gebhart/patrepeval huggingface dataset:
Isolation of motile and non‐motile insertional mutants of Campylobacter jejuni: the role of motility in adherence and invasion of eukaryotic cells (1994)

A method of insertional mutagenesis for naturally transformable organisms has been adapted from Haemophilus influenzae and applied to the study of the pathogenesis of Campylobacter jejuni. A series of kanamycin-resistant insertional mutants of C. jejuni 81-176 has been generated and screened for loss of ability to invade INT407 cells. Eight noninvasive mutants were identified which showed 18-200-fold reductions in the level of invasion compared with the parent. Three of these eight show defects in motility, and five are fully motile. The three mutants with motility defects were further characterized to evaluate the method. One mutant, K2-32, which is non-adherent and non-invasive, has an insertion of the kanamycin-resistance cassette into the flaA flagellin gene and has greatly reduced motility and a truncated flagellar filament typical of flaA mutants. The adherent non-invasive mutants K2-37 and K2-55 are phenotypically paralysed, i.e. they have a full-length flagellar filament but are non-motile. All three mutants show an aberration in flagellar structure at the point at which the filament attaches to the cell. Mutants K2-37 and K2-55 represent overlapping deletions affecting the same gene, termed pflA (paralysed flagella). This gene encodes a predicted protein of 788 amino acid residues and a molecular weight of 90,977 with no significant homology to known proteins. Site-specific insertional mutants into this open reading frame result in the same paralysed flagellar phenotype and the same invasion defects as the original mutants. The differences in adherence between the two classes of flagellar mutant suggest that flagellin can serve as a secondary adhesion, although other adhesins mediate a motility-dependent internalization process. Characterization of the mutants at the molecular level and in animal models should further contribute to our understanding of the pathogenicity of these organisms.
C-Nucleoside Analogues of Nicotinamide Mononucleotide (NMN) (1988)

Abstract 5-(β-D-Ribofuranosyl)nicotinamide (lie) and 6-(β-D-ribo-furanosyDpicolinamide (IId) and their corresponding o-isomers (III) were synthesized from ribonolactone. The C-nucleoside lie was further converted to its 5′-monophosphate Up which is isosteric to NMN Ip).
Role of pseudorabies virus glycoprotein gI in virus release from infected cells (1987)

The Bartha vaccine strain of pseudorabies virus has a deletion in the short unique (Us) region of its genome which includes the genes that code for glycoproteins gI and gp63 (E. Petrovskis, J. G. Timmins, T. M. Gierman, and L. E. Post, J. Virol. 60:1166-1169, 1986). Restoration of an intact Us to the Bartha strain enhances its ability to be released from infected rabbit kidney cells and increases the size of the plaques formed on these cells (T. Ben-Porat, J. M. DeMarchi, J. Pendrys, R. A. Veach, and A. S. Kaplan, J. Virol. 57:191-196, 1986). To determine which gene function plays a role in virus release from rabbit kidney cells, deletions were introduced into the genomes of both wild-type virus and the "rescued" Bartha strain (Bartha strain to which an intact Us had been restored) that abolish the expression of either the gI gene alone or both gI and gp63 genes. The effect of these deletions on the phenotype of the viruses was studied. Deletion mutants of wild-type virus defective in either gI or gI and gp63 behave like wild-type virus with respect to virus release and plaque size on rabbit kidney cells. Deletion of gI from the rescued Bartha strain, however, strongly affects virus release and causes a decrease in plaque size. We conclude that gI affects virus release but that at least one other viral function also affects this process. This function is defective in the Bartha strain but not in wild-type virus; in its absence gI is essential to efficient release of the virus from rabbit kidney cells.
Defective Oxidative Phosphorylation in Thyroid Oncocytic Carcinoma Is Associated with Pathogenic Mitochondrial DNA Mutations Affecting Complexes I and III (2006)

