Structure-activity relationship and bioactivity studies of neurotrophic trans-banglene.

The synthesis and bioactivity of neurotrophic banglenes and derivatives is described, establishing a structure-activity relationship which enables future mechanistic studies. Neuritogenesis assays indicate that (-) trans-banglene is the active enantiomer. Assays performed with and without NGF protein suggest that neurotrophic activity and potentiation of NGF activity by (-) trans-banglene might be distinct unassociated processes. Interestingly, (-) trans-banglene potentiation of NGF-induced neuritogenesis is unaffected by the presence of Erk1/2, Akt and Pkc inhibitors.

Lewis or Brønsted? A Rectification of the Acidic and Aromatic Nature of Boranol-Containing Naphthoid Heterocycles.

Boron-containing heterocycles are important in a variety of applications from drug discovery to materials science; therefore a clear understanding of their structure and reactivity is desirable to optimize these functions. Although the boranol (B-OH) unit of boronic acids behaves as a Lewis acid to form a tetravalent trihydroxyborate conjugate base, it has been proposed that pseudoaromatic hemiboronic acids may possess sufficient aromatic character to act as Brønsted acids and form a boron oxy conjugate base, thereby avoiding the disruption of ring aromaticity that would occur with a tetravalent boronate anion. Until now no firm evidence existed to ascertain the structure of the conjugate base and the aromatic character of the boron-containing ring of hemiboronic "naphthoid" isosteres. Here, these questions are addressed with a combination of experimental, spectroscopic, X-ray crystallographic, and computational studies of a series of model benzoxazaborine and benzodiazaborine naphthoids. Although these hemiboronic heterocycles are unambiguously shown to behave as Lewis acids in aqueous solutions, boraza derivatives possess partial aromaticity provided their nitrogen lone electron pair is sufficiently available to participate in extended delocalization. As demonstrated by dynamic exchange and crossover experiments, these heterocycles are stable in neutral aqueous medium, and their measured pKa values are consistent with the ability of the endocyclic heteroatom substituent to stabilize a partial negative charge in the conjugate base. Altogether, this study corrects previous inaccuracies and provides conclusions regarding the properties of these compounds that are important toward the methodical application of hemiboronic and other boron heterocycles in catalysis, bioconjugation, and medicinal chemistry.

Intraspecific and intragenomic variability of ITS rDNA sequences reveals taxonomic problems in Ceratocystis fimbriata sensu stricto.

Fourteen new species in the Latin American Clade (LAC) of the Ceratocystis fimbriata complex recently were distinguished from C. fimbriata sensu stricto largely based on variation in ITS rDNA sequences. Among the 116 isolates representing the LAC, there were 41 ITS haplotypes. Maximum parsimony (MP) analysis of ITS sequences produced poorly resolved trees. In contrast, analyses of mating-type genes (MAT1-1-2 and MAT1-2-1) resolved a single MP tree with branches of high bootstrap and posterior probability support. Four isolates showed intragenomic variation in ITS sequences. Cloning and sequencing of PCR products from a single haploid strain identified two or more ITS sequences differing at up to 16 base positions and representing two described species. Isolates from introduced populations that appeared to be clonal based on microsatellite markers varied at up to 14 bp in ITS sequence. Strains of seven Brazilian ITS haplotypes and an isolate from Ipomoea batatas (on which the species name C. fimbriata was based) were fully interfertile in sexual crosses. These analyses support three phylogenetic species that differ in pathogenicity: C. platani, C. cacaofunesta and C. colombiana. Five ITS species (C. manginecans, C. mangicola, C. mangivora, C. acaciivora, C. eucalypticola) appear to be ITS haplotypes that have been moved from or within Brazil on nursery stock. The taxonomic status of other species delineated primarily by ITS sequences (C. diversiconidia, C. papillata, C. neglecta, C. ecuadoriana, C. fimbriatomima, C. curvata) needs further study, but they are considered doubtful species.

Expanding the Role of Boron in New Drug Chemotypes: Properties, Chemistry, Pharmaceutical Potential of Hemiboronic Naphthoids.

