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Supplementary Figures S1-S4

We assessed the contribution of IL1 signaling molecules to malignant tumor growth using IL1β−/−, IL1α−/−, and IL1R1−/− mice. Tumors grew progressively in IL1R−/− and IL1α−/− mice but were often absent in IL1β−/− mice. This was observed whether tumors were implanted intradermally or injected intravenously and was true across multiple distinct tumor lineages. Antibodies to IL1β prevented tumor growth in wild-type (WT) mice but not in IL1R1−/− or IL1α−/− mice. Antibodies to IL1α promoted tumor growth in IL1β−/− mice and reversed the tumor-suppressive effect of anti-IL1β in WT mice. Depletion of CD8+ T cells and blockade of lymphocyte mobilization abrogated the IL1β−/− tumor suppressive effect, as did crossing IL1β−/− mice to SCID or Rag1−/− mice. Finally, blockade of IL1β synergized with blockade of PD-1 to inhibit tumor growth in WT mice. These results suggest that IL1β promotes tumor growth, whereas IL1α inhibits tumor growth by enhancing T-cell–mediated antitumor immunity.

Background Early diagnosis and treatment of Buruli ulcer is critical in order to avoid the debilitating effects of the disease. In this regard, the development of new diagnostic and point of care tools is encouraged. The loop-mediated isothermal amplification for the detection of Mycobacterium ulcerans represents one of the new tools with a good potential of being developed into a point of care test. There is however the need to standardize the assays, reduce sample preparation times, improve the detection/visualization system and optimize them for high-throughput screening, adaptable to low resourced laboratories. Methods In this study, we assessed two DNA extraction protocols (modified Boom and EasyNAT methods), three previously published LAMP primer sets (BURULI, MU 2404 and BU-LAMP), and compared the sensitivity and specificity of LAMP assays on three DNA amplification platforms. Results Our results show that Buruli ulcer diagnosis using primers targeting IS2404 for the LAMP method is sensitive (73.75–91.49%), depending on the DNA extraction method used. Even though the modified Boom DNA extraction method provided the best results, its instrumentation requirement prevent it from being field applicable. The EasyNAT method on the other hand is simpler and may represent the best method for DNA extraction in less resourced settings. Conclusions For further work on the development and use of LAMP tests for Buruli diagnosis, it is recommended that the BURULI sets of primers be used, as these yielded the best results in terms of sensitivity (87.50–91.49%) and specificity (89.23–100%), depending on the DNA extraction methods used.

The presence of mosquitoes in an area is dependent on the availability of suitable breeding sites that are influenced by several environmental factors. Identification of breeding habitats for vector surveillance and larval source management is key to disease control programs. We investigated water quality parameters and microbial composition in selected mosquito breeding sites in urban Accra, Ghana and associated these with abundance of Anopheles (Diptera: Culicidae) and Culex (Diptera: Culicidae) larvae. Physicochemical parameters and microbial composition explained up to 72% variance among the breeding sites and separated Anopheles and Culex habitats (P < 0.05). Anopheles and Culex abundances were commonly influenced by water temperature, pH, nitrate, and total hardness with contrasting impacts on the two mosquito species. In addition, total dissolved solids, biochemical oxygen demand, and alkalinity uniquely influenced Anopheles abundance, while total suspended solids, phosphate, sulphate, ammonium, and salinity were significant determinants for Culex. The correlation of these multiple parameters with the occurrence of each mosquito species was high (R2 = 0.99, P < 0.0001). Bacterial content assessment of the breeding ponds revealed that the most abundant bacterial phyla were Patescibacteria, Cyanobacteria, and Proteobacteria, constituting >70% of the total bacterial richness. The oligotrophic Patescibacteria was strongly associated with Anopheles suggestive of the mosquito’s adaptation to environments with less nutrients, while predominance of Cyanobacteria, indicative of rich nutritional source was associated with Culex larval ponds. We propose further evaluation of these significant abiotic and biotic parameters in field identification of larval sources and how knowledge of these can be harnessed effectively to reduce conducive breeding sites for mosquitoes.

We assessed the contribution of IL1 signaling molecules to malignant tumor growth using IL1β−/−, IL1α−/−, and IL1R1−/− mice. Tumors grew progressively in IL1R−/− and IL1α−/− mice but were often absent in IL1β−/− mice. This was observed whether tumors were implanted intradermally or injected intravenously and was true across multiple distinct tumor lineages. Antibodies to IL1β prevented tumor growth in wild-type (WT) mice but not in IL1R1−/− or IL1α−/− mice. Antibodies to IL1α promoted tumor growth in IL1β−/− mice and reversed the tumor-suppressive effect of anti-IL1β in WT mice. Depletion of CD8+ T cells and blockade of lymphocyte mobilization abrogated the IL1β−/− tumor suppressive effect, as did crossing IL1β−/− mice to SCID or Rag1−/− mice. Finally, blockade of IL1β synergized with blockade of PD-1 to inhibit tumor growth in WT mice. These results suggest that IL1β promotes tumor growth, whereas IL1α inhibits tumor growth by enhancing T-cell–mediated antitumor immunity.

