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Phagocytic cells are critical to host defense against Pseudomonas aeruginosa, a Gram-negative bacterium that is an opportunistic pathogen. Accordingly, susceptible individuals frequently have impaired innate immune responses, including those with cystic fibrosis or neutropenia. Previous studies identified that the downregulation, or loss, of bacterial flagellar motility enables bacteria to evade interactions with phagocytic cells that result in phagocytic uptake of the bacteria. However, the mechanistic bases for motility-dependent interactions between P. aeruginosa and host cell surfaces that lead to phagocytic uptake of the bacteria are poorly understood. A recent insight is that exogenous addition of a negatively charged phospholipid, phosphatidylinositol-(3,4,5)-triphosphate (PIP(3)), promotes the engagement of non-motile strains of P. aeruginosa with phagocytes leading to uptake of the bacteria. Thus, we hypothesized that the engagement of P. aeruginosa by phagocytic cells is mediated by motility-dependent interactions with cell-surface polyanions. Here we report that endogenous polyanionic N-linked glycans and heparan sulfate mediate bacterial binding of P. aeruginosa by human monocytic cells. These specific interactions resulted in P. aeruginosa phagocytosis, bacterial type 3 secretion system (T3SS)-mediated cellular intoxication and the IL-1β response of host innate immune cells. Importantly, the bacterial interactions with the glycans were motility-dependent and could be recapitulated with purified, immobilized glycans. Therefore, this work describes novel interactions of P. aeruginosa with specific phagocyte cell-surface glycans that modulate relevant host innate immune responses to the bacteria, including phagocytosis, inflammation and cytotoxicity.

Highlights • CIS-platin chemotherapy leads to an increase in the percentage of monocytes within the ascites in a murine model of epithelial ovarian cancer. • Ascites derived CD11b+ cells elicit an increased response from T-cells after treatment with CIS-platin. • CIS-platin chemotherapy increases tumor-antigen -specific IFN-ɣ responses from T-cells., Epithelial ovarian cancer (EOC) is an immunologically active malignancy, but thus far immune therapy has had limited success in clinical trials. One barrier to implementation of efficacious immune therapies is a lack of knowledge of the effect of chemotherapy on the monocyte-derived component of the immune infiltrate within the tumor. We utilized the ID8 murine EOC model to investigate alterations within tumor ascites that occur following administration of platinum chemotherapy. Cisplatin treatment resulted in a significant increase in monocytes within the ascites of tumor bearing mice. We identified that CD11b+ cells from the ascites of mice that have been treated with cisplatin elicits an increase in IFN-ɣ expression from CD8+T-cells compared to CD11b+ cells from a mouse treated with vehicle control (604.0 pg/mL v. 4328.0 pg/mL; p .05). These results indicate that treatment with cisplatin leads to an increase of anti-tumor activity within the ascites related to alterations in the ascites monocytes. Further investigation of these findings in humans is necessary to identify how these cells behave in different patient subgroups and if there is a role for monocyte directed therapy in conjunction with T-cell directed therapy and/or chemotherapy.