New chemotypes and bioisosteres can open a new chemical space in drug discovery and help meet an urgent demand for novel agents to fight infections and other diseases. With the aim of identifying new boron-containing drug chemotypes, this article details a comprehensive evaluation of the pseudoaromatic hemiboronic naphthoids, benzoxaza- and benzodiazaborines. Relevant physical properties in aqueous media (acidity, solubility, log P, and stability) of prototypic members of four subclasses were determined. Both scaffolds are amenable to common reactions used in drug discovery, such as chemoselective Suzuki-Miyaura, Chan-Lam, and amidation reactions. Small model libraries were prepared to assess the scope of these transformations, and the entire collection was screened for antifungal (Candida albicans) and antibacterial activity (MRSA, Escherichia coli), unveiling promising benzoxazaborines with low micromolar minimum inhibitory concentration values. Select DMPK assays of representative compounds suggest promising drug-like behavior for all four subclasses. Moreover, several drug isosteres were evaluated for anti-inflammatory and anticancer activity as appropriate.

Autologous stem cell transplantation for multiple myeloma patients with chronic kidney disease: a safe and effective option.

Chronic Kidney Disease (CKD) is a frequent complication in patients with multiple myeloma (MM) and is associated with adverse outcomes. The use of autologous stem cell transplantation (ASCT) has improved disease outcomes, however, the safety and efficacy of ASCT in patients with CKD has been the subject of debate. To investigate this, we conducted a retrospective analysis of 370 MM patients who underwent their first ASCT, including those with mild, moderate and severe CKD as well as normal renal function at the time of transplant. No significant difference in ASCT-related mortality, Progression-Free or Overall Survival was noted between the different renal function groups. A decline in estimated glomerular filtration rate (eGFR) at 1-year of >8.79% was associated with poorer overall survival (p < 0.001). The results of this study show that ASCT is a safe and effective option for myeloma patients with CKD, including those on dialysis. Patients who demonstrate renal deterioration at 1-year post-transplant should be closely monitored as this is a predictor for poor survival.

ABVD and BEACOPP regimens' effects on fertility in young males with Hodgkin lymphoma.

PURPOSE: Considering the increased cancer patient survivorship, the focus is now on addressing the impacts of treatment on quality of life. In young people, altered reproductive function is a major issue and its effects in young males are largely neglected by novel research. To improve clinician awareness, we systematically reviewed side effects of chemotherapy for Hodgkin lymphoma (HL) in young males. METHODS: The review was prospectively registered (PROSPERO N. CRD42019122868). Three databases (Medline via PUBMED, SCOPUS, and Cochrane Library) were searched for studies featuring males aged 13-51-years who underwent chemotherapy for HL using ABVD (Adriamycin® (doxorubicin), bleomycin, vinblastine, and dacarbazine) or BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisolone) regimens. These chemotherapy regimens were compared against each other using sperm characteristics, FSH, and inhibin B levels to measure fertility levels. RESULTS: Data were extracted from five studies featuring 1344 patients. 6 months post-ABVD saw marked deterioration in sperm count, further reduced by more cycles (P = 0.05). Patients treated with BEACOPP rather than ABVD were more prone to oligospermia. Receiving fewer cycles of both regimens increased the likelihood of sperm production recovering. Patients treated with 6-8 cycles of BEACOPP did not recover spermiogenesis. CONCLUSIONS: ABVD and BEACOPP regimens significantly reduce fertility function to varying effects depending on treatment duration. ABVD temporarily causes significant reductions in male fertility, whereas BEACOPP's effects are more permanent. Therefore, clinicians should discuss fertility preservation with male patients receiving infertility-inducing gonadotoxic therapy. Further high-quality studies are required to more adequality describe the risk to fertility by chemotherapy.

Thalidomide induced remission of refractory diffuse large B-Cell Lymphoma post-allogeneic SCT.

Patients who relapse after High dose therapy and autologous stem cell transplant (ASCT) for Diffuse large B cell Lymphoma (DLBCL) have a poor prognosis with a median survival of only 3-6 month.1-2 This case demonstrates the ability of thalidomide at low doses to induce durable response in a patient with DLBCL who relapsed after full intensity allogeneic transplantation.

