD contribution appears to be complex with roles for both KD activity (e.g., Yck2-mediated phosphorylation) and structure (e.g., Akr1 recognition elements). KD and CCTP mutations are strongly synergistic, suggesting that, like the CCTP, the KD may also participate at the Yck2-Akr1 recognition step. The long, glutamine-rich domain, which is located between the KD and CCTP, is predicted to be intrinsically disordered and may function as a flexible, interdomain linker, allowing a coupled interaction of the KD and CCTP with Akr1. Multipart palmitoylation signals may prove to be a general feature of this large class of palmitoylation substrates. These soluble proteins have no clear means of accessing membranes and thus may require active capture out of the cytoplasm for palmitoylation by their membrane-localized transferases.

Protein aggregation induced during glass bead lysis of yeast.

Yeast cell lysates produced by mechanical glass bead disruption are widely used in a variety of applications, including for the analysis of native function, e.g. protein-protein interaction, enzyme assays and membrane fractionations. Below, we report a striking case of protein denaturation and aggregation that is induced by this lysis protocol. Most of this analysis focuses on the type 1 casein kinase Yck2, which normally tethers to the plasma membrane through C-terminal palmitoylation. Surprisingly, when cells are subjected to glass bead disruption, non-palmitoylated, cytosolic forms of the kinase denature and aggregate, while membrane-associated forms, whether attached through their native palmitoyl tethers or through a variety of artificial membrane-tethering sequences, are wholly protected from denaturation and aggregation. A wider look at the yeast proteome finds that, while the majority of proteins resist glass bead-induced aggregation, a significant subset does, in fact, succumb to such denaturation. Thus, yeast researchers should be aware of this potential artifact when embarking on biochemical analyses that employ glass bead lysates to look at native protein function. Finally, we demonstrate an experimental utility for glass bead-induced aggregation, using its fine discrimination of membrane-associated from non-associated Yck2 forms to discern fractional palmitoylation states of Yck2 mutants that are partially defective for palmitoylation.

Neural palmitoyl-proteomics reveals dynamic synaptic palmitoylation.

Palmitoylation regulates diverse aspects of neuronal protein trafficking and function. Here a global characterization of rat neural palmitoyl-proteomes identifies most of the known neural palmitoyl proteins-68 in total, plus more than 200 new palmitoyl-protein candidates, with further testing confirming palmitoylation for 21 of these candidates. The new palmitoyl proteins include neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins, as well as SNAREs and other vesicular trafficking proteins. Of particular interest is the finding of palmitoylation for a brain-specific Cdc42 splice variant. The palmitoylated Cdc42 isoform (Cdc42-palm) differs from the canonical, prenylated form (Cdc42-prenyl), both with regard to localization and function: Cdc42-palm concentrates in dendritic spines and has a special role in inducing these post-synaptic structures. Furthermore, assessing palmitoylation dynamics in drug-induced activity models identifies rapidly induced changes for Cdc42 as well as for other synaptic palmitoyl proteins, suggesting that palmitoylation may participate broadly in the activity-driven changes that shape synapse morphology and function.

Palmitoylated proteins: purification and identification.

This proteomic protocol purifies and identifies palmitoylated proteins (i.e., S-acylated proteins) from complex protein extracts. The method relies on an acyl-biotinyl exchange chemistry in which biotin moieties are substituted for the thioester-linked protein acyl-modifications through a sequence of three in vitro chemical steps: (i) blockade of free thiols with N-ethylmaleimide; (ii) cleavage of the Cys-palmitoyl thioester linkages with hydroxylamine; and (iii) labeling of thiols, newly exposed by the hydroxylamine, with biotin-HPDP (Biotin-HPDP-N-[6-(Biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide. The biotinylated proteins are then affinity-purified using streptavidin-agarose and identified by multi-dimensional protein identification technology (MuDPIT), a high-throughput, tandem mass spectrometry (MS/MS)-based proteomic technology. MuDPIT also affords a semi-quantitative analysis that may be used to assess the gross changes induced to the global palmitoylation profile by mutation or drugs. Typically, 2-3 weeks are required for this analysis.

Proteomic identification of palmitoylated proteins.

A proteomic method that purifies and identifies palmitoylated proteins from complex protein extracts is described. Using the fatty acid exchange labeling chemistry (described in the preceding report), palmitoyl modifications are exchanged for biotinylated compounds, allowing the subset of palmitoyl-proteins to be affinity-purified and then identified by mass spectroscopic protein identification technologies. The advantages and pitfalls of this new technology are discussed within the context of the recent application of this method in the yeast Saccharomyces cerevisiae.