Humoral immunity is reliant on efficient recruitment of circulating naive B cells from blood into peripheral lymph nodes (LN) and timely transition of naive B cells to high affinity antibody (Ab)-producing cells. Current understanding of factor(s) coordinating B cell adhesion, activation and differentiation within LN, however, is incomplete. Prior studies on naive B cells reveal remarkably strong binding to putative immunoregulator, galectin (Gal)-9, that attenuates BCR activation and signaling, implicating Gal-9 as a negative regulator in B cell biology. Here, we investigated Gal-9 localization in human tonsils and LNs and unearthed conspicuously high expression of Gal-9 on high endothelial and post-capillary venules. Adhesion analyses showed that Gal-9 can bridge human circulating and naive B cells to vascular endothelial cells (EC), while decelerating transendothelial migration. Moreover, Gal-9 interactions with naive B cells induced global transcription of gene families related to regulation of cell signaling and membrane/cytoskeletal dynamics. Signaling lymphocytic activation molecule F7 (SLAMF7) was among key immunoregulators elevated by Gal-9-binding, while SLAMF7's cytosolic adapter EAT-2, which is required for cell activation, was eliminated. Gal-9 also activated phosphorylation of pro-survival factor, ERK. Together, these data suggest that Gal-9 promotes B cell - EC interactions while delivering anergic signals to control B cell reactivity.

Leukocytes are coated with a layer of heterogeneous carbohydrates (glycans) that modulate immune function, in part by governing specific interactions with glycan-binding proteins (lectins). Although nearly all membrane proteins bear glycans, the identity and function of most of these sugars on leukocytes remain unexplored. Here, we characterize the N-glycan repertoire (N-glycome) of human tonsillar B cells. We observe that naive and memory B cells express an N-glycan repertoire conferring strong binding to the immunoregulatory lectin galectin-9 (Gal-9). Germinal center B cells, by contrast, show sharply diminished binding to Gal-9 due to upregulation of I-branched N-glycans, catalyzed by the β1,6-N-acetylglucosaminyltransferase GCNT2. Functionally, we find that Gal-9 is autologously produced by naive B cells, binds CD45, suppresses calcium signaling via a Lyn-CD22-SHP-1 dependent mechanism, and blunts B cell activation. Thus, our findings suggest Gal-9 intrinsically regulates B cell activation and may differentially modulate BCR signaling at steady state and within germinal centers., Leukocytes are coated with glycans that modulate immune function through interactions with lectins. Here, the authors characterize the N-glycan repertoire of human tonsillar B cells. They report that Gal-9 is an intrinsic regulator of B cell activation that may differentially modulate BCR signaling at steady state and within germinal centers due to expression of I-branched glycans.

The main malaria vectors in sub-Saharan Africa, the Anopheles gambiae (Giles; Diptera: Culicidae), normally breed in clean water sources. However, evidence suggests an on-going adaptation of Anopheline species to polluted breeding habitats in urban settings. This study aimed at understanding the adaptation to breeding in water bodies with different qualities, in five selected mosquito breeding sites in urban Accra, Ghana. The study sites were also evaluated for the WHO water-quality parameters as a measure of pollution, and insecticide residues. Field mosquitoes were evaluated for five genes; CYP6P3, CYP4H19, CYP4H24, GSTD1-4, and ABCC11-associated with insecticide detoxification-using quantitative RT-PCR, as well as Mono-oxygenase, Alpha Esterase, Glutathione S-transferase, and insensitive acetylcholinesterase (AChE) using biochemical enzyme assays. The lab-reared, insecticide susceptible An. gambiae Kisumu strain was bred in the most polluted water source for 10 generations and evaluated for the same genes and enzymes. The results revealed that the fold expression of the genes was higher in the larvae compared with the adults. The results also suggest that detoxification enzymes could be involved in the adaptation of An. gambiae to polluted breeding sites. Correlation analysis revealed a highly positive significant correlation between calcium levels and all five genes (P < 0.05). Stepwise linear regression to understand which of the variables predicted the expression of the genes revealed that sulphate was responsible for ABCC11 and CYP4H24, alkalinity for GSTD1-4, and calcium for CYP4H19 and CYP6P3. The detailed genetic basis of this adaptation need to be further investigated. A further understanding of this adaptation may provide outlooks for controlling malaria and other disease vectors adapted to polluted breeding water sources.

The IL-1 superfamily of cytokines and receptors has been studied extensively. However, the specific roles of IL-1 elements in host immunity to cutaneous viral infection remain elusive. In this study, we applied vaccinia virus (VACV) by scarification to IL-1R1 knockout mice (IL-1R1−/−) and found that these mice developed markedly larger lesions with higher viral genome copies in skin than did wild-type mice. The phenotype of infected IL-1R1−/− mice was similar to eczema vaccinatum, a severe side effect of VACV vaccination that may develop in humans with atopic dermatitis. Interestingly, the impaired cutaneous response of IL-1R1−/− mice did not reflect a systemic immune deficiency, because immunized IL-1R1−/− mice survived subsequent lethal VACV intranasal challenge, or defects of T cell activation or T cell homing to the site of inoculation. Histologic evaluation revealed that VACV infection and replication after scarification were limited to the epidermal layer of wild-type mice, whereas lack of IL-1R1 permitted extension of VACV infection into dermal layers of the skin. We explored the etiology of this discrepancy and determined that IL-1R1−/− mice contained significantly more macrophages and monocyte-derived dendritic cells in the dermis after VACV scarification. These cells were vulnerable to VACV infection and may augment the transmission of virus to adjacent skin, thus leading to larger skin lesions and satellite lesions in IL-1R1−/− mice. These results suggest new therapeutic strategies for treatment of eczema vaccinatum and inform assessment of risks in patients receiving IL-1 blocking Abs for treatment of chronic inflammatory disorders.