ABVD and BEACOPP regimens’ effects on fertility in young males with Hodgkin lymphoma

Purpose Considering the increased cancer patient survivorship, the focus is now on addressing the impacts of treatment on quality of life. In young people, altered reproductive function is a major issue and its effects in young males are largely neglected by novel research. To improve clinician awareness, we systematically reviewed side effects of chemotherapy for Hodgkin lymphoma (HL) in young males. Methods The review was prospectively registered (PROSPERO N. CRD42019122868). Three databases (Medline via PUBMED, SCOPUS, and Cochrane Library) were searched for studies featuring males aged 13-51-years who underwent chemotherapy for HL using ABVD (Adriamycin® (doxorubicin), bleomycin, vinblastine, and dacarbazine) or BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisolone) regimens. These chemotherapy regimens were compared against each other using sperm characteristics, FSH, and inhibin B levels to measure fertility levels. Results Data were extracted from five studies featuring 1344 patients. 6 months post-ABVD saw marked deterioration in sperm count, further reduced by more cycles (P = 0.05). Patients treated with BEACOPP rather than ABVD were more prone to oligospermia. Receiving fewer cycles of both regimens increased the likelihood of sperm production recovering. Patients treated with 6-8 cycles of BEACOPP did not recover spermiogenesis. Conclusions ABVD and BEACOPP regimens significantly reduce fertility function to varying effects depending on treatment duration. ABVD temporarily causes significant reductions in male fertility, whereas BEACOPP’s effects are more permanent. Therefore, clinicians should discuss fertility preservation with male patients receiving infertility-inducing gonadotoxic therapy. Further high-quality studies are required to more adequality describe the risk to fertility by chemotherapy (AU)

SCT for severe autoimmune diseases: consensus guidelines of the European Society for Blood and Marrow Transplantation for immune monitoring and biobanking.

Over the past 15 years, SCT has emerged as a promising treatment option for patients with severe autoimmune diseases (ADs). Mechanistic studies recently provided the proof-of-concept that restoration of immunological tolerance can be achieved by haematopoietic SCT in chronic autoimmunity through eradication of the pathologic, immunologic memory and profound reconfiguration of the immune system, that is, immune 'resetting'. Nevertheless, a number of areas remain unresolved and warrant further investigation to refine our understanding of the underlying mechanisms of action and to optimize clinical SCT protocols. Due to the low number of patients transplanted in each centre, it is essential to adequately collect and analyse biological samples in a larger cohort of patients under standardized conditions. The European society for blood and marrow transplantation Autoimmune Diseases and Immunobiology Working Parties have, therefore, undertaken a joint initiative to develop and implement guidelines for 'good laboratory practice' in relation to procurement, processing, storage and analysis of biological specimens for immune reconstitution studies in AD patients before, during and after SCT. The aim of this document is to provide practical recommendations for biobanking of samples and laboratory immune monitoring in patients with ADs undergoing SCT, both for routine supportive care purposes and investigational studies.

Inverted repeats are necessary for circularization of the mouse testis Sry transcript.

Circular non-polyadenylated RNA molecules have been identified as stable transcription products of the human ETS-1 and mouse Sry genes. RNA circularization has been proposed to require two steps. The first step utilizes intramolecular base pairing to produce a transient stem-loop structure. The second step involves splicing a downstream donor splice site (DSS) to a now closely appositioned upstream acceptor splice site (ASS) within the loop. We demonstrate that the presence of long inverted repeats (IR) flanking the mouse Sry gene leads to the formation of the Sry circular transcript in cultured cells. Circularization requires the presence of both IR. As few as 400 complementary nt are necessary for this process. The presence of the IR does not significantly stimulate intermolecular annealing and trans-splicing in vivo.

High-level inducible expression of visual pigments in transfected cells.

A method for high-level expression of a functionally active, recombinant human red cone opsin was developed by adding the coding sequence for the C-terminal epitope of bovine rhodopsin onto the C terminus of the cone opsin and cloning the resulting construct into the vector pMEP4 beta. The recombinant pMEP4 beta vector was transfected stably into 293-EBNA cells, and expression of the cone opsin was induced by the addition of CdCl2 into the medium. The recombinant cone opsin was reconstituted with 11-cis retinal and purified by immunoaffinity chromatography. Spectral analysis prior to and following photobleaching confirmed its identity as a red cone opsin. The protein was targeted to the cell membrane and activated bovine transducin.