Palmitoylation by the DHHC protein Pfa4 regulates the ER exit of Chs3.

The yeast chitin synthase Chs3 provides a well-studied paradigm for polytopic membrane protein trafficking. In this study, high-throughput analysis of the yeast deletion collection identifies a requirement for Pfa4, which is an uncharacterized protein with protein acyl transferase (PAT) homology, in Chs3 transport. PATs, which are the enzymatic mediators of protein palmitoylation, have only recently been discovered, and few substrates have been identified. We find that Chs3 is palmitoylated and that this modification is Pfa4-dependent, indicating that Pfa4 is indeed a PAT. Chs3 palmitoylation is required for ER export, but not for interaction with its dedicated ER chaperone, Chs7. Nonetheless, both palmitoylation and chaperone association are required to prevent the accumulation of Chs3 in high-molecular mass aggregates at the ER. Our data indicate that palmitoylation is necessary for Chs3 to attain an export-competent conformation, and suggest the possibility of a more general role for palmitoylation in the ER quality control of polytopic membrane proteins.

Global analysis of protein palmitoylation in yeast.

Protein palmitoylation is a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Palmitoylation has proven to be a difficult study: Specifying consensuses for predicting palmitoylation remain unavailable, and first-example palmitoylation enzymes--i.e., protein acyltransferases (PATs)--were identified only recently. Here, we use a new proteomic methodology that purifies and identifies palmitoylated proteins to characterize the palmitoyl proteome of the yeast Saccharomyces cerevisiae. Thirty-five new palmitoyl proteins are identified, including many SNARE proteins and amino acid permeases as well as many other participants in cellular signaling and membrane trafficking. Analysis of mutant yeast strains defective for members of the DHHC protein family, a putative PAT family, allows a matching of substrate palmitoyl proteins to modifying PATs and reveals the DHHC family to be a family of diverse PAT specificities responsible for most of the palmitoylation within the cell.

Elevation in plasma Abeta42 in geriatric depression: a pilot study.

Elevated plasma amyloid beta 1-42 (Abeta42) level has been linked to increased risk for incident AD in cognitively-intact elderly. However, plasma Abeta levels in individuals with late-life depression (LLMD), especially those with a late age of onset of first depressive episode, who are at a particularly increased risk for Alzheimer's disease, have not been studied. We compared plasma Abeta in 47 elderly with LLMD with 35 controls and examined its relationships to age of onset of first depressive episode, antidepressant treatment (paroxetine or nortriptyline), and indices of platelet activation (platelet factor 4 and beta-thromboglobulin) and brain abnormalities. Results indicated that plasma Abeta42 levels and the Abeta42/40 ratio were elevated in the LLMD group relative to controls in the overall group analyses and in the age- and gender-matched groups. MRI data indicated that higher Abeta42/40 ratio was associated with greater severity of total white matter hyperintensity burden in LLMD. Plasma Abeta levels in LLMD were not influenced by age of onset of first depressive episode or antidepressant treatment and were not related to indices of platelet activation. Our preliminary results suggest that increased plasma Abeta42 and Abeta42/40 ratio are present in geriatric depression, and future studies should be done to confirm these findings and to determine their relationship to cognitive decline and brain abnormalities associated with LLMD.

Dose-dependent retrograde facilitation of verbal memory in healthy elderly after acute oral lorazepam administration.

RATIONALE: Retrograde facilitation (RF) refers to a paradoxical phenomenon in which recall of information presented before acute administration of agents generally associated with anterograde amnestic and sedative effects, such as benzodiazepines, is enhanced relative to a placebo condition. However, it is unclear whether this effect occurs in elderly individuals and if it is influenced by plasma drug levels, baseline cognitive function, or genetic factors such as the APOE e-4 allele, that may modulate drug-induced cognitive toxicity. OBJECTIVES: To determine if acute oral doses of lorazepam (0.5 mg, 1 mg) produced RF in elderly individuals exposed to interference tasks, and the variables associated with RF. MATERIALS AND METHODS: Sixty-four cognitively intact and highly educated (>12 years) older adults (mean age, 66.09 years) participated in a placebo-controlled double-blind crossover study. The Buschke Selective Reminding Test was used to assess RF and amnestic effects for verbal memory. Self-ratings of mood states were also obtained. RESULTS: Lorazepam administration resulted in significant dose-dependent RF, i.e., better recall of pre-drug word lists compared to placebo [F(1,63)= 15.358; p<0.001 and F(1,63)= 46.163; p<0.001 for 0.5 and 1 mg lorazepam, respectively]. Also, more of the pre-drug words were recalled in the 1.0-mg-lorazepam condition relative to the 0.5-mg-lorazepam condition [F(1,63)=29.498; p<0.001]. In both the 0.5 and 1 mg lorazepam conditions, participants who exhibited high RF experienced significantly greater lorazepam-induced memory impairments over time [F(3,61)=2.901; p<0.05; F(3,61)=2.698; p<0.05; 0.5 and 1 mg lorazepam, respectively]. Also, in the 1-mg-lorazepam condition, participants who exhibited high RF reported significantly greater drowsiness relative to participants who showed less RF [t(62)=-2.521; p<0.05]. RF was not significantly associated with age, the APOE epsilon4 allele, years of education, global cognitive status, vocabulary scores, or a memory index score. CONCLUSION: In healthy elderly, acute oral lorazepam administration resulted in dose-dependent RF, which was associated with greater anterograde amnestic and sedative effects.

