Exploring pre-implementation perceptions of integrated care in a university setting.

Objective: To examine providers' perceptions of integrated care prior to the merger of a university's student health center and counseling services. Participants: Seventeen providers across student health services (n = 9) and counseling (n = 8) agreed to participate in the qualitative study. Method: Semi-structured individual interviews that focused on the perceived benefits and challenges of the merger were conducted in December 2019. Following the interviews, a thematic analysis was completed. Results: The perceived benefits noted by providers centered on the ability of an interdisciplinary team to improve the coordination of, access to, and quality of care delivered to students. However, more anticipated challenges were reported by providers (e.g., differences in training and care protocols, losing one's autonomy as a provider). Conclusions: This qualitative study provides a more in-depth analysis of providers' perceptions of integrated care prior to implementation in a university setting and may have implications for model adoption.

The invertebrate B(0) system transporter, D. melanogaster NAT1, has unique d-amino acid affinity and mediates gut and brain functions.

The CG3252 gene product, DmNAT1, represents the first Nutrient Amino acid Transporter cloned from Drosophila. It absorbs a broader set of neutral amino acids versus earlier characterized insect NATs and mammalian NATs-B(0) system transporters from the Sodium Neurotransmitter symporter Family (SNF, a.k.a. solute carrier family 6, SLC6). In addition to B(0)-specific l-substrates, DmNAT1 equally or more effectively transports d-amino acids with sub-millimolar affinities and 1:1 sodium:amino acid transport stoichiometry. DmNAT1 is strongly transcribed in the absorptive and secretory regions of the larval alimentary canal and larval brain, revealing its roles in the primary absorption and redistribution of large neutral l-amino acids as well as corresponding d-isomers. The absorption of d-amino acids via DmNAT1 may benefit the acquisition of fermented and symbiotic products, and may support the unique capacity of fruit fly larvae to utilize a diet with substitution of essential amino acids by d-isomers. It also suggests a remarkable adaptive plasticity of NAT-SLC6 mechanisms via alterations of a few identifiable sites in the substrate-binding pocket. The strong transcription in the brain suggests roles for DmNAT1 in neuronal nutrition and clearance of l-neutral amino acids from the fly brain. In addition, neuronal DmNAT1 may absorb synaptic d-serine and modulate NMDA receptor-coupled signal transduction. The characterization of the first invertebrate B(0)-like transporter extends the biological roles of the SLC6 family, revealing adaptations for the absorption of d-isomers of the essential amino acids. These findings suggest that some members of the NAT-SLC6 subfamily are evolving specific properties which contribute to nutrient symbiotic relationships and neuronal functions.

A novel eukaryotic Na+ methionine selective symporter is essential for mosquito development.

AeNAT5 (NCBI, ABZ81822), an orphan member of the insect-specific Nutrient Amino acid Transporter subfamily of SoLute Carrier family 6 (NAT-SLC6) and the first representative of a novel eukaryotic methionine-selective transport system (M), was cloned from cDNA of the vector mosquito, Aedes aegypti. It has orphan orthologs throughout several mosquito genomes, but not in Drosophila or outside Diptera. It shows the highest apparent affinity to L-Met (K(0.5) = 0.021 mM) and its metabolites Homocysteine and Cysteine (K(0.5) = 0.89 and 2.16 mM), but weakly interact with other substrates. It has a Na(+) - coupled mechanism (K(0.5) Na(+) ∼ 46 mM) with 1AA:1Na(+) stoichiometry that maintains ∼60% activity in Cl(-) - free media. In situ hybridization showed accumof AeNAT5 transcript in the absorptive and secretory epithelia, as well as in specific peripheral neurons and the central ganglia of mosquito larvae. The labeling pattern is distinct from that of the previously characterized AeNAT1. RNAi of AeNAT5 increases larval mortality during ecdysis and dramatically suppresses adult emergence. Our results showed that in addition to previously characterized broad spectra and aromatic amino acid selective transport systems, the mosquito NAT-SLC6 subfamily evolved a unique mechanism for selective absorption of sulfur-containing substrates. We demonstrated specific patterns of alimentary and neuronal transcription of AeNAT5 in mosquito larvae that is collateral with the indispensable function of this transporter in mosquito development.

Cloning and functional expression of the first eukaryotic Na+-tryptophan symporter, AgNAT6.