Mutation of a conserved cysteine in the X-linked cone opsins causes color vision deficiencies by disrupting protein folding and stability.

PURPOSE: To test the effects of disruption of a conserved cysteine in the green cone opsin molecule on light-activated isomerization, transducin activation, folding, transport, and protein half-life. METHODS: Stable cell lines were established by transfecting 293-EBNA cells with a plasmid containing wild-type or mutant (C203R, C203S, C126S, C126S/C203S) green opsin cDNA molecules. The proteins were induced by culturing the cells in the presence of cadmium chloride and analyzed by spectra, transducin activation, Western blotting, pulse-labeling with immunoprecipitation, and immunocytochemistry. RESULTS: The C203R mutation disrupts the folding and half-life of the green opsin molecule and its abilities to absorb light at the appropriate wavelength and to activate transducin. Similar disruption of folding, half-life, and light activation occurs when Cys203 or its presumed partner for formation of a disulfide bond (Cys126) is replaced by serine residues. CONCLUSIONS: Like rhodopsin, the folding of the cone opsins appears to be dependent on the formation of a disulfide bond between the third transmembrane helix and the second extracellular loop. Disruption of this disulfide bond represents a cause of color vision deficiencies that is unrelated to spectral shifts of the photopigment.

Mutation of a conserved proline disrupts the retinal-binding pocket of the X-linked cone opsins.

PURPOSE: To test the effects of disruption of a conserved proline in the green cone opsin molecule on light-activated isomerization, transducin activation, protein accumulation, glycosylation, and transport. METHODS: Stable cell lines were established by transfecting EBNA-293 cells with a plasmid containing wild-type or mutant (P307L) green opsin cDNA molecules. The proteins were induced by culturing the cells in the presence of CdCl2 and analyzed by spectra, transducin activation, Western blotting, and immunocytochemistry. RESULTS: The P307L mutation diminished ability of the visual pigment to absorb light at the appropriate wavelength and to activate transducin. Protein glycosylation and transport to the cell membrane were unaffected. Although there was some diminution in the accumulation of the opsin, this was insufficient to account for the observed effect. CONCLUSIONS: Like rhodopsin, the formation of the cone opsins visual pigments is dependent on the binding of retinal into a hydrophobic pocket that is formed by the second and fourth transmembranous loops. Disruption of a conserved proline near the retinal binding site represents a cause of color vision deficiency that is unrelated to spectral shifts of the photopigment.

Glycosylation and palmitoylation are not required for the formation of the X-linked cone opsin visual pigments.

PURPOSE: This study was designed to test whether palmitoylation and glycosylation are required for the formation of the green opsin visual pigment. METHODS: Stable cell lines were established by transfecting EBNA-293 cells with a pMEP4ss recombinant plasmid containing wild-type bovine rhodopsin or wild-type or mutant (N32S) green opsin cDNA molecules that included a tag for the eight amino acid residues located at the C-terminus of rhodopsin. The opsins were induced by addition of CdCl2 into the medium and then reconstituted with 11-cis-retinal. The reconstituted opsins were purified by immunoaffinity chromatography, then analyzed by difference spectra, and by binding 35S-GTP in the presence of bovine transducin. Non-reconstituted opsins were analyzed by Western blotting and by pulse-labeling with 3H-palmitic acid followed by immunoprecipitation. RESULTS: Elimination of glycosylation by mutagenesis of the N-linked glycosylation site did not impair the ability of the resulting cone opsin to absorb light at the appropriate wavelength nor to activate transducin. Furthermore, as judged by pulse-labeling with 3H-palmitic acid and immunoprecipitation and by gas chromatography-mass spectroscopy, the wild type green opsin differs from rhodopsin by not being palmitoylated. CONCLUSIONS: Glycosylation and palmitoylation are not required for the formation of cone opsin visual pigments. For the previously described green opsin C203R mutation, disruption of folding and transport, rather than altered glycosylation is sufficient to explain the associated color vision deficiency.