Estradiol (E2) and progesterone (P) have potent effects on immune function in the human uterine endometrium which is essential for creating an environment conducive for successful reproduction. Type III/lambda (λ) interferons (IFN) are implicated in immune defense of the placenta against viral pathogens, which occurs against the backdrop of high E2 and P levels. However, the effect of E2 and P in modulating the expression and function of IFNλ1 in the non-pregnant human uterine endometrium is unknown. We generated purified in vitro cultures of human uterine epithelial cells and stromal fibroblast cells recovered from hysterectomy specimens. Poly (I:C), a viral dsRNA mimic, potently increased secretion of IFNλ1 by both epithelial cells and fibroblasts. The secretion of IFNλ1 by epithelial cells significantly increased with increasing age following poly (I:C) stimulation. Stimulation of either cell type with E2 (5x10-8M) or P (1x10-7M) had no effect on expression or secretion of IFNλ1 either alone or in the presence of poly (I:C). E2 suppressed the IFNλ1-induced upregulation of the antiviral IFN-stimulated genes (ISGs) MxA, OAS2 and ISG15 in epithelial cells, but not fibroblasts. Estrogen receptor alpha (ERα) blockade using Raloxifene indicated that E2 mediated its inhibitory effects on ISG expression via ERα. In contrast to E2, P potentiated the upregulation of ISG15 in response to IFNλ1 but had no effect on MxA and OAS2 in epithelial cells. Our results demonstrate that the effects of E2 and P on IFNλ1-induced ISGs are cell-type specific. E2-mediated suppression, and selective P-mediated stimulation, of IFNλ1-induced ISG expression in uterine epithelial cells suggest that the effects of IFNλ1 varies with menstrual cycle stage, pregnancy, and menopausal status. The suppressive effect of E2 could be a potential mechanism by which ascending pathogens from the lower reproductive tract can infect the pregnant and non-pregnant endometrium.

BACKGROUND: Acetaminophen (APAP) is a widely self-prescribed analgesic for mild to moderate pain, but overdose or repeat doses can lead to liver injury and death. Kalyra Pharmaceuticals has developed a novel APAP analog, KP-1199, currently in Phase 1 clinical studies, which lacks hepatotoxicity. In this study, the authors evaluated the antinociceptive effect of KP-1199 on thermal injury-induced nociceptive behaviors as well as hemostatic parameters using human blood samples. METHODS: Full-thickness thermal injury was induced in anesthetized adult male Sprague-Dawley rats. On day 7 post-injury, KP-1199 (30 and 60 mg/kg) or APAP (60 mg/kg) was administered orally. Antinociception of KP-1199 and APAP were assessed at multiple time points using Hargreaves' test. In separate experiments, human whole blood was collected and treated with either KP-1199, APAP, or Vehicle (citrate buffer) at 1× (214 µg/ml) and 10× (2140 µg/ml) concentrations. The treated blood samples were assessed for: clotting function, thrombin generation, and platelet activation. RESULTS: APAP did not produce antinociceptive activity. KP-1199 treatment significantly increased the nociceptive threshold, and the antinociceptive activity persisted up to 3 h post-treatment. In human samples, 10× APAP caused significantly prolonged clotting times and increased platelet activation, whereas KP-1199 had caused no negative effects on either parameter tested. CONCLUSION: These results suggest that KP-1199 possesses antinociceptive activity in a rat model of thermal injury. Since KP-1199 does not induce platelet activation or inhibit coagulation, it presents an attractive alternative to APAP for analgesia, especially for battlefield or surgical scenarios where blood loss and blood clotting are of concern.

Harnessing social media data for social science research entails creating measures out of the largely unstructured, noisy data that users generate on different platforms. This harnessing, particularly of data at scale, requires using methods developed in computer science. But it also typically requires integrating these methods with assessments of measurement quality along social science criteria -- reliability, validity and unbiasedness. In this paper, we outline measurement issues that arise when using social media data. We show examples of how to construct measures and discuss different measurement considerations and best practices. We conclude with a discussion of ways to accelerate research in this space, highlighting contributions that can be made by both social scientists and computer scientists.