A general method for the N-arylation of amino acid esters with aryl triflates is described. Both α- and β-amino acid esters, including methyl, tert-butyl, and benzyl esters, are viable substrates. Reaction optimization was carried out by design of experiment (DOE) analysis using JMP software. The mild reaction conditions, which use t-BuBrettPhos Pd G3 or G4 precatalyst, result in minimal racemization of the amino acid ester. This method is the first synthetic application of the t-BuBrettPhos Pd G4 precatalyst. Mechanistic studies show that the observed erosion in enantiomeric excess is due to racemization of the amino acid ester starting material and not of the product.

Ghana has been implementing mass drug administration (MDA) of ivermectin and albendazole for the elimination of lymphatic filariasis (LF) since the year 2000, as part of the Global Programme to Eliminate Lymphatic Filariasis (GPELF). It was estimated that 5&ndash, 6 years of treatment would be sufficient to eliminate the disease. Tremendous progress has been made over the years, and treatment has stopped in many disease endemic districts. However, despite the successful implementation of MDA, there are districts with persistent transmission. In this study we assessed the epidemiology of LF in three adjoining districts that have received at least 16 years of MDA. The assessments were undertaken one year after the last MDA. 1234 adults and 182 children below the age of 10 years were assessed. The overall prevalence of circulating filarial antigen in the study participants was 8.3% (95% CI: 6.9&ndash, 9.9), with an estimated microfilaria prevalence of 1.2%. The microfilarial intensity in positive individuals ranged from 1 to 57 microfilariae/mL of blood. Higher antigen prevalence was detected in males (13.0%, 95% CI: 10.3&ndash, 16.2) compared to females (5.5%, 95% CI: 4.1&ndash, 7.2). The presence of infection was also highest in individuals involved in outdoor commercial activities, with the risks of infection being four- to five-fold higher among farmers, fishermen, drivers and artisans, compared to all other occupations. Using bednets or participating in MDA did not significantly influence the risk of infection. No children below the age of 10 years were found with infection. Detection of Wb123 antibodies for current infections indicated a prevalence of 14.4% (95% CI: 8.1&ndash, 23.0) in antigen-positive individuals above 10 years of age. No antibodies were detected in children 10 years or below. Assessment of infection within the An. gambiae vectors of LF indicated an infection rate of 0.9% (95% CI: 0.3&ndash, 2.1) and infectivity rate of 0.5% (95% CI: 0.1&ndash, 1.6). These results indicate low-level transmission within the districts, and suggest that it will require targeted interventions in order to eliminate the infection.

Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment. Keywords: dendritic cells; homeostasis; differentiation; IFNγ; tumor microenvironment; melanoma tolerance; immunotherapy; suppressor-of-cytokine-signaling 2 (SOCS2); tissue mononuclear phagocytes

A N-arylation of α- and β-amino acid esters with aryl triflates is presented utilizing a Pd catalyst under mild reaction conditions with minimal racemization.

We describe a general strategy to prepare the hasubanan and acutumine alkaloids, a large family of botanical natural products that display antitumor, antiviral, and memory-enhancing effects. The absolute stereochemistry of the targets is established by an enantioselective Diels-Alder reaction between 5-(trimethylsilyl)cyclopentadiene (36) and 5-(2-azidoethyl)-2,3-dimethoxybenzoquinone (24). The Diels-Alder adduct 38 is transformed to the tetracyclic imine 39 by a Staudinger reduction-aza-Wittig sequence. The latter serves as a universal precursor to the targets. Key carbon-carbon bond constructions include highly diastereoselective acetylide additions to the N-methyliminium ion derived from 39 and Friedel-Crafts and Hosomi-Sakurai cyclizations to construct the carbocyclic skeleton of the targets. Initially, this strategy was applied to the syntheses of (-)-acutumine (4), (-)-dechloroacutumine (5), and four hasubanan alkaloids (1, 2, 3, and 8). Herein, the synthetic route is adapted to the syntheses of six additional hasubanan alkaloids (12, 13, 14, 15, 18, and 19). The strategic advantage of 5-(trimethylsilyl)cyclopentadiene Diels-Alder adducts is demonstrated by site-selective functionalization of distal carbon-carbon π-bonds in the presence of an otherwise reactive norbornene substructure. Evaluation of the antiproliferative properties of the synthetic metabolites revealed that four hasubanan alkaloids are submicromolar inhibitors of the N87 cell line.

Ly6G is a glycosylphosphatidylinositol (GPI)–anchored protein of unknown function that is commonly targeted to induce experimental neutrophil depletion in mice. In the present study, we found that doses of anti-Ly6G Abs too low to produce sustained neutropenia remained capable of inhibiting experimental arthritis, leaving joint tissues free of infiltrating neutrophils. Thioglycollate-stimulated peritonitis was also attenuated. No alteration in neutrophil apoptosis was observed, implicating impaired recruitment. Indeed, Ly6G ligation abrogated neutrophil migration toward LTB4 and other chemoattractants in a transwell system. Exploring the basis for this blockade, we identified colocalization of Ly6G and β2-integrins by confocal microscopy and confirmed close association by both coimmunoprecipitation and fluorescence lifetime imaging microscopy. Anti-Ly6G Ab impaired surface expression of β2-integrins in LTB4-stimulated neutrophils and mimicked CD11a blockade in inhibiting both ICAM-1 binding and firm adhesion to activated endothelium under flow conditions. Correspondingly, migration of β2-integrin–deficient neutrophils was no longer inhibited by anti-Ly6G. These results demonstrate that experimental targeting of Ly6G has functional effects on the neutrophil population and identify a previously unappreciated role for Ly6G as a modulator of neutrophil migration to sites of inflammation via a β2-integrin–dependent mechanism.