Second autologous transplant with cyclosporin/interferon alpha-induced graft versus host disease for patients who have failed first-line consolidation.

The prognosis for patients with non-Hodgkin's lymphoma (NHL) and advanced Hodgkin's disease (HD) who relapse following autologous transplant is poor. We report on a pilot study designed to evaluate the feasibility of using Cyclosporin A and interferon alpha to induce autologous GVHD following a second autologous transplant for relapsed lymphoma. In all, 10 patients entered the study with median age 46.5 years. Diagnosis was NHL (n=7) or Hodgkin's lymphoma (n=3). All had relapsed from a prior autologous transplant. The second transplant was well tolerated by all patients. Histological changes consistent with cutaneous GVHD developed in 30% of patients at a median of 22.5 days from transplant and settled spontaneously in all cases. Five patients have died (four from progressive disease) at a median 7 months from second transplant. Five patients are still alive and in complete remission at a median of 20 months from transplant. Median overall survival for the group is 13.5 months and median relapse-free survival has not been reached at 42 months. This is a well-tolerated regimen for use in this poor-risk group of patients with lymphoma. The overall survival and event-free survival are encouraging, however further studies are necessary.

Acquired haemophilia A: errors in the diagnosis.

The distinction between a specific factor inactivator and a non-specific inhibitor is important when confronted by a patient with a history of bleeding and abnormal in-vitro coagulation tests. We report on two patients who presented with bleeding and a prolonged activated partial thromboplastin time. Initial factor assays suggested combined deficiency of factors VIII and IX as a result of the presence of inactivators. The use of dilution studies, chromogenic assays, a novel in-house enzyme-linked-immunosorbent-assay-based technique and phospholipid neutralization, demonstrated that Case 1 had a genuine factor VIII inactivator resulting in factor VIII levels of less than 1 IU/dl but no factor IX deficiency. Case 2 had normal levels of factor VIII on further testing and no specific inactivator to either factor VIII or IX but a potent antiphospholipid antibody which had interfered with the phospholipid-dependent in-vitro assays. Care must be taken in the interpretation of laboratory assays in the presence of antiphospholipid antibodies to ensure that the correct diagnosis is made and inappropriate treatment avoided.

Autologous stem cell transplantation in severe treatment-resistant Crohn's disease: long-term follow-up of UK patients treated on compassionate basis.

BACKGROUND: Although autologous stem cell transplantation (ASCT) may achieve disease control in severe treatment-resistant Crohn's disease (CD), relapse is frequent, and there is little information regarding long-term outcomes in terms of response to subsequent treatments and complications of ASCT. DESIGN: Retrospective evaluation of UK patients treated on a compassionate basis from three UK tertiary centres. METHODS: We summarize long-term outcomes of six previously unreported patients with severe treatment-resistant CD treated with ASCT according to international guidelines between 2003 and 2009. Median duration of CD before ASCT was 14 (7-22) years. Following stem cell mobilization, patients were treated with high-dose cyclophosphamide (200 mg/kg) and rabbit anti-thymocyte globulin (7.5 mg/kg) followed by ASCT. RESULTS: All patients tolerated ASCT with routine toxicities and no treatment-related mortality and are alive at 50-123 months post-ASCT. Clinical and endoscopic remissions of CD were confirmed at 3 months post-ASCT in five patients, although median time to next treatment for inflammatory disease was 10 months (range: 3-16 months). Subsequently, disease control was achieved with previously ineffective and newer treatments, with surgery performed predominantly for pre-existing fibrotic strictures. Two patients became independent of home total parenteral nutrition (TPN). Reported late complications of ASCT included hypothyroidism and ovarian failure. CONCLUSION: Long-term follow-up supports the safety and feasibility of ASCT as a means of achieving short-term control of severe CD whilst potentially re-sensitizing the disease to medical therapy and reducing requirements for surgery and TPN. Given the inevitability of relapse, pre-emptive salvage and/or maintenance treatments post-ASCT should be the focus of future trials.