Phagocytosis is the key mechanism for host control of Pseudomonas aeruginosa, a motile Gram-negative, opportunistic bacterial pathogen which frequently undergoes adaptation and selection for traits that are advantageous for survival. One such clinically relevant adaptation is the loss of bacterial motility, observed within chronic infections, that is associated with increased antibiotic tolerance and phagocytic resistance. Previous studies using phagocytes from a leukocyte adhesion deficiency type 1 (LAD-I) patient identified CD18 as a putative cell surface receptor for uptake of live P. aeruginosa. However, how bacterial motility alters direct engagement with CD18-containing integrins remains unknown. Here we demonstrate, with the use of motile and isogenic nonmotile deletion mutants of two independent strains of P. aeruginosa and with CRISPR-generated CD18-deficient cell lines in human monocytes and murine neutrophils, that CD18 expression facilitates the uptake of both motile and nonmotile P. aeruginosa. However, unexpectedly, mechanistic studies revealed that CD18 expression was dispensable for the initial attachment of the bacteria to the host cells, which was validated with ectopic expression of complement receptor 3 (CR3) by CHO cells. Our data support that surface N-linked glycan chains (N-glycans) likely facilitate the initial interaction of bacteria with monocytes and cooperate with CD18 integrins in trans to promote internalization of bacteria. Moreover, talin-1 and kindlin-3 proteins promote uptake, but not binding, of P. aeruginosa by murine neutrophils, which supports a role for CD18 integrin signaling in this process. These findings provide novel insights into the cellular determinants for phagocytic recognition and uptake of P. aeruginosa.

Pseudomonas aeruginosa is a bacterial pathogen that causes severe chronic infections in immunocompromised individuals. This bacterium is highly adaptable to its environments, which frequently select for traits that promote bacterial persistence. A clinically significant temporal adaptation is the formation of surface- or cell-adhered bacterial biofilms that are associated with increased resistance to immune and antibiotic clearance. Extensive research has shown that bacterial flagellar motility promotes formation of such biofilms, whereupon the bacteria subsequently become nonmotile. However, recent evidence shows that antibiotic-tolerant nonattached bacterial aggregates, distinct from surface-adhered biofilms, can form, and these have been reported in the context of lung infections, otitis media, nonhealing wounds, and soft tissue fillers. It is unclear whether the same bacterial traits are required for aggregate formation as for biofilm formation. In this report, using isogenic mutants, we demonstrate that P. aeruginosa aggregates in liquid cultures are spontaneously formed independent of bacterial flagellar motility and independent of an exogenous scaffold. This contrasts with the role of the flagellum to initiate surface-adhered biofilms. Similarly to surface-attached biofilms, these aggregates exhibit increased antibiotic tolerance compared to planktonic cultures. These findings provide key insights into the requirements for aggregate formation that contrast with those for biofilm formation and that may have relevance for the persistence and dissemination of nonmotile bacteria found within chronic clinical infections. IMPORTANCE In this work, we have investigated the role of bacterial motility with regard to antibiotic-tolerant bacterial aggregate formation. Previous work has convincingly demonstrated that P. aeruginosa flagellar motility promotes the formation of surface-adhered biofilms in many systems. In contrast, aggregate formation by P. aeruginosa was observed for nonmotile but not for motile cells in the presence of an exogenous scaffold. Here, we demonstrate that both wild-type P. aeruginosa and mutants that genetically lack motility spontaneously form antibiotic-tolerant aggregates in the absence of an exogenously added scaffold. Additionally, we also demonstrate that wild-type (WT) and nonmotile P. aeruginosa bacteria can coaggregate, shedding light on potential physiological interactions and heterogeneity of aggregates.

A 34-year-old man presented with a history of bilaterally undescended testes since birth. A bilateral orchidectomy was performed. Macroscopic examination of the specimen demonstrated multiple pale nodules within both testes, the largest of which measured 3 mm in diameter. Review the high-quality, interactive digital Aperio slide at http://virtualacp.com/JCPCases/jclinpath-2017-204691/ and consider your diagnosis. 1. Germ cell neoplasia in situ (GCNIS) 2. Leydig cell hyperplasia 3. Seminoma 4. Sertoli cell nodule 5. Sertoli cell tumour The submitted section demonstrates features of cryptorchidism, with marked hyalinisation of the tubular basement membrane, germ cell atrophy and Leydig cell hyperplasia (figure 1A,B). Most of the seminiferous tubules are lined only by Sertoli cells. However, there are also multiple small nodular aggregates of Sertoli cells that correspond to the macroscopically described abnormality (figure 1A,C,D). These have been occasionally referred to by the misnomer ‘Pick’s Adenomas’ but have been shown to be polyclonal and best referred to as Sertoli cell nodules. Figure 1 The testis (A) shows germ cell atrophy with basement membrane thickening and Leydig cell hyperplasia in keeping with cryptorchidism (B) as well as Sertoli cell nodules (C,D). (A–D: H&E). On low-power examination some of the atrophic tubules contain peripherally located cells with clear cytoplasm that on closer examination show cytological atypia with prominent nucleoli (figure 2). This represents in situ malignancy that has been referred to as ‘carcinoma in situ’ or ‘intratubular germ cell neoplasia’. However, these cells are germ cells rather …