One strategy adopted by vaccinia virus (VV) to evade the host immune system is to encode homologs of TNF receptors (TNFRs) that block TNF-α function. The response to VV skin infection under conditions of TNF-α deficiency, however, has not been reported. We found that TNFR1-/- mice developed larger primary lesions, numerous satellite lesions, and higher skin virus levels after VV scarification. Following their recovery, VV-scarified TNFR1-/- mice were fully protected against challenge with a lethal intranasal dose of VV, suggesting these mice had developed an effective memory immune response. A functional systemic immune response was further demonstrated by enhanced production of VV-specific IFN-γ and VV-specific CD8(+) T cells in spleens and draining lymph nodes. Interestingly, bone marrow (BM)-reconstitution studies using wild-type (WT) BM in TNFR1-/- host mice, but not TNFR1-/- BM in WT host mice, reproduced the original results seen in TNFR1-/- mice, indicating that TNFR1 deficiency in resident skin cells, rather than hematopoietic cells, accounts for the impaired cutaneous immune response. Our data suggest that lack of TNFR1 leads to a skin-specific immune deficiency, and that resident skin cells have a crucial role in mediating an optimal immune defense to VV cutaneous infection via TNF-α/TNFR1 signaling.

Several scholars, most notably judges, have called for judicial reform in the selection process of appellate and supreme court justices in Texas. However, not much attention has been placed on the selection process of Texas trial court judges. This Article focuses on the genealogy of district courts in Texas, with an emphasis on Texas's family court system, an area of the law that decides the fate of thousands of children who, for the most part, are unable to speak for themselves and that comprises a majority of civil cases within the state of Texas. As the majority of family court cases are decided by the trial court, judicial review by an appellate or higher court is virtually non-existent. It is therefore necessary to have associate and district court family judges who are educated and adequately staffed to handle the multifarious complex issues involving children and families. It is also vital that citizens better educate themselves about our judicial candidates and the judiciary in general until such time as the Texas legislature gets serious and enacts fundamental change pertaining to the selection of state judges at all levels.

Our objective for this project was to determine if we could find, collect, and tissue culture local native plants which grow directly on acid, heavy metal contaminated (AHM) soil. These plants are potentially useful for mine land reclamation and may possess acid soil, heavy metal tolerance. Explants (small actively growing plant tissue segments) were collected from fifteen plant species growing directly on degraded sites heavily impacted from past hard rock mining activities in Western Montana. These plants, when collected were rooted, surviving and growing in soils characterized by very low soil pH (about 4.0) and high concentrations of various heavy metals. Of the 15 species collected, six were successfully propagated using plant tissue culture, also known as micro- propagation. We concentrated on three of the six species and have attained commercial rates of multiplication and rooting in-vitro. These three species have been successfully acclimated and grown under commercial greenhouse conditions. Results comparing our potentially AHMT tissue cultured plants to non-selected plants of the same species (raised from seed) in test soils under greenhouse conditions will be presented. Future plans for field tests will be described and discussed.

In order to ensure sufficient plants for replanting threatened populations of Physaria didymocarpa var. lanata (Common Twinpod, Wooly Twinpod), in vitro propagation of this species was attempted. Seeds from three plants of Physaria didymocarpa var. lanata were cultured on a Murashige and Skoog salts base medium supplemented with 6-Benzylaminopurine. Both immature and mature seeds survived and germinated in culture. Each seedling culture produced several new shoots every four to six weeks. Each of these shoots was transferred to fresh medium to start new cultures. Roots were produced on excised shoots both in vitro on gelled medium and ex vitro on moistened coco fiber. Six months after initiating 100 seeds, 2,000 plants were ready for shipment to the nursery greenhouse and enough multiplying cultures were established to produce 1,000 new plants every two months. Though further work needs to be done on acclimatization to greenhouse conditions, survivors grew into normal robust plants, flowering in the pots.

Research in the hasubanan and acutumine alkaloid fields up to 1970 was discussed under "morphine alkaloids" in Volume 13 of this chapter. Advances in the field of hasubanan alkaloids from 1971 to 1975 were reviewed in Volume 16 and from 1976 to 1986 in Volume 33. This chapter extends the information in the three preceding reviews to hasubanan alkaloid literature published from 1987 to June 2013. This chapter covers acutumine alkaloid literature since (-)-acutumine (3) was isolated in 1929. This chapter includes occurrence and physical constants, new alkaloids, synthesis, biosynthesis, and pharmacology. Section 1 introduces the foremost alkaloids, (-)-hasubanonine (1) and (-)-acutumine (3), and the numbering systems of the hasubanan (2) and acutumine (4) skeletons. Section 2 details the occurrence and physical constants of 29 new hasubanan and 15 acutumine alkaloids. The isolation and structural determination of these new alkaloids are described in Section 3. Section 4 summarizes total syntheses and synthetic studies toward hasubanan and acutumine alkaloids. Completed syntheses of the hasubanan alkaloids (+)-cepharamine (ent-71), (-)-hasubanonine (1), (-)-runanine (8), (-)-delavayine (6), (+)-periglaucine B (19), and (-)-8-demethoxyrunanine (12) are reviewed. Completed syntheses of (-)-acutumine (3) and (-)-dechloroacutumine (52) are also described. Section 5 details biosyntheses of (-)-acutumine (3) advanced by Barton, Wipf, and Sugimoto. Section 6 summarizes pharmacological studies of hasubanan and acutumine alkaloids. Opioid receptor affinity, anti-HBV activity, and antimicrobial activity of hasubanan alkaloids are reported. Antiamnesic properties, cytotoxicity, and anti-HBV activity of acutumine alkaloids are described.

