T-bet+ B cells are activated by and control endogenous retroviruses through TLR-dependent mechanisms.

Endogenous retroviruses (ERVs) are an integral part of the mammalian genome. The role of immune control of ERVs in general is poorly defined as is their function as anti-cancer immune targets or drivers of autoimmune disease. Here, we generate mouse-strains where Moloney-Murine Leukemia Virus tagged with GFP (ERV-GFP) infected the mouse germline. This enables us to analyze the role of genetic, epigenetic and cell intrinsic restriction factors in ERV activation and control. We identify an autoreactive B cell response against the neo-self/ERV antigen GFP as a key mechanism of ERV control. Hallmarks of this response are spontaneous ERV-GFP+ germinal center formation, elevated serum IFN-γ levels and a dependency on Age-associated B cells (ABCs) a subclass of T-bet+ memory B cells. Impairment of IgM B cell receptor-signal in nucleic-acid sensing TLR-deficient mice contributes to defective ERV control. Although ERVs are a part of the genome they break immune tolerance, induce immune surveillance against ERV-derived self-antigens and shape the host immune response.

Convergent Biocatalytic Mediated Synthesis of siRNA.

New technologies are required to combat the challenges faced with manufacturing commercial quantities of oligonucleotide drug substances which are required for treating large patient populations. Herein we report a convergent biocatalytic synthesis strategy for an Alnylam model siRNA. The siRNA chemical structure includes several of the unnatural modifications and conjugations typical of siRNA drug substances. Using Almac's 3-2-3-2 hybrid RNA ligase enzyme strategy that sequentially ligates short oligonucleotide fragments (blockmers), the target siRNA was produced to high purity at 1 mM concentration. Additional strategies were investigated including the use of polynucleotide kinase phosphorylation and the use of crude blockmer starting materials without chromatographic purification. These findings highlight a path toward a convergent synthesis of siRNAs for large-scale manufacture marrying both enzymatic liquid and classical solid-phase synthesis.

Locoregional lung ventilation distribution in girls with adolescent idiopathic scoliosis and healthy adolescents. The immediate effect of Schroth 'derotational breathing' exercise in a controlled-trial.

BACKGROUND: Scoliosis curves present transverse plane deviations due to vertebral rotation. The Schroth method supports thoracic derotation by training patients to exert "derotational" breathing based on assumed enhanced ventilation in areas called "humps" in scoliosis and a patient's ability to voluntarily direct ventilation in less ventilated areas called "flats." OBJECTIVE: To assess the asymmetric ventilation distribution and the ability of patients to direct their ventilation to perform derotational breathing. METHODS: Twelve girls with adolescent idiopathic scoliosis and 12 healthy girls performed 3 × 3 min of rest, maximal, and derotational breathing. Electrical impedance tomography was used to record locoregional lung ventilation distribution (LLVD) within 4 thoracic regions of interest: anterior right (ROI 1), anterior left (ROI 2), posterior right (ROI 3), and posterior left (ROI 4) quadrants. Humps and flats were the sums of ROI '2 + 3' and ROI '1 + 4,' respectively. RESULTS: Overall, no difference in LLVD was observed in the flats and humps between groups. At rest, the LLVD in the humps was more elevated than that in the flats (51.5 ± 8.1% versus 43.6 ± 7.9%; p = .021) when considering both groups. Maximal and derotational breathing led to a more homogeneous LLVD between the humps and flats. CONCLUSION: The postulated derotational breathing effect was not confirmed.

Rural health systems' perceptions of referral to community pharmacists during transitions of care.

OBJECTIVES: To identify rural health systems' perceptions of value, benefits, barriers, and opportunities associated with community pharmacist involvement in patient transitions of care. SETTING: Rural health systems in northwest and central Missouri. PRACTICE DESCRIPTION AND INNOVATION: Qualitative descriptive study of key informant interviews with self-identified decision makers of rural health systems within a 50-mile radius of 15 independent community pharmacy chain locations. EVALUATION: Interviews were recorded, transcribed, and coded to evaluate themes in participant responses. RESULTS: Fifteen interviews were conducted at 8 rural health systems. Participants expressed significant value in community pharmacist involvement in transitions of care and highlighted several benefits, barriers, and opportunities related to potential collaboration. Benefits that were identified included medication monitoring, resource for patient information, and desire among health care providers to work with community pharmacists. Barriers included legal and regulatory issues with referral, communication, and prescriber utilization. Opportunities described included: patient education, monitoring, and follow-up; targeted interventions; medication access assistance; bedside medication delivery; and collaboration between community pharmacies and health care entities. CONCLUSION: Rural health system informants perceived community pharmacy involvement to be valuable and were receptive to collaboration during transitional care to improve patient outcomes. They highlighted barriers to overcome to truly incorporate community pharmacists into the transitional care arena. Understanding these rural health systems' perceptions can guide community pharmacies in developing collaborative relationships and patient care services to assist with care transitions.