Oncocytic tumors are characterized by cells with an aberrant accumulation of mitochondria. To assess mitochondrial function in neoplastic oncocytic cells, we studied the thyroid oncocytic cell line XTC.UC1 and compared it with other thyroid non-oncocytic cell lines. Only XTC.UC1 cells were unable to survive in galactose, a condition forcing cells to rely solely on mitochondria for energy production. The rate of respiration and mitochondrial ATP synthesis driven by complex I substrates was severely reduced in XTC.UC1 cells. Furthermore, the enzymatic activity of complexes I and III was dramatically decreased in these cells compared with controls, in conjunction with a strongly enhanced production of reactive oxygen species. Osteosarcoma-derived transmitochondrial cell hybrids (cybrids) carrying XTC.UC1 mitochondrial DNA (mtDNA) were generated to discriminate whether the energetic failure depended on mitochondrial or nuclear DNA mutations. In galactose medium, XTC.UC1 cybrid clones showed reduced viability and ATP content, similarly to the parental XTC.UC1, clearly pointing to the existence of mtDNA alterations. Sequencing of XTC.UC1 mtDNA identified a frameshift mutation in ND1 and a nonconservative substitution in cytochrome b, two mutations with a clear pathogenic potential. In conclusion, this is the first demonstration that mitochondrial dysfunction of XTC.UC1 is due to a combined complex I/III defect associated with mtDNA mutations, as proven by the transfer of the defective energetic phenotype with the mitochondrial genome into the cybrids.
Retroviral infection coupled with tissue transplantation limits gene transfer in the chicken embryo. (1993)

Gene transfer into early embryos is a powerful methodology for unraveling the molecular bases of developmental processes. One can attempt to minimize widespread effects of an exogenous gene by using tissue- or region-specific promoters in the few instances where they are available. We have developed a method that bypasses the requirement for specific targeting sequences to achieve regionally restricted gene transfer. Intraspecific chimeras have been created by transplantation of restricted portions of a chicken embryo from a donor strain to a host strain. The donor cells are infectable with a recombinant retroviral vector that carries the exogenous gene, whereas the host cells are not. We have demonstrated the feasibility of this approach using a histochemically distinct reporter gene, human placental alkaline phosphatase. The expression of retrovirally transduced alkaline phosphatase was limited to a transplanted hemiprosencephalon (forebrain and eye) in embryonic chickens. This technique can be applied to many other organ systems during avian embryogenesis to test the function(s) of molecules that are normally controlled through spatial and/or temporal regulation, such as many of the growth factor receptors or homeobox-containing proteins.
Host-centric Proteomics of Stool: A Novel Strategy Focused on intestinal Responses to the Gut Microbiota (2013)

The diverse community of microbes that inhabits the human bowel is vitally important to human health. Host-expressed proteins are essential for maintaining this mutualistic relationship and serve as reporters on the status of host-microbiota interaction. Therefore, unbiased and sensitive methods focused on host proteome characterization are needed. Herein we describe a novel method for applying shotgun proteomics to the analysis of feces, focusing on the secreted host proteome. We have conducted the most complete analysis of the extracellular mouse gut proteome to date by employing a gnotobiotic mouse model. Using mice colonized with defined microbial communities of increasing complexity or a complete human microbiota ('humanized'), we show that the complexity of the host stool proteome mirrors the complexity of microbiota composition. We further show that host responses exhibit signatures specific to the different colonization states. We demonstrate feasibility of this approach in human stool samples and provide evidence for a "core" stool proteome as well as personalized host response features. Our method provides a new avenue for noninvasive monitoring of host-microbiota interaction dynamics via host-produced proteins in stool.
Qualitative and quantitative changes of human tenascin expression in transformed lung fibroblast and lung tumor tissues: comparison with fibronectin. (1991)

Qualitative and quantitative alterations of human tenascin (TN) expression in virally transformed lung fibroblasts and in lung tumor tissues were investigated using S1 nuclease protection analysis in comparison with those of fibronectin (FN). Transformed fibroblasts and fetal lung tissues expressed more TN mRNA with an extra sequence encoding the sixth FN type III repeat than did normal cells and adult tissues. The splicing pattern of TN mRNA was also altered in many lung cancer tissues, showing increased or sometimes decreased expression of the TN mRNA with the extra sequence when compared with their surrounding normal tissues. These results provide additional evidence for the oncodevelopmental regulation of alternative RNA splicing in human lung tissues, first observed with FN mRNA (F. Oyama, et al., Cancer Res., 50: 1075-1078, 1990). Quantitative analysis of the levels of TN and FN mRNAs showed that the ratio of TN mRNA to FN mRNA was significantly increased in transformed fibroblasts and in some lung tumor tissues, when compared with their normal counterparts. Among different types of lung tumors, a significant increase of the TN/FN ratio was observed with most squamous cell carcinoma but with only a small fraction of adenocarcinoma. Since TN has been shown to inhibit cell adhesion to FN, the altered ratio of TN mRNA to FN mRNA may well affect the adhesive and migratory properties of tumor cells in lung cancer tissues.
Mobilization of allogeneic peripheral blood stem cell donors with intravenous plerixafor mobilizes a unique graft (2017)