Background\ud \ud The Global Programme to Eliminate Lymphatic Filariasis was launched in 2000 with the goal of interrupting transmission of lymphatic filariasis (LF) through multiple rounds of mass drug administration (MDA). In Guinea, there is evidence of ongoing LF transmission, but little is known about the most densely populated parts of the country, including the capital Conakry. In order to guide the LF control and elimination efforts, serological and entomological surveys were carried out to determine whether or not LF transmission occurs in Conakry.\ud \ud Methods\ud \ud The prevalence of circulating filarial antigen (CFA) of Wuchereria bancrofti was assessed by an immuno-chromatography test (ICT) in people recruited from all five districts of Conakry. Mosquitoes were collected over a 1-year period, in 195 households in 15 communities. A proportion of mosquitoes were analysed for W. bancrofti, using dissection, loop-mediated isothermal amplification (LAMP) assay and conventional polymerase chain reaction (PCR).\ud \ud Results\ud \ud CFA test revealed no infection in the 611 individuals examined. A total of 14,334 mosquitoes were collected; 14,135 Culex (98.6 %), 161 Anopheles (1.1 %) and a few other species. Out of 1,312 Culex spp. (9.3 %) and 51 An. gambiae (31.7 %) dissected, none was infected with any stage of the W. bancrofti parasite. However, the LAMP assay revealed that 1.8 % of An. gambiae and 0.31 % of Culex spp. were positive, while PCR determined respective prevalences of 0 % and 0.19 %.\ud \ud Conclusions\ud \ud This study revealed the presence of W. bancrofti DNA in mosquitoes, despite the apparent absence of infection in the human population. Although MDA interventions are not recommended where the prevalence of ICT is below 1 %, the entomological results are suggestive of the circulation of the parasite in the population of Conakry. Therefore, rigorous surveillance is still warranted so that LF transmission in Conakry would be identified rapidly and adequate responses being implemented.

Functional group taxonomy provides a powerful conceptual framework to classify and predict the chemical reactivity of molecular structures. These principals are most effective in monofunctional settings, wherein individual functional groups can be analyzed without complications. In more complex settings, the predictive value of these analyses decreases as alternative reaction pathways, promoted by neighboring substituents and aggregate molecular properties, emerge. We refer to this phenomenon as substrate-modified functional group reactivity. In this Perspective, we explain how substrate-modified functional group reactivity molded our synthetic routes to the hasubanan and acutumine alkaloids. These investigations underscore the potential for discovery and insight that can only be gained by studying the reactivity of complex multifunctional structures.

Human memory T cells associated with cutaneous inflammatory responses are characterized by their expression of cutaneous lymphocyte-associated Ag (CLA), a carbohydrate determinant differentially expressed on P-selectin glycoprotein ligand-1 (PSGL-1). Although expression of the CLA epitope on PSGL-1 (CLA+ PSGL-1) by memory T cells is associated with acquisition of E-selectin ligand activity, it is not known whether CLA+ PSGL-1, itself, is a ligand for E-selectin on human T cells or whether other glycoproteins, with or without CLA modification, support E-selectin-dependent rolling in shear flow. To address this issue, we developed a method for real-time analysis of functional adhesive interactions between selectin-bearing cells in shear flow with leukocyte ligands resolved by SDS-PAGE and immobilized on standard Western blots. The results of these studies provide direct evidence that CLA+ PSGL-1 is a functional ligand for both E- and P-selectin, confirm that the P-selectin ligand activity of PSGL-1 is independent of CLA modification, and identify a distinct, non-PSGL-1 E-selectin ligand on CLA-positive human memory T cells.

A general method for the selective hydrogenation of alkenyl halides to alkyl halides is described. Fluoro, chloro, bromo, iodo, and gem-dihaloalkenes are viable substrates for the transformation. The selectivity of the hydrogenation is consistent with reduction by a hydrogen atom transfer pathway.

Research in the hasubanan and acutumine alkaloid fields up to 1970 was discussed under "morphine alkaloids" in Volume 13 of this chapter. Advances in the field of hasubanan alkaloids from 1971 to 1975 were reviewed in Volume 16 and from 1976 to 1986 in Volume 33. This chapter extends the information in the three preceding reviews to hasubanan alkaloid literature published from 1987 to June 2013. This chapter covers acutumine alkaloid literature since (-)-acutumine (3) was isolated in 1929. This chapter includes occurrence and physical constants, new alkaloids, synthesis, biosynthesis, and pharmacology. Section 1 introduces the foremost alkaloids, (-)-hasubanonine (1) and (-)-acutumine (3), and the numbering systems of the hasubanan (2) and acutumine (4) skeletons. Section 2 details the occurrence and physical constants of 29 new hasubanan and 15 acutumine alkaloids. The isolation and structural determination of these new alkaloids are described in Section 3. Section 4 summarizes total syntheses and synthetic studies toward hasubanan and acutumine alkaloids. Completed syntheses of the hasubanan alkaloids (+)-cepharamine (ent-71), (-)-hasubanonine (1), (-)-runanine (8), (-)-delavayine (6), (+)-periglaucine B (19), and (-)-8-demethoxyrunanine (12) are reviewed. Completed syntheses of (-)-acutumine (3) and (-)-dechloroacutumine (52) are also described. Section 5 details biosyntheses of (-)-acutumine (3) advanced by Barton, Wipf, and Sugimoto. Section 6 summarizes pharmacological studies of hasubanan and acutumine alkaloids. Opioid receptor affinity, anti-HBV activity, and antimicrobial activity of hasubanan alkaloids are reported. Antiamnesic properties, cytotoxicity, and anti-HBV activity of acutumine alkaloids are described.