Pseudomonas aeruginosa is a Gram-negative bacterium that, as an opportunistic pathogen, substantially contributes to high morbidity and mortality rates in susceptible individuals such as those with cystic fibrosis or neutropenia. Previous studies identified that the downregulation or loss of bacterial flagellar motility, typically observed within chronic infections, enables bacteria to evade interactions with phagocytic cells that would result in phagocytic uptake. Our recent work demonstrated that exogenous addition of a negatively charged lipid, PIP3, induces binding and phagocytosis of non-motile strains of P. aeruginosa. Based on this work, we hypothesized that the engagement of P. aeruginosa by host innate cells, and subsequent phagocytosis, is mediated by motility-dependent interactions with cell-surface polyanions. We now report that endogenous polyanionic N-linked glycans and heparan sulfate mediate bacterial binding of P. aeruginosa by human monocytic cells. These specific cell-surface interactions result in P. aeruginosa phagocytosis, bacterial type 3 secretion system (T3SS)-mediated cellular intoxication and the IL-1β inflammatory response of the host innate immune cells. Concomitantly, inhibition of host cell N-glycan synthesis reduces T3SS-mediated cytotoxicity and the IL-1β response induced by the bacteria. Importantly, the bacterial interactions with the glycans were motility-dependent and could be recapitulated with purified, immobilized glycans. Therefore, this work describes novel interactions of P. aeruginosa with specific phagocyte cell-surface glycans that modulate relevant host innate immune responses to the bacteria, including phagocytosis, inflammation and cytotoxicity.

Pathogenic bacteria that establish chronic infections in immunocompromised patients frequently undergo adaptation or selection for traits that are advantageous for their growth and survival. Clinical isolates of Pseudomonas aeruginosa, a Gram-negative, opportunistic bacterial pathogen, exhibit a temporal transition from a motile to a nonmotile phenotype through loss of flagellar motility during the course of chronic infection. This progressive loss of motility is associated with increased resistance to both antibiotic and immune clearance. We have previously shown that loss of bacterial motility enables P. aeruginosa to evade phagocytic clearance both in vitro and in vivo and fails to activate the phosphatidylinositol 3-kinase (PI3K)/Akt-dependent phagocytic pathway. Therefore, we tested the hypothesis that clearance of phagocytosis-resistant bacteria could be induced by exogenously pretreating innate immune cells with the Akt-activating molecule phosphatidylinositol-(3,4,5)-trisphosphate (PIP3). Here, we demonstrate that PIP3 induces the uptake of nonmotile P. aeruginosa by primary human neutrophils >25-fold, and this effect is phenocopied with the use of murine phagocytes. However, surprisingly, mechanistic studies revealed that the induction of phagocytosis by PIP3 occurs because polyphosphoinositides promote bacterial binding by the phagocytes rather than bypassing the requirement for PI3K. Moreover, this induction was selective since the uptake of other nonmotile Gram-negative, but not Gram-positive, bacteria can also be induced by PIP3 Since there is currently no treatment that effectively eradicates chronic P. aeruginosa infections, these findings provide novel insights into a potential methodology by which to induce clearance of nonmotile pathogenic bacteria and into the endogenous determinants of phagocytic recognition of P. aeruginosa.