A single subcutaneous (SC) injection of plerixafor results in rapid mobilization of hematopoietic progenitors, but fails to mobilize 33% of normal allogeneic sibling donors in 1 apheresis. We hypothesized that changing the route of administration of plerixafor from SC to IV may overcome the low stem cell yields and allow collection in 1 day. A phase 1 trial followed by a phase 2 efficacy trial was conducted in allogeneic sibling donors. The optimal dose of IV plerixafor was determined to be 0.32 mg/kg. The primary outcome of reducing the failure to collect ≥2 × 10<sup>6</sup> CD34<sup>+</sup>/kg recipient weight in 1 apheresis collection to ≤10% was not reached. The failure rate was 34%. Studies evaluating the stem cell phenotype and gene expression revealed a novel plasmacytoid dendritic cell precursor preferentially mobilized by plerixafor with high interferon-α producing ability. The observed cytomegalovirus (CMV) viremia rate for patients at risk was low (15%), as were the rates of acute grade 2-4 graft-versus-host disease (GVHD) (21%). Day 100 treatment related mortality was low (3%). In conclusion, plerixafor results in rapid stem cell mobilization regardless of route of administration and resulted in novel cellular composition of the graft and favorable recipient outcomes. These trials were registered at clinicaltrials.gov as #NCT00241358 and #NCT00914849.
Annexins in Cell Membrane Dynamics (2000)

The sarcolemma of smooth muscle cells is composed of alternating stiff actin-binding, and flexible caveolar domains. In addition to these stable macrodomains, the plasma membrane contains dynamic glycosphingolipid- and cholesterol-enriched microdomains, which act as sorting posts for specific proteins and are involved in membrane trafficking and signal transduction. We demonstrate that these lipid rafts are neither periodically organized nor exclusively confined to the actin attachment sites or caveolar regions. Changes in the Ca(2+) concentration that are affected during smooth muscle contraction lead to important structural rearrangements within the sarcolemma, which can be attributed to members of the annexin protein family. We show that the associations of annexins II, V, and VI with smooth muscle microsomal membranes exhibit a high degree of Ca(2+) sensitivity, and that the extraction of annexins II and VI by detergent is prevented by elevated Ca(2+) concentrations. Annexin VI participates in the formation of a reversible, membrane-cytoskeleton complex (Babiychuk, E.B., R.J. Palstra, J. Schaller, U. Kämpfer, and A. Draeger. 1999. J. Biol. Chem. 274:35191-35195). Annexin II promotes the Ca(2+)-dependent association of lipid raft microdomains, whereas annexin V interacts with glycerophospholipid microcompartments. These interactions bring about a new configuration of membrane-bound constituents, with potentially important consequences for signaling events and Ca(2+) flux.
Mechanism of Action of N -Butyl Deoxynojirimycin in Inhibiting HIV-1 Infection and Activity in Combination with Nucleoside Analogs (1993)

The effects on HIV-1 infection of a glucosidase inhibitor, N-butyl deoxynojirimycin (N-buDNJ), were examined. The combinations of N-buDNJ and nucleoside analogs dideoxyinosine (DDI), dideoxycytidine (DDC), or azidothymidine (AZT) were examined in an acute infection assay. The combination of N-buDNJ and nucleoside analog reduced the yield of reverse transcriptase activity more than did either agent alone, and the effects on the number of infectious virus particles were additive or synergistic. In studies of the mechanism whereby N-buDNJ alters HIV-1 envelope fusion activity, no effects on CD4 binding were detected. However, cleavage within the V3 loop of gp120 was reduced by N-buDNJ treatment, possibly reflecting an altered conformation of this region of the envelope protein.
Safety and efficacy of plerixafor dose escalation for the mobilization of CD34 + hematopoietic progenitor cells in patients with sickle cell disease: interim results (2018)