Functional group taxonomy provides a powerful conceptual framework to classify and predict the chemical reactivity of molecular structures. These principals are most effective in monofunctional settings, wherein individual functional groups can be analyzed without complications. In more complex settings, the predictive value of these analyses decreases as alternative reaction pathways, promoted by neighboring substituents and aggregate molecular properties, emerge. We refer to this phenomenon as substrate-modified functional group reactivity. In this Perspective, we explain how substrate-modified functional group reactivity molded our synthetic routes to the hasubanan and acutumine alkaloids. These investigations underscore the potential for discovery and insight that can only be gained by studying the reactivity of complex multifunctional structures.

A general method for the selective hydrogenation of alkenyl halides to alkyl halides is described. Fluoro, chloro, bromo, iodo, and gem-dihaloalkenes are viable substrates for the transformation. The selectivity of the hydrogenation is consistent with reduction by a hydrogen atom transfer pathway.

A general strategy for the preparation of ten hasubanan and two acutumine alkaloids is described.

Research in the hasubanan and acutumine alkaloid fields up to 1970 was discussed under "morphine alkaloids" in Volume 13 of this chapter. Advances in the field of hasubanan alkaloids from 1971 to 1975 were reviewed in Volume 16 and from 1976 to 1986 in Volume 33. This chapter extends the information in the three preceding reviews to hasubanan alkaloid literature published from 1987 to June 2013. This chapter covers acutumine alkaloid literature since (-)-acutumine (3) was isolated in 1929. This chapter includes occurrence and physical constants, new alkaloids, synthesis, biosynthesis, and pharmacology. Section 1 introduces the foremost alkaloids, (-)-hasubanonine (1) and (-)-acutumine (3), and the numbering systems of the hasubanan (2) and acutumine (4) skeletons. Section 2 details the occurrence and physical constants of 29 new hasubanan and 15 acutumine alkaloids. The isolation and structural determination of these new alkaloids are described in Section 3. Section 4 summarizes total syntheses and synthetic studies toward hasubanan and acutumine alkaloids. Completed syntheses of the hasubanan alkaloids (+)-cepharamine (ent-71), (-)-hasubanonine (1), (-)-runanine (8), (-)-delavayine (6), (+)-periglaucine B (19), and (-)-8-demethoxyrunanine (12) are reviewed. Completed syntheses of (-)-acutumine (3) and (-)-dechloroacutumine (52) are also described. Section 5 details biosyntheses of (-)-acutumine (3) advanced by Barton, Wipf, and Sugimoto. Section 6 summarizes pharmacological studies of hasubanan and acutumine alkaloids. Opioid receptor affinity, anti-HBV activity, and antimicrobial activity of hasubanan alkaloids are reported. Antiamnesic properties, cytotoxicity, and anti-HBV activity of acutumine alkaloids are described.

Echoviruses (EV) 1 and 8 were originally considered to be distinct serotypes, but more recently have been considered strains of the same virus. In experiments with chimeric recombinant fusion proteins, both viruses bound to the I domain of the integrin VLA-2, and both required the same receptor residues for attachment. A full-length, infectious cDNA clone encoding EV1 was obtained; its nucleotide sequence was determined, as were the sequences encoding the EV8 capsid. EV1 and 8 show 94% amino acid identity within the capsid region and are more similar to each other than to any other human picornavirus.

The conceptual literature on family businesses suggests that family businesses have difficulty managing their human resources, especially when it concerns a family member or the transition from the founder to the successor. The authors empirically examined the assumptions raised in the conceptual literature regarding whether family businesses were experiencing human resource problems in growing their business and what factors enabled or constrained the ability of their businesses to grow. The authors used in-depth interviewing to collect data in order to emphasize the depth of the issue. Using content analysis with subject matter experts coding the data, the authors sought to mine the richness of data. Finally, the authors analyzed the data using Elliot Jaques' Stratified Systems Theory as a model to examine the strategic human resource issues and to draw some tentative conclusions.

This multisite investigation assessed subjective, behavioral, and objective balance function in 30 blast survivors. Subjects with vertigo, dizziness, or imbalance were screened (n = 6) or evaluated (n = 27) during 1 year. Tests included a questionnaire, electronystagmography (ENG), and computerized dynamic posturography (CDP). Ninety-seven percent of subjects were located inside a building during the blast, and 63 percent of subjects experienced dys-equilibrium within 48 hours. Forty-three percent of symptoms could not be attributed to head injury. Sixty percent of subjects had abnormal ENG and/or CDP; ENG abnormalities mostly were peripheral or nonlocalizing, whereas CDP patterns were “vestibular,” “surface dependent,” and “physiologically inconsistent.” At 1-year postblast, 55 percent of initially abnormal CDP results were normal, and 72 percent of subjects said symptoms were unchanged or occurred intermittently. A serial, test battery approach is recommended to assess symptoms. Blast-related dysequilibrium had clinically significant manifestations and should be considered a valid component of aural blast injury. Abbreviations: BPPV = benign paroxysmal positioning vertigo, CDP = computerized dynamic posturography, dB pSPL = decibel re: peak sound pressure level, ENG = electronystagmography, MCT = motor control test, psi = air pressure measured in pounds per square inch, Q = quarterly evaluations, SHA = smooth harmonic acceleration, SOT = sensory organization test, TNT = trinitrotoluene