The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. Previously, we have reported the glycoengineering of this organism to produce human-like N-linked glycans but up to now no one has addressed engineering the O-linked glycosylation pathway. Typically, O-linked glycans produced by wild-type P. pastoris are linear chains of four to five α-linked mannose residues, which may be capped with β- or phospho-mannose. Previous genetic engineering of the N-linked glycosylation pathway of P. pastoris has eliminated both of these two latter modifications, resulting in O-linked glycans which are linear α-linked mannose structures. Here, we describe a method for the co-expression of an α-1,2-mannosidase, which reduces these glycans to primarily a single O-linked mannose residue. In doing so, we have reduced the potential of these glycans to interact with carbohydrate-binding proteins, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin. Furthermore, the introduction of the enzyme protein-O-linked-mannose β-1,2-N-acetylglucosaminyltransferase 1, resulted in the capping of the single O-linked mannose residues with N-acetylglucosamine. Subsequently, this glycoform was extended into human-like sialylated glycans, similar in structure to α-dystroglycan-type glycoforms. As such, this represents the first example of sialylated O-linked glycans being produced in yeast and extends the utility of the P. pastoris production platform beyond N-linked glycosylated biotherapeutics to include molecules possessing O-linked glycans.

Pichia pastoris is a methylotropic yeast that has gained great importance as an organism for protein expression in recent years. Here, we report the expression of recombinant human erythropoietin (rhEPO) in glycoengineered P. pastoris. We show that glycosylation fidelity is maintained in fermentation volumes spanning six orders of magnitude and that the protein can be purified to high homogeneity. In order to increase the half-life of rhEPO, the purified protein was coupled to polyethylene glycol (PEG) and then compared to the currently marketed erythropoiesis stimulating agent, Aranesp(®) (darbepoetin). In in vitro cell proliferation assays the PEGylated protein was slightly, and the non-PEGylated protein was significantly more active than comparator. Pharmacodynamics as well as pharmacokinetic activity of PEGylated rhEPO in animals was comparable to that of Aranesp(®). Taken together, our results show that glycoengineered P. pastoris is a suitable production host for rhEPO, yielding an active biologic that is comparable to those produced in current mammalian host systems.

The methylotrophic yeast Pichia pastoris is an attractive expression system due to its ability to secrete large amounts of recombinant protein, with the potential for glycosylation. Advances in glycoengineering of P. pastoris have successfully demonstrated the humanization of both the N- and O-linked glycosylation pathways in this organism. However, in certain cases, the presence of O-linked glycans on a therapeutic protein may not be desirable. Recently, we have reported the in vitro utility of jack bean α-1,2/3/6-mannosidase to remove O-linked mannose from intact undenatured glycoproteins produced in glycoengineered P. pastoris. However, one caveat of this strategy is that jack bean mannosidase has yet to be cloned and as such is only available as crude cellular extracts. This raises several concerns for using this reagent to treat large preparations of therapeutic proteins generated in P. pastoris. Therefore, we postulated that lysosomal mannosidases which have been cloned and demonstrated to have similar activities to jack bean mannosidase on N-linked glycans would also process O-linked glycans in a similar fashion. To this end, we screened a panel of recombinant lysosomal mannosidases from different organisms and identified several which cannot only reduce extended O-linked mannose chains but which can also hydrolyze the Man-α-O-Ser/Thr glycosidic bond on intact glycoproteins. As such, not only do we show for the first time the utility of lysosomal mannosidase for O-linked mannose processing, but since this is a recombinant enzyme, it has several benefits over the use of crude jack bean mannosidase extracts.