Transmembrane topology of the protein palmitoyl transferase Akr1.

The two recently identified protein acyl transferases (PATs), Akr1p and Erf2p/Erf4p, point toward the DHHC protein family as a likely PAT family. The DHHC protein family, defined by the novel, zinc finger-like DHHC cysteine-rich domain (DHHC-CRD), is a diverse collection of polytopic membrane proteins extending through all eukaryotes. To define the PAT domains that are oriented to the cytoplasm and are thus available to effect the cytoplasmically limited palmitoyl modification, we have determined the transmembrane topology of the yeast PAT Akr1p. Portions of the yeast protein invertase (Suc2p) were inserted in-frame at 10 different hydrophilic sites within the Akr1 polypeptide. Three of the Akr1-Suc2-Akr1 insertion proteins were found to be extensively glycosylated, indicating that the invertase segment inserted at these Akr1p sites is luminally oriented. The remaining seven insertion proteins were not glycosylated, consistent with a cytoplasmic orientation for these sites. The results support a model in which the Akr1 polypeptide crosses the bilayer six times with the bulk of its hydrophilic domains disposed toward the cytoplasm. Cytoplasmic domains include both the relatively large, ankyrin repeat-containing N-terminal domain and the DHHC-CRD, which maps to a cytosolic loop segment. Functionality of the different Akr1-Suc2-Akr1 proteins also was examined. Insertions at only 4 of the 10 sites were found to disrupt Akr1p function. Interestingly, these four sites all map cytoplasmically, suggesting key roles for these cytoplasmic domains in Akr1 PAT function. Finally, extrapolating from the Akr1p topology, topology models are proposed for other DHHC protein family members.

Blinded laboratory comparison of the in situ enzyme immunoassay, the VecTest wicking assay, and a reverse transcription-polymerase chain reaction assay to detect mosquitoes infected with West Nile and St. Louis encephalitis viruses.

A blinded laboratory evaluation compared the accuracy, sensitivity, and specificity of an in situ enzyme immunoassay (EIA), VecTest wicking assay, and reverse transcription-polymerase chain reaction (RT-PCR) to detect and distinguish West Nile (WN) and St. Louis encephalitis (SLE) viruses in pools of 50 mosquitoes. Adult female Culex tarsalis Coquillett were inoculated with either WN or SLE viruses, held for 0-11 d at 28 degrees C, killed by freezing, and then were added to 49 or 48 uninfected mosquitoes to make up 14 pools positive for WN virus, 14 positive for SLE virus, 14 positive for both WN and SLE viruses, and 14 negative for virus. Pools were number coded and tested blindly. Virus was not detected in known negative pools. VecTest and RT-PCR assays were comparably sensitive and accurate, detecting virus in pools containing females held for 3 d postinoculation; only RT-PCR detected SLE virus in pools on days 0-1. The VecTest and RT-PCR produced a single false-positive result for WN and SLE, respectively. RT-PCR detected RNA in samples positive by the VecTest, indicating that the detergent in the wicking buffer did not prevent RT-PCR from confirming VecTest results. Detector antibodies used in the in situ EIA cross-reacted between SLE and WN viruses, reducing accuracy. Both the VecTest and RT-PCR provided rapid and specific results, but they detected only those viruses known to be present. Plaque assay on Vero cells was comparably sensitive and had the added benefit of detecting newly emerging viruses, but this method required virus culture followed by identification, thereby delaying reporting.

The yeast casein kinase Yck3p is palmitoylated, then sorted to the vacuolar membrane with AP-3-dependent recognition of a YXXPhi adaptin sorting signal.