This longitudinal study evaluated subjective, behavioral, and objective auditory function in 83 explosion survivors. Subjects were evaluated quarterly for 1 year with conventional pure-tone and extended high-frequencies audiometry, otoscopic inspections, immittance and speech audiometry, and questionnaires. There was no obvious relationship between subject location and symptoms or test results. Tinnitus, distorted hearing, loudness sensitivity, and otalgia were common symptoms. On average, 76 percent of subjects had predominantly sensorineural hearing loss at one or more frequencies. Twenty-four percent of subjects required amplification. Extended high frequencies showed evidence of acoustic trauma even when conventional frequencies fell within the normal range. Males had significantly poorer responses than females across frequencies. Auditory status of the group was significantly compromised and unchanged at the end of 1–year postblast. Abbreviations: ANOVA= analysis of variance, CF = conventional frequencies (0.25–8 kHz), EHF = extended high frequencies (10–20 kHz), LSD = least significant difference, psi = air pressure measured in pounds per square inch, dB pSPL = decibel re: peak sound pressure level, Q = quarterly evaluations, TNT = trinitrotoluene

Our objective was to determine the validity of the SensorMedics 2900 Metabolic Measurement Cart® (SensorMedics Corp., Yorba Linda, CA) for use at pediatric ventilator volumes by designing an in vitro validation study. The study was performed using a previously described nitrogen dilution, carbon dioxide infusion technique in which known amounts of nitrogen (N2) and carbon dioxide (CO2) are infused while a test lung is ventilated. Metabolic measurements were made at tidal volumes (Vt) varying from 50–500 mL at a peak end-expiratory pressure (PEEP) of 0 and 12 cm H2O and at inspiratory oxygen fractions (Fs) ranging from 0.21–0.60. Each study was repeated six times to assess reproducibility of measurements. At a PEEP of 0 cm H2O, the measurement of oxygen consumption (V′), carbon dioxide production (V′), and minute ventilation (V′E) were all within ±10% of predicted values at all Fs except when Vtt was 50 mL. When PEEP was increased to 12 cm H2O, error in measurement of V′ increased and became unacceptably large as F was increased. Each study consisted of six trials, and results showed acceptable reproducibility as demonstrated by narrow 95% confidence intervals. For Vts of 100–500 mL, the metabolic monitor provided accurate and reproducible results under in vitro testing conditions when PEEP was low. Accurate results could not be obtained with a Vt of 50 mL or at elevated PEEP. Pediatr Pulmonol. 1998; 26:405–411. © 1998 Wiley-Liss, Inc.

This study followed 44 subjects who had been diagnosed as extremely dangerous during placement in an institution for adjudicated juvenile delinquents. Criteria included post-release criminal behavior, sentences imposed for such behavior, and type of crime. Follow-up for periods ranging between two years post-release and more than ten years post-release found that the vast majority had been re-involved in crime; of those who had recidivated, only one had not been involved in a violent offense. Results suggest that for some proportion of subjects diagnosed as extremely dangerous, the likelihood of re-offending criminally, particularly violently, is very high.

We have determined the high resolution crystal structure of the I domain from the α-subunit of the integrin α2β1, a cell surface adhesion receptor for collagen and the human pathogen echovirus-1. The domain, as expected, adopts the dinucleotide-binding fold, and contains a metal ion-dependent adhesion site motif with bound Mg2+ at the top of the β-sheet. Comparison with the crystal structures of the leukocyte integrin I domains reveals a new helix (the C-helix) protruding from the metal ion-dependent adhesion site face of the domain which creates a groove centered on the magnesium ion. Modeling of a collagen triple helix into the groove suggests that a glutamic acid side chain from collagen can coordinate the metal ion, and that the C-helix insert is a major determinant of binding specificity. The binding site for echovirus-1 maps to a distinct surface of the α2-I domain (one edge of the β-sheet), consistent with data showing that virus and collagen binding occur by different mechanisms. Comparison with the homologous von Willebrand factor A3 domain, which also binds collagen, suggests that the two domains bind collagen in different ways.

The human integrin very late antigen (VLA)-2 (CD49b/CD29) mediates interactions with collagen and is the receptor for echovirus 1. Binding sites for both collagen and echovirus 1 have been mapped to the I domain within the alpha2 subunit of the VLA-2 alpha2beta1 heterodimer. Although murine VLA-2 interacts with collagen, it does not bind virus. We have used isolated human-murine chimeric I domains expressed as glutathione S-transferase fusion proteins in Escherichia coli to identify two groups of amino acids, 199-201 and 212-216, independently involved in virus attachment. These residues are distinct from the metal ion-dependent adhesion site previously demonstrated to be essential for VLA-2 interactions with collagen. Mutations in three metal ion-dependent adhesion site residues that abolish adhesion to collagen had no effect on virus binding. These results confirm that different sites within the I domain are responsible for VLA-2 interaction with extracellular matrix proteins and with viral ligands.

This study analyzed the accuracy of predictions of criminality as a measure of future violent behavior by two psychologists at a juvenile correctional facility. The purpose of the study was to look at how effectively predictions of future dangerousness could be made under certain conditions. While other studies have looked at how accurately predictions of violence could be made for large populations, this study looked at whether a subgroup within a general population could accurately be labeled as very likely to reoffend violently. A comparison between the accuracy of the psychologists∗ prediction of future dangerousness and a sample of the institution population was made. The results indicated that the psychologists accurately identified a subpopulation significantly more likely to engage in future acts of violence within a one-year follow-up period and were able to select a group significantly more likely to reofTend than the general population.