Yeast are important production platforms for the generation of recombinant proteins. Nonetheless, their use has been restricted in the production of therapeutic proteins due to differences in their glycosylation profile with that of higher eukaryotes. The yeast strain Pichia pastoris is an industrially important organism. Recent advances in the glycoengineering of this strain offer the potential to produce therapeutic glycoproteins with sialylated human-like N- and O-linked glycans. However, like higher eukaryotes, yeast also express numerous proteases, many of which are either localized to the secretory pathway or pass through it en route to their final destination. As a consequence, nondesirable proteolysis of some recombinant proteins may occur, with the specific cleavage being dependent on the class of protease involved. Dipeptidyl aminopeptidases (DPP) are a class of proteolytic enzymes which remove a two-amino acid peptide from the N-terminus of a protein. In P. pastoris, two such enzymes have been identified, Ste13p and Dap2p. In the current report, we demonstrate that while the knockout of STE13 alone may protect certain proteins from N-terminal clipping, other proteins may require the double knockout of both STE13 and DAP2. As such, this understanding of DPP activity enhances the utility of the P. pastoris expression system, thus facilitating the production of recombinant therapeutic proteins with their intact native sequences.

The cartographic record of Denmark's one-hundred and-eighty-year colonial adventure on the Guinea Coast is completely dominated by more than a dozen manuscript versions and copies of a single map of the Danish African enclave sent in to the central colonial administration from Africa in 1802. This unusually rich and large-scale map was compiled by a natural historian named Peter Thonning, who had no prior experience with making maps and no connection to Danish cartographic institutions. Thonning's career carried him into the colonial administration in Copenhagen, and he deployed his map repeatedly, over the course of decades, in support of his efforts to persuade the government to expand the Danish colonial presence in West Africa. The map can be traced through the Danish archives into the hands of kings, ministers, diplomats, and colonial officers on the ground in Africa. Upon the death of King Frederik VI, Denmark abandoned its African colonial ambitions: Peter Thonning's map was finally laid before the British government, and the Danish colonial possessions in Africa passed under British rule in 1850.

Yeast is a widely used recombinant protein expression system. We expanded its utility by engineering the yeast Pichia pastoris to secrete human glycoproteins with fully complex terminally sialylated N-glycans. After the knockout of four genes to eliminate yeast-specific glycosylation, we introduced 14 heterologous genes, allowing us to replicate the sequential steps of human glycosylation. The reported cell lines produce complex glycoproteins with greater than 90% terminal sialylation. Finally, to demonstrate the utility of these yeast strains, functional recombinant erythropoietin was produced.

The rich archival record of Denmark's nineteenth-century African colonial undertakings, and particularly the work of the natural historian and colonial administrator Peter Thonning of the Guinea Commission, opens fresh perspectives on the broader history and geography of European colonialism.

The rich archival record of Denmark's nineteenth-century African colonial undertakings, and particularly the work of the natural historian and colonial administrator Peter Thonning of the Guinea Commission, opens fresh perspectives on the broader history and geography of European colonialism.

The rich archival record of Denmark's nineteenth-century African colonial undertakings, and particularly the work of the natural historian and colonial administrator Peter Thonning of the Guinea Commission, opens fresh perspectives on the broader history and geography of European colonialism.

The rich archival record of Denmark's nineteenth-century African colonial undertakings, and particularly the work of the natural historian and colonial administrator Peter Thonning of the Guinea Commission, opens fresh perspectives on the broader history and geography of European colonialism.

The rich archival record of Denmark's nineteenth-century African colonial undertakings, and particularly the work of the natural historian and colonial administrator Peter Thonning of the Guinea Commission, opens fresh perspectives on the broader history and geography of European colonialism.

The rich archival record of Denmark's nineteenth-century African colonial undertakings, and particularly the work of the natural historian and colonial administrator Peter Thonning of the Guinea Commission, opens fresh perspectives on the broader history and geography of European colonialism.

The rich archival record of Denmark's nineteenth-century African colonial undertakings, and particularly the work of the natural historian and colonial administrator Peter Thonning of the Guinea Commission, opens fresh perspectives on the broader history and geography of European colonialism.

The rich archival record of Denmark's nineteenth-century African colonial undertakings, and particularly the work of the natural historian and colonial administrator Peter Thonning of the Guinea Commission, opens fresh perspectives on the broader history and geography of European colonialism.