The nutrient amino acid transporter (NAT) subfamily of the neurotransmitter sodium symporter family (NSS, also known as the solute carrier family 6, SLC6) represents transport mechanisms with putative synergistic roles in the absorption of essential and conditionally essential neutral amino acids. It includes a large paralogous expansion of insect-specific genes, with seven genes from the genome of the malaria mosquito, Anopheles gambiae. One of the An. gambiae NATs, AgNAT8, was cloned, functionally expressed and characterized in X. laevis oocytes as a cation-coupled symporter of aromatic amino acids, preferably l-phenylalanine, l-tyrosine and l-DOPA. To explore an evolutionary trend of NAT-SLC6 phenotypes, we have cloned and characterized AgNAT6, which represents a counterpart of AgNAT8 descending from a recent gene duplication (53.1% pairwise sequence identity). In contrast to AgNAT8, which preferably mediates the absorption of phenol-branched substrates, AgNAT6 mediates the absorption of indole-branched substrates with highest apparent affinity to tryptophan (K(0.5)(Trp)=1.3 micromol l(-1) vs K(0.5)(Phe)=430 micromol l(-1)) and [2 or 1 Na(+) or K(+)]:[aromatic substrate] stoichiometry. AgNAT6 is highly transcribed in absorptive and secretory regions of the alimentary canal and specific neuronal structures, including the neuropile of ventral ganglia and sensory afferents. The alignment of AgNATs and LeuT(Aa), a bacterial NAT with a resolved 3D structure, reveals three amino acid differences in the substrate-binding pocket that may be responsible for the indole- vs phenol-branch selectivity of AgNAT6 vs AgNAT8. The identification of transporters with a narrow selectivity for essential amino acids suggests that basal expansions in the SLC6 family involved duplication and retention of NATs, improving the absorption and distribution of under-represented essential amino acids and related metabolites. The identified physiological and expression profiles suggest unique roles of AgNAT6 in the active absorption of indole-branched substrates that are used in the synthesis of the neurotransmitter serotonin as well as the key circadian hormone and potent free-radical scavenger melatonin.

Synergy and specificity of two Na+-aromatic amino acid symporters in the model alimentary canal of mosquito larvae.

The nutrient amino acid transporter (NAT) subfamily is the largest subdivision of the sodium neurotransmitter symporter family (SNF; also known as SLC6; HUGO). There are seven members of the NAT population in the African malaria mosquito Anopheles gambiae, two of which, AgNAT6 and AgNAT8, preferably transport indole- and phenyl-branched substrates, respectively. The relative expression and distribution of these aromatic NATs were examined with transporter-specific antibodies in Xenopus oocytes and mosquito larval alimentary canal, representing heterologous and tissue expression systems, respectively. NAT-specific aromatic-substrate-induced currents strongly corresponded with specific accumulation of both transporters in the plasma membrane of oocytes. Immunolabeling revealed elevated expressions of both transporters in specific regions of the larval alimentary canal, including salivary glands, cardia, gastric caeca, posterior midgut and Malpighian tubules. Differences in relative expression densities and spatial distribution of the transporters were prominent in virtually all of these regions, suggesting unique profiles of the aromatic amino acid absorption. For the first time reversal of the location of a transporter between apical and basal membranes was identified in posterior and anterior epithelial domains corresponding with secretory and absorptive epithelial functions, respectively. Both aromatic NATs formed putative homodimers in the larval gut whereas functional monomers were over-expressed heterologously in Xenopus oocytes. The results unequivocally suggest functional synergy between substrate-specific AgNAT6 and AgNAT8 in intracellular absorption of aromatic amino acids. More broadly, they suggest that the specific selectivity, regional expression and polarized membrane docking of NATs represent key adaptive traits shaping functional patterns of essential amino acid absorption in the metazoan alimentary canal and other tissues.

Molecular cloning, phylogeny and localization of AgNHA1: the first Na+/H+ antiporter (NHA) from a metazoan, Anopheles gambiae.

We have cloned a cDNA encoding a new ion transporter from the alimentary canal of larval African malaria mosquito, Anopheles gambiae Giles sensu stricto. Phylogenetic analysis revealed that the corresponding gene is in a group that has been designated NHA, and which includes (Na+ or K+)/H+ antiporters; so the novel transporter is called AgNHA1. The annotation of current insect genomes shows that both AgNHA1 and a close relative, AgNHA2, belong to the cation proton antiporter 2 (CPA2) subfamily and cluster in an exclusive clade of genes with high identity from Aedes aegypti, Drosophila melanogaster, D. pseudoobscura, Apis mellifera and Tribolium castaneum. Although NHA genes have been identified in all phyla for which genomes are available, no NHA other than AgNHA1 has previously been cloned, nor have the encoded proteins been localized or characterized. The AgNHA1 transcript was localized in An. gambiae larvae by quantitative real-time PCR (qPCR) and in situ hybridization. AgNHA1 message was detected in gastric caeca and rectum, with much weaker transcription in other parts of the alimentary canal. Immunolabeling of whole mounts and longitudinal sections of isolated alimentary canal showed that AgNHA1 is expressed in the cardia, gastric caeca, anterior midgut, posterior midgut, proximal Malpighian tubules and rectum, as well as in the subesophageal and abdominal ganglia. A phylogenetic analysis of NHAs and KHAs indicates that they are ubiquitous. A comparative molecular analysis of these antiporters suggests that they catalyze electrophoretic alkali metal ion/hydrogen ion exchanges that are driven by the voltage from electrogenic H+ V-ATPases. The tissue localization of AgNHA1 suggests that it plays a key role in maintaining the characteristic longitudinal pH gradient in the lumen of the alimentary canal of An. gambiae larvae.

RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction.

RBP16 is an abundant RNA binding protein from Trypanosoma brucei mitochondria that affects both RNA editing and stability. We report here experiments aimed at elucidating the mechanism of RBP16 function in RNA editing. In in vitro RNA editing assays, recombinant RBP16 is able to significantly stimulate insertion editing of both CYb and A6 pre-mRNAs. Enhancement of in vitro editing activity occurs at, or prior to, the step of pre-mRNA cleavage, as evidenced by increased accumulation of pre-mRNA 3' cleavage products in the presence of RBP16. Mutated RBP16 that is severely compromised in cold shock domain (CSD)-mediated RNA binding was able to enhance editing to levels comparable to the wild-type protein in some assays at the highest RBP16 levels tested. However, at low RBP16 concentrations or in assays with native, oligo(U)-tail-bearing gRNAs, editing stimulation by mutant RBP16 was somewhat compromised. Together, these results indicate that both the N-terminal CSD and C-terminal RGG RNA binding domains of RBP16 are required for maximal editing stimulation. Finally, the relaxed specificity of RBP16 for stimulation of both CYb and A6 editing in vitro implicates additional specificity factors that account for the strict CYb specificity of RBP16 action in editing in vivo. Our results constitute the first report of any putative RNA editing accessory factor eliciting an effect on editing in vitro. Overall, these results support a novel accessory role for RBP16 in U insertion editing.

Molecular characterization of the first aromatic nutrient transporter from the sodium neurotransmitter symporter family.

Nutrient amino acid transporters (NATs, subfamily of sodium neurotransmitter symporter family SNF, a.k.a. SLC6) represent a set of phylogenetically and functionally related transport proteins, which perform intracellular absorption of neutral, predominantly essential amino acids. Functions of NATs appear to be critical for the development and survival in organisms. However, mechanisms of specific and synergetic action of various NAT members in the amino acid transport network are virtually unexplored. A new transporter, agNAT8, was cloned from the malaria vector mosquito Anopheles gambiae (SS). Upon heterologous expression in Xenopus oocytes it performs high-capacity, sodium-coupled (2:1) uptake of nutrients with a strong preference for aromatic catechol-branched substrates, especially phenylalanine and its derivatives tyrosine and L-DOPA, but not catecholamines. It represents a previously unknown SNF phenotype, and also appears to be the first sodium-dependent B(0) type transporter with a narrow selectivity for essential precursors of catecholamine synthesis pathways. It is strongly and specifically transcribed in absorptive and secretory parts of the larval alimentary canal and specific populations of central and peripheral neurons of visual-, chemo- and mechano-sensory afferents. We have identified a new SNF transporter with previously unknown phenotype and showed its important role in the accumulation and redistribution of aromatic substrates. Our results strongly suggest that agNAT8 is an important, if not the major, provider of an essential catechol group in the synthesis of catecholamines for neurochemical signaling as well as ecdysozoan melanization and sclerotization pathways, which may include cuticle hardening/coloring, wound curing, oogenesis, immune responses and melanization of pathogens.

Trypanosoma brucei: functions of RBP16 cold shock and RGG domains in macromolecular interactions.

The RNA binding protein RBP16 regulates mitochondrial RNA editing and stability in Trypanosoma brucei. To aid in understanding the biochemical mechanisms of RBP16 function, we analyzed the RNA and protein binding capacity of RBP16 and its individual cold shock (CSD) and RGG domains. Both recombinantly expressed domains possess RNA binding activity. However, the specificity and affinity of RBP16 for gRNA is mediated predominantly through the interaction of the CSD with poly(U). The RGG domain contributes to the association between full length RBP16 and gRNA, as it was required for maximal binding. We further demonstrate that both domains contribute to maximal binding of RBP16 to the mitochondrial p22 protein. However, p22 can interact with the CSD alone and stimulate its gRNA binding activity. Thus, the CSD is primary in RBP16 interactions, while the RGG domain enhances the capacity of the CSD to bind both RNA and protein. These results suggest a model for RBP16 molecular interactions.