Gene therapy for sickle cell disease is limited by the yield of hematopoietic progenitor cells that can be harvested for transduction or gene editing. We therefore performed a phase I dose-escalation study of the hematopoietic progenitor cell mobilizing agent plerixafor to evaluate the efficacy and safety of standard dosing on peripheral blood CD34<sup>+</sup> cell mobilization. Of 15 patients enrolled to date, only one was chronically transfused and ten were on hydroxyurea. Of eight patients who achieved a CD34<sup>+</sup> cell concentration >30 cells/μL, six were on hydroxyurea. There was no clear dose response to increasing plerixafor dosage. There was a low rate of serious adverse events; two patients developed vaso-occlusive crises, at the doses of 80 μg/kg and 240 μg/kg. Hydroxyurea may have contributed to the limited CD34<sup>+</sup> mobilization by affecting baseline peripheral blood CD34 counts, which correlated strongly with peak peripheral blood CD34 counts. Plerixafor administration did not induce significant increases in the fraction of activated neutrophils, monocytes, or platelets. However, increased neutrophils positive for activated β2 integrin and Mac-1 were associated with serious adverse events. In summary, plerixafor was well tolerated but did not achieve consistent CD34<sup>+</sup> cell mobilization in this cohort of patients, most of whom were being actively treated with hydroxyurea and only one was chronically transfused. The study will continue with escalation of the dose of plerixafor and modification of hydroxyurea administration. <i>Clinicaltrials.gov identifier: NCT02193191</i>.
Localization of protein disulfide isomerase to the external surface of the platelet plasma membrane (1995)

Protein disulfide isomerase (PDI) is an enzyme that catalyzes the formation as well as the isomerization of disulfide bonds. In this study, antibodies against PDI were used to show PDI antigen on the platelet surface by indirect immunofluorescence microscopy and by flow cytometry. The platelets were not activated, as evidenced by the absence of staining by an antibody against P-selectin. Permeabilized platelets showed little cytosolic PDI by indirect immunofluorescence microscopy, suggesting that the majority of platelet PDI is localized to the platelet surface. PDI activity against "scrambled" RNase was shown with intact platelets. The activity was inhibited by inhibitors of PDI and by an antibody against PDI. Other blood cells showed little PDI. Platelet surface PDI may play a role in the various physiological and pathophysiologic processes in which platelets are involved.
Reaction of 2‐dimethylaminomethylene‐1,3‐diones with dinucleophiles. VIII. Synthesis of ethyl and methyl 2,4‐disubstituted 5‐pyrimidinecarboxylates (1990)

Abstract Reaction of ethyl or methyl 2‐dimethylaminomethylene‐3‐oxoalkanoates with N‐C‐N dinucleophiles such as guanidine, acetamidine or benzamidine afforded in high yields the relative esters of 4‐substituted 2‐amino‐, 2‐methyl‐ or 2‐phenyl‐5‐pyrimidinecarboxylic acids, respectively. These esters were hydrolyzed to the corresponding carboxylic acids, which were converted by heating to 4‐substituted 2‐pyrimidinamines, 2‐methyl or 2‐phenylpyrimidines, respectively, generally in excellent yields. The 4‐unsubstituted ethyl 2‐amino‐, 2‐methyl‐ and 2‐phenyl‐5‐pyrimidinecarboxylates were obtained in moderate yields by reaction of the above dinucleophiles with ethyl 2,2‐diformylacetate. These esters were hydrolyzed and the corresponding acids (with the exception of the 2‐methyl derivative) were decarboxylated to give 2‐pyrimidinamine and 2‐phenylpyrimidine in satisfactory yields.
Human macrophage colony stimulating factor (M-CSF): alternate RNA splicing generates three different proteins that are expressed on the cell surface and secreted. (1988)

Human macrophage colony stimulating factor (M-CSF) cDNA clones were isolated from a pancreatic carcinoma cell line. Three different classes of M-CSF precursor protein (256, 554 and 438 amino acids in length) were predicted to be encoded by these cDNAs. Two of these, that we designate M-CSF alpha and M-CSF beta have already been described. The third, M-CSF gamma represents a novel class of M-CSF cDNA. All three precursors share a 32 amino acid signal sequence and the first 149 amino acids of the mature protein. At this position, M-CSF beta and gamma have insertions of 298 and 182 amino acids relative to M-CSF alpha. The first 182 amino acids of these insertions are shared between M-CSF beta and gamma. All three precursors share the C-terminal 75 amino acids that encode the transmembrane and cytoplasmic domains. Expression of all three cDNAs in COS-7 monkey kidney cells gave rise to soluble M-CSF activity, associated with proteins of subunit molecular weight 44 Kda (beta and gamma) or 28 Kda (alpha). In addition, M-CSF proteins could be detected on the surface of the transfected cells by indirect immunofluorescence. When the transmembrane and cytoplasmic domains of M-CSF alpha were removed by introducing a stop codon after amino acid 190, no membrane-bound M-CSF could be detected, but the truncated protein was secreted efficiently and was biologically active. This suggests that all three forms of M-CSF can exist as cell surface proteins, anchored by their hydrophobic transmembrane domains, and can be processed to soluble forms by proteolytic digestion. Although all soluble forms of M-CSF were biologically active in murine bone marrow colony and proliferation assays, they showed greatly reduced or no activity in similar assays using human bone marrow.
Clinical Experience with Autologous Tumor Cell Lines for Patient-Specific Vaccine Therapy in Metastatic Melanoma (1998)