The rich archival record of Denmark's nineteenth-century African colonial undertakings, and particularly the work of the natural historian and colonial administrator Peter Thonning of the Guinea Commission, opens fresh perspectives on the broader history and geography of European colonialism.

The rich archival record of Denmark's nineteenth-century African colonial undertakings, and particularly the work of the natural historian and colonial administrator Peter Thonning of the Guinea Commission, opens fresh perspectives on the broader history and geography of European colonialism.

The rich archival record of Denmark's nineteenth-century African colonial undertakings, and particularly the work of the natural historian and colonial administrator Peter Thonning of the Guinea Commission, opens fresh perspectives on the broader history and geography of European colonialism.

The rich archival record of Denmark's nineteenth-century African colonial undertakings, and particularly the work of the natural historian and colonial administrator Peter Thonning of the Guinea Commission, opens fresh perspectives on the broader history and geography of European colonialism.

The rich archival record of Denmark's nineteenth-century African colonial undertakings, and particularly the work of the natural historian and colonial administrator Peter Thonning of the Guinea Commission, opens fresh perspectives on the broader history and geography of European colonialism.

The rich archival record of Denmark's nineteenth-century African colonial undertakings, and particularly the work of the natural historian and colonial administrator Peter Thonning of the Guinea Commission, opens fresh perspectives on the broader history and geography of European colonialism.

Previous studies have shown that glycoproteins expressed in wild-type Pichia pastoris bind to Dendritic cell-SIGN (DC-Specific Intercellular adhesion molecule-3 Grabbing Nonintegrin), a mannose-binding receptor found on dendritic cells in peripheral tissues which is involved in antigen presentation and the initiation of an immune response. However, the binding of DC-SIGN to glycoproteins purified from P. pastoris strains engineered to express humanized N- and O-linked glycans has not been tested to date. In this study, the binding of glycoproteins with specific high-mannose or human N- and O-linked glycan structures to DC-SIGN was tested. Proteins with humanized N-glycans including Man5 structures and O-glycans (up to as many as 24) with single mannose chain length showed DC-SIGN binding that was comparable to that measured for a CHO-produced IgG1 which lacks O-linked mannose. Glycoproteins with wild-type N-glycans and mannotriose and higher O-glycans bound to DC-SIGN in a manner that was strongly inhibited by either the use of enzymatic N-deglycosylation or sodium meta-periodate oxidation. Mannan purified from humanized P. pastoris also showed lower ability to inhibit DC-SIGN binding to glycoproteins with wild type fungal glycosylation than mannan purified from wild type strains. This study shows that humanized P. pastoris can produce glycoproteins that do not bind to DC-SIGN.

The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, such as those containing β-mannose (Man) linkages, can elicit an immune response or bind to Man receptors, thus reducing their efficacy. Recent studies have confirmed that P. pastoris has four genes from the β-mannosyl transferase (BMT) family and that Bmt2p is responsible for the majority of β-Man linkages on glycans. While expressing recombinant human erythropoietin (rhEPO) in a developmental glycoengineered strain devoid of BMT2 gene expression, cross-reactivity was observed with an antibody raised against host cell antigens. Treatment of the rhEPO with protein N-glycosidase F eliminated cross-reactivity, indicating that the antigen was associated with the glycan. Thorough analysis of the glycan profile of rhEPO demonstrated the presence of low amounts of α-1,2-mannosidase resistant high-Man glycoforms. In an attempt to eliminate the α-mannosidase resistant glycoforms, we used a systemic approach to genetically knock-out the remaining members of the BMT family culminating in a quadruple bmt2,4,1,3 knock-out strain. Data presented here conclude that the additive elimination of Bmt2p, Bmt3p and Bmt1p activities are required for total abolition of β-Man-associated glycans and their related antigenicity. Taken together, the elimination of β-Man containing glycoforms represents an important step forward for the Pichia production platform as a suitable system for the production of therapeutic glycoproteins.