Our previous work found the two yeast plasma membrane-localized casein kinases Yck1p and Yck2p to be palmitoylated on C-terminal Cys-Cys sequences by the palmitoyl transferase Akr1p. The present work examines a third casein kinase, Yck3p, which ends with the C-terminal sequence Cys-Cys-Cys-Cys-Phe-Cys-Cys-Cys. Yck3p is palmitoylated and localized to the vacuolar membrane. While the C-terminal cysteines are required for this palmitoylation, Akr1p is not. Palmitoylation requires the C-terminal Yck3p residues 463-524, whereas information for vacuolar sorting maps to the 409-462 interval. Vacuolar sorting is disrupted in cis through deletion of the 409-462 sequences and in trans through mutation of the AP-3 adaptin complex; both cis- and trans-mutations result in Yck3p missorting to the plasma membrane. This missorted Yck3p restores 37 degrees C viability to yck1Delta yck2-ts cells. yck1Delta yck2-ts suppressor mutations isolated within the YCK3 gene identify the Yck3p vacuolar sorting signal-the tetrapeptide YDSI, a perfect fit to the YXXPhi adaptin-binding consensus. Although YXXPhi signals have a well-appreciated role in the adaptin-mediated sorting of mammalian cells, this is the first signal of this class to be identified in yeast.

Vertical transmission of West Nile Virus by three California Culex (Diptera: Culicidae) species.

Three California Culex species previously identified as efficient laboratory vectors of West Nile (WN) virus were tested for their capability to vertically transmit WN virus. Wild-caught Culex pipiens pipiens L., Culex pipiens quinquefasciatus Say, and two populations of Culex tarsalis Coquillett females were inoculated intrathoracically with 10(2.7 +/- 0.1) plaque-forming units of WN virus. F1 progeny were reared at 18 degrees C and subsequently tested as adults for infectious virus on Vero cell culture. Virus was not detected in 197 pools comprising 4,884 Cx. p. pipiens. The minimum filial infection rate (MFIR) for Cx. p. quinquefasciatus was approximately 3.0/1,000 for 665 progeny tested in 28 pools. There was no virus detected in 102 pools of 2,453 progeny from Cx. tarsalis collected in Riverside County. The MFIR for Cx. tarsalis collected in Yolo County was approximately 6.9/1,000 for 2,165 progeny tested in 86 pools. Mosquito progeny infected vertically during the fall could potentially serve as a mechanism for WN virus to overwinter and initiate horizontal transmission the following spring.

Vector competence of California mosquitoes for West Nile virus.

To identify the mosquito species competent for West Nile virus (WNV) transmission, we evaluated 10 California species that are known vectors of other arboviruses or major pests: Culex tarsalis, Cx. pipiens pipiens, Cx. p. quinquefasciatus, Cx. stigmatosoma, Cx. erythrothorax, Ochlerotatus dorsalis, Oc. melanimon, Oc. sierrensis, Aedes vexans, and Culiseta inornata. All 10 became infected and were able to transmit WNV at some level. Ochlerotatus, Culiseta, and Aedes were low to moderately efficient vectors. They feed primarily on mammals and could play a secondary role in transmission. Oc. sierrensis, a major pest species, and Cx. p. quinquefasciatus from southern California were the least efficient laboratory vectors. Cx. tarsalis, Cx. stigmatosoma, Cx. erythrothorax, and other populations of Cx. pipiens complex were the most efficient laboratory vectors. Culex species are likely to play the primary role in the enzootic maintenance and transmission of WNV in California.

The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase.

Protein palmitoylation has been long appreciated for its role in tethering proteins to membranes, yet the enzymes responsible for this modification have eluded identification. Here, experiments in vivo and in vitro demonstrate that Akr1p, a polytopic membrane protein containing a DHHC cysteine-rich domain (CRD), is a palmitoyl transferase (PTase). In vivo, we find that the casein kinase Yck2p is palmitoylated and that Akr1p function is required for this modification. Akr1p, purified to near homogeneity from yeast membranes, catalyzes Yck2p palmitoylation in vitro, indicating that Akr1p is itself a PTase. Palmitoylation is stimulated by added ATP. Furthermore, during the reaction, Akr1p is itself palmitoylated, suggesting a role for a palmitoyl-Akr1p intermediate in the overall reaction mechanism. Mutations introduced into the Akr1p DHHC-CRD eliminate both the trans- and autopalmitoylation activities, indicating a central participation of this conserved sequence in the enzymatic reaction. Finally, our results indicate that palmitoylation within the yeast cell is controlled by multiple PTase specificities. The conserved DHHC-CRD sequence, we propose, is the signature feature of an evolutionarily widespread PTase family.