Because of their patient specificity and proliferative capacity, tumor cell lines established from autologous metastatic melanoma tumor samples may be an excellent immunogen for patient-specific vaccine therapy. Between October 1990 and July 1996, the Hoag Cancer Center cell biology laboratory received 136 fresh metastatic melanoma samples from 122 different patients. Tumor cell lines were successfully established for 92 of 136 samples (68%), for 87 of 122 patients (71%). Successful cultures were expanded to 10(8) cells (total culture time about 8 weeks), confirmed to be sterile, irradiated, and stored frozen in aliquots of 10(7) cells. Vaccines were prepared from 72 lines, and 62 vaccines were used in 57 different patients. Subcutaneous vaccination took place on weeks 1, 2 and 3, and then monthly for a total of 6 months. A delayed tumor hypersensitivity skin test (DTH) was administered at week zero and week 4. Various adjuvants were co-administered including BCG, alpha- or gamma-interferon, and GM-CSF. Patients were monitored for failure-free survival (FFS) and overall survival (OS) from the date of the first vaccination. Follow-up data is available for 52 patients, 27 who had no evident disease (NED) at the time of vaccination and 25 who had metastatic disease at the time of treatment. There were two partial responses which persisted 11.9 and 39.8+ months among the 25 patients who had detectable metastatic disease whün treatment was initiated (8%, 1 to 26%, 95%-Ci). Twenty patients had negative skin tests at week 0 and week 4; six were positive both times, and 13 converted their DTH from negative to positive, for a conversion rate of 13 of 33 (39%). Patients who received interferon-gamma and/or GM-CSF as an adjuvant had a higher rate of DTH conversion compared to patients who received other adjuvants (13 of 20 v 2 of 13, P = 0.003). For patients who were NED, nine of 19 (47%) converted their DTH test compared to four of 14 (29%) patients with metastatic disease (p = 0.33). For patients whose DTH converted from negative to positive after 3 weeks of vaccination, median FFS and OS were superior compared to patients whose DTH remained negative (19.4 v 4.0 months FFS, p = 0.0052 and 39.6 v 18.3 months OS, p = 0.0602). The autologous cell line approach to active specific immunotherapy is feasible for patients who have resectable foci of metastatic disease. Administration of such patient-specific vaccines improves survival for those patients who are NED at the time of vaccination and convert their DTH skin test, compared to those whose DTH test remains negative.
Early recognition and management of sepsis in adults: the first six hours. (2013)

Sepsis is a complication of severe infection characterized by a systemic inflammatory response. Mortality rates from sepsis range between 25% to 30% for severe sepsis and 40% to 70% for septic shock. The clinical presentation of sepsis is highly variable depending on the etiology. The most common sites of infection are the respiratory, genitourinary, and gastrointestinal systems, as well as the skin and soft tissue. Fever is often the first manifestation of sepsis, with pneumonia being the most common presentation leading to sepsis. Early goal-directed therapy completed within the first six hours of sepsis recognition significantly decreases in-hospital mortality. Initial management includes respiratory stabilization followed by aggressive fluid resuscitation. Vasopressor therapy is indicated when fluid resuscitation fails to restore adequate mean arterial pressure and organ perfusion. Early antibiotic therapy can improve clinical outcomes, and should be given within one hour of suspected sepsis. Blood product therapy may be required in some cases to correct coagulopathy and anemia, and to improve the central venous oxygen saturation. Insulin therapy may be required to maintain serum glucose levels less than 180 mg per dL. Initiation of low-dose corticosteroids may further improve survival in patients with septic shock that does not respond to vasopressor therapy. Timely initiation of evidence-based protocols should improve sepsis outcomes.