Microsatellites of the dystrophin gene have been used extensively in the genetic analysis of Duchenne and Becker muscular dystrophy families. The microsatellites that have been reported to date are clustered within disparate regions of the dystrophin gene, specifically at the 5'-end and in the central rod-domain. YACs encompassing the gene were screened for further microsatellites to improve the density of available genetic markers. Four microsatellites were localized to defined regions of the dystrophin gene by the analysis of patient DNA samples, somatic cell hybrids and YACs. In addition, varying combinations of microsatellite loci were amplified in multiplex PCRs, which complement those loci that have been studied to date.

The F-BAR domain containing protein CIP4 (Cdc42 interacting protein 4) interacts with Cdc42 and WASP/N-WASP and is thought to participate in the assembly of filamentous actin. CIP4 −/− mice had normal T- and B-lymphocyte development but impaired T cell-dependent antibody production, IgG antibody affinity maturation, and germinal center (GC) formation, despite an intact CD40L–CD40 axis. CIP4 −/− mice also had impaired contact hypersensitivity (CHS) to haptens, and their T cells failed to adoptively transfer CHS. Ovalbumin-activated CD4 + effector T cells from CIP4 −/− /OT-II mice migrated poorly to antigen-challenged skin. Activated CIP4 −/− T cells exhibited impaired adhesion and polarization on immobilized VCAM-1 and ICAM-1 and defective arrest and transmigration across murine endothelial cell monolayers under shear flow conditions. These results demonstrate an important role for CIP4 in integrin-dependent T cell-dependent antibody responses and GC formation and in integrin-mediated recruitment of effector T cells to cutaneous sites of antigen-driven immune reactions.

The recruitment of memory T cells from blood into tissues is a central element of immune surveillance and adaptive immune responses and a key feature of chronic cutaneous inflammatory diseases such as psoriasis and atopic dermatitis. Human memory T cells that infiltrate skin express the carbohydrate epitope cutaneous lymphocyte-associated antigen (CLA). Expression of the CLA epitope on T cells has been described on P-selectin glycoprotein ligand-1 (PSGL-1) and associated with the acquisition of both E-selectin and P-selectin ligand functions. In this report, we show that CD43, a sialomucin expressed constitutively on T cells, can also be decorated with the CLA epitope and serve as an E-selectin ligand. CLA expressed on CD43 was found exclusively on the high-molecular-weight (125 kDa) glycoform bearing core-2-branched O-linked glycans. CLA+ CD43 purified from human T cells supported tethering and rolling in shear flow via E-selectin but did not support binding of P-selectin. The identification and characterization of CD43 as a T-cell E-selectin ligand distinct from PSGL-1 expands the role of CD43 in the regulation of T-cell trafficking and provides new targets for the modulation of immune functions in skin.

Background/Purpose: Traditionally, immunologic memory was thought to be maintained by populations of central (TCM) and effector (TEM) memory lymphocytes that circulate in the blood and lymphatics, only temporarily extravagating into peripheral tissue to execute immunologic responses. Recently, this theory of adaptive memory has been challenged by the discovery of long-lived and stable populations of tissue-resident memory T cells (TRM). While TRM have been implicated in the pathogenesis of skin, intestinal, and lung inflammatory diseases, little information is available about TRM in synovium. The study of TRM in arthritis has been impaired by the scarcity of synovium samples coupled with the meager yield of lymphocytes from this tissue using conventional tissue digestion protocols. We have employed novel culturing techniques, previously used to examine TRM in skin, to further characterize TRM in inflammatory arthritis. Methods: Discard synovium samples were collected from patients with rheumatoid arthritis (RA) who were undergoing joint replacement surgery. A 3-dimensional explant method was used to isolated synovial T cells. Synovial tissue was cut into pieces with scissors, loaded on titanium matrices, and cultured in 24 well plates with T cell media enriched with IL-2, 15. After 3 weeks, T cells were harvested. Cell surface markers were studied with flow cytometry. Intracellular cytokine expression was assessed by flow cytometry after T cells were stimulated, fixed, and permeabilized, while Foxp3 expression was evaluated following fixation and permeabilization. Results: Seven RA synovial samples were obtained from 5 women and 2 men. All patients were receiving immunomodulatory medications (prednisone n = 5, methotrexate n = 2, tumor necrosis factor inhibitors n = 1). Synovial samples were small, ranging from 0.5 mm2 to 2 mm2; yet, a substantial number of mononuclear cells were harvested (mean: 7.5 × 106). The isolated lymphocytes were phenotypically similar to prior published reports of synovial T cell populations. The majority of CD3+ lymphocytes were CD4+ (Mean ± SEM: 73.5 ± 7.1%) compared to CD8+ cells (25.5 ± 7.3%). In the CD4+ subset, interferon gamma expression was common (39.5 ± 9.0%), IL-17+ expression was rare (range: 0.1 to 3.4%), and T regulatory cells (CD4+CD25+Foxp3+) were present (8.9 ± 3.2%). A memory phenotype characterized by CD45RO+ expression predominated in both CD4+ (94.5 ± 2.4%) and CD8+ (78.0 ± 6.8%) lymphocytes. A TRM population that lacked the TCM cell surface markers CCR7+ and CD62L+ was identified in CD4+ (47.5 + 10.6%) and CD8+ (36.8 ± 8.1%) cells. Conclusion: The 3-dimensional explant culturing method is a novel tool, which can be employed to study lymphocytes that are resident in synovium. We confirmed that the T cells isolated through this technique were phenotypically similar to populations of RA synovial lymphocytes previously assessed by immunohistochemistry and tissue digestion. A population of TRM was identified in all synovial samples, suggesting these T cells may be important in the pathogenesis of inflammatory arthritis, though further study of this population is needed.