Insight into determinants of substrate binding and transport in a multidrug efflux protein.

Multidrug resistance arising from the activity of integral membrane transporter proteins presents a global public health threat. In bacteria such as Escherichia coli, transporter proteins belonging to the major facilitator superfamily make a considerable contribution to multidrug resistance by catalysing efflux of myriad structurally and chemically different antimicrobial compounds. Despite their clinical relevance, questions pertaining to mechanistic details of how these promiscuous proteins function remain outstanding, and the role(s) played by individual amino acid residues in recognition, binding and subsequent transport of different antimicrobial substrates by multidrug efflux members of the major facilitator superfamily requires illumination. Using in silico homology modelling, molecular docking and mutagenesis studies in combination with substrate binding and transport assays, we identified several amino acid residues that play important roles in antimicrobial substrate recognition, binding and transport by Escherichia coli MdtM, a representative multidrug efflux protein of the major facilitator superfamily. Furthermore, our studies suggested that 'aromatic clamps' formed by tyrosine and phenylalanine residues located within the substrate binding pocket of MdtM may be important for antimicrobial substrate recognition and transport by the protein. Such 'clamps' may be a structurally and functionally important feature of all major facilitator multidrug efflux proteins.

A single-component multidrug transporter of the major facilitator superfamily is part of a network that protects Escherichia coli from bile salt stress.

Resistance to high concentrations of bile salts in the human intestinal tract is vital for the survival of enteric bacteria such as Escherichia coli. Although the tripartite AcrAB-TolC efflux system plays a significant role in this resistance, it is purported that other efflux pumps must also be involved. We provide evidence from a comprehensive suite of experiments performed at two different pH values (7.2 and 6.0) that reflect pH conditions that E. coli may encounter in human gut that MdtM, a single-component multidrug resistance transporter of the major facilitator superfamily, functions in bile salt resistance in E. coli by catalysing secondary active transport of bile salts out of the cell cytoplasm. Furthermore, assays performed on a chromosomal ΔacrB mutant transformed with multicopy plasmid encoding MdtM suggested a functional synergism between the single-component MdtM transporter and the tripartite AcrAB-TolC system that results in a multiplicative effect on resistance. Substrate binding experiments performed on purified MdtM demonstrated that the transporter binds to cholate and deoxycholate with micromolar affinity, and transport assays performed on inverted vesicles confirmed the capacity of MdtM to catalyse electrogenic bile salt/H(+) antiport.

Nucleic acid-sensing Toll-like receptors are essential for the control of endogenous retrovirus viremia and ERV-induced tumors.

The genome of vertebrates contains endogenous retroviruses (ERVs) that are largely nonfunctional relicts of ancestral germline infection by exogenous retroviruses. However, in some mouse strains ERVs are actively involved in disease. Here we report that nucleic acid-recognizing Toll-like receptors 3, 7, and 9 (TLR 3, TLR7, and TLR9) are essential for the control of ERVs. Loss of TLR7 function caused spontaneous retroviral viremia that coincided with the absence of ERV-specific antibodies. Importantly, additional TLR3 and TLR9 deficiency led to acute T cell lymphoblastic leukemia, underscoring a prominent role for TLR3 and TLR9 in surveillance of ERV-induced tumors. Experimental ERV infection induced a TLR3-, TLR7-, and TLR9-dependent group of "acute-phase" genes previously described in HIV and SIV infections. Our study suggests that in addition to their role in innate immunity against exogenous pathogens, nucleic acid-recognizing TLRs contribute to the immune control of activated ERVs and ERV-induced tumors.