In 1733, the Danish West India and Guinea Company, in which the upper levels of Danish politics and commerce were involved, purchased the Caribbean island of St. Croix. The Company was for a surprising time unable to marshall the cartographic and organizational expertise and resources needed to make satisfactory maps of its important new colonial acquisition. One result was a drastic discrepancy between the actual area of the island and calculated returns from land revenues. The communication between colony and metropolis reached a cartographic impasse when the Danish colonial authorities ordered the superimposition of the emerging cadastral pattern on a quite materially inaccurate 17th-century base map - an impossible undertaking. L'Ile de Sainte-Croix, dans les Antilles, fut achetee en 1733 par la Compagnie danoise de l'Inde occidentale et de la Guinee, dans laquelle etaient impliquees les plus hautes couches de la politique et du commerce danois. La Compagnie fut, pour une periode surprenante, incapabl...

Plantations in the Danish West Indies, now the United States Virgin Islands, were for a century or more registered and taxed in Danish acres, a unit of measure not used in Denmark itself. This “acre” was derived from the round arithmetic subdivision of the uniform rectangular plantation lots into which the island of St Croix was laid out in the 1730s and '40s. The measure was a cultural compromise between Danish administrative practices and the customs of the predominantly English planter class on the island. Its derivation from the locally unprecedented grid survey system of St Croix is directly comparable to Thomas Jefferson's unrealized proposals in the 1790s for the incorporation of a geodetic decimal system of measures into the legislation for the survey of the public lands of the United States. Both of these New World cases exemplify the developing faith—naive, tentative, and demonstrably misplaced, given the technical and administrative limitations of the age—in rational ideals of measurement that at the end of the century produced the revolutionary French metric system.

Perplexing land disputes arise in the U.S. Virgin Islands, formerly the Danish West Indies, not only because of the archival and linguistic inaccessibility of the original Danish land records, but also because of an unusual experiment with the forms of land tenure and administration in the early 18th century. A neat grid survey system established on the island of St. Croix proved surprisingly intractable, and in 1759, a generation or so after the Danes took possession of the island, an island-wide cadastral audit was conducted. The manuscript record of this inventory, itself a historical reconstruction of the island's land system, is an invaluable indicator of contemporary understandings and practice. The proceedings of the audit, with the associated colonial correspondence, throw light on the economic, intellectual, and lasting legal significance of this early exercise in the elaboration and application of rationalist ideals of order in the administration of land in a New World slave-plantation society.

In Saccharomyces cerevisiae, Kin28 is a member of the cyclin-dependent kinase family. Kin28 is a subunit of the basal transcription factor holo-TFIIH and its trimeric sub-complex TFIIK. Kin28 is the primary kinase that phosphorylates the RNA polymerase II (RNA pol II) C-terminal domain (CTD) within a transcription initiation complex. Mediator, a global transcriptional co-activator, dramatically enhances the phosphorylation of the CTD of RNA pol II by holo-TFIIH in vitro. Using purified proteins we have determined that the subunits of TFIIK are sufficient for Mediator to enhance Kin28 CTD kinase activity and that Mediator enhances phosphorylation of a glutathione S-transferase-CTD fusion protein, despite the absence of multiple Mediator and/or TFIIH interactions with polymerase. Mediator does not stimulate the activity of several other CTD kinases, suggesting that the specific enhancement of TFIIH kinase activity results in Kin28 being the primary CTD kinase at initiation. In addition, we have found that Kin28 phosphorylates Mediator subunit Med4 in an assay, including purified holo-TFIIH, and either Mediator or recombinant Med4 alone. Furthermore, Kin28 appears to be, at least in part, responsible for the phosphorylation of Med4 in vivo. We have identified Thr-237 as the site of phosphorylation of Med4 by Kin28 in vitro. The mutation of Thr-237 to Ala has no effect on the growth of a yeast strain under normal conditions but confirms that Thr-237 is also the site of Med4 phosphorylation in vivo.