Pregnancy-associated growth factor. II. A T-dependent polyclonal activator of human adult peripheral blood lymphocytes (PBL)

This study examined the ability of pregnancy-associated growth factor (PAGF), a substance found in crude human chorionic gonadotropin (hCG), to induce plaque-forming cells (PFC) in cultured human peripheral blood lymphocytes (PBL). PAGF, 0.25 to 1 mg/ml, induced maximal PFC at 6 to 7 days as measured by the staphylococcal protein A-coupled SRBC reverse hemolytic plaque assay with a rabbit anti-human Ig antiserum. PAGF-induced PFC/culture ranged from 1800 to 39,000 with a mean of 11,524 in unfractionated PBL (N = 24), as compared to 540 to 77,840 with a mean of 17,303 for pokeweed (PWM) (N = 22). Comparison of PAGF- and PWM-induced PFC showed that both induced specific IgG, IgA, and IgM PFC. In most individuals, PAGF induced more IgM and PWM more IgG PFC. The kappa: lambda ratio was 1.5 for unstimulated PBL, and approximately 3.5 for PAGF and PWM. To see if PAGF was a T-dependent polyclonal activator of B cells, T and non-T populations were obtained by SRBC rosettes and negatively selected T4 and T8 cells by complement-mediated lysis of SRBC+(T) cells. Only the recombined subsets which included T4 cells and non-T cells supported PAGF- and PWM-induced PFC. These data indicate that PAGF, a substance derived from commercial extracts of pregnancy urine, is a T4-dependent polyclonal activator of normal human B cells.     

Freeze-fracturing in normal vacuum reveals ringlike yeast plasmalemma structures

The fine structure of the regular arrays of subunits seen on both plasmalemma fracture faces in resting and starved Saccharomyces cerevisiae (baker's yeast) has been compared using different freeze-fracture replication methods. Freeze-cleaving was carried out at 173 degrees, 133 degrees, and 108 degrees K under a vacuum of 2 X 10(-7) torr (2.6 X 10(- 7)mbar) or under liquid nitrogen at atmosphereic pressure. Independent of the preparation conditions (fracturing temperature, and whether cleaved under vacuum or liquid nitrogen), resting and starved yeast show a significant difference in the morphology of the subunits forming the regular arrays. The regularly arranged particles of the P face of the plasmalemma of starved yeast have a clear craterlike structure which has previously been reported to be demonstrated only by freeze-etching at very low temperatures in ultrahigh vacuum. A complementary structure is seen on the plasmalemma E face. Prolonged exposures of fracture faces under the protection of liquid nitrogen-cooled shrouds have shown that, because of the consequent drastic reduction of condensable gases in the specimen area, no detectable condensation contamination of exposed fracture faces occurs within 15 min at a specimen temperature of 108 degrees K. This shows that a complicated ultrahigh vacuum technology is not required for high resolution freeze- etching.     

Combined ipilimumab and nivolumab first‐line and after BRAF‐targeted therapy in advanced melanoma

The combination of ipilimumab and nivolumab is a highly active systemic therapy for metastatic melanoma but can cause significant toxicity. We explore the safety and efficacy of this treatment in routine clinical practice, particularly in the setting of serine/threonine‐protein kinase B‐Raf (BRAF)‐targeted therapy. Consecutive patients with unresectable stage IIIC/IV melanoma commenced on ipilimumab and nivolumab across 10 tertiary melanoma institutions in Australia were identified retrospectively. Data collected included demographics, response and survival outcomes. A total of 152 patients were included for analysis, 39% were treatment‐naïve and 22% failed first‐line BRAF/MEK inhibitors. Treatment‐related adverse events occurred in 67% of patients, grade 3–5 in 38%. The overall objective response rate was 41%, 57% in treatment‐naïve and 21% in BRAF/MEK failure patients. Median progression‐free survival was 4.0 months (95% CI, 3.0–6.0) in the whole cohort, 11.0 months (95% CI, 6.0‐NR) in treatment‐naïve and 2.0 months (95% CI, 1.4–4.6) in BRAF/MEK failure patients. The combination of ipilimumab and nivolumab can be used safely and effectively in a real‐world population. While first‐line efficacy appears comparable to trial populations, BRAF‐mutant patients failing prior BRAF/MEK inhibitors show less response.     

Monosaccharide precursors for boosting chondroitin-like capsular polysaccharide production

Chondroitin sulfate is a well-known bioactive molecule, widely used as an anti-osteoarthritis drug, that is nowadays mainly produced by animal tissue sources with unsafe extraction procedures. Recent studies have explored an integrated biotechnological–chemical strategy to obtain a chondroitin sulfate precursor from Escherichia coli K4 capsular polysaccharide, demonstrating the influence of environmental and growth conditions on capsule synthesis. In this research work, the flexibility of the strain biosynthetic machinery was investigated to enhance the K4 capsular polysaccharide production by supplementing the growth medium with the monosaccharides (glucuronic acid, galactosamine and fructose) that constitute the chain. Shake flask experiments were performed by adding the sugars singularly or together, by testing monosaccharide different concentrations and times of addition and by observing the bacterial sugar consumption. A K4 capsular polysaccharide production enhancement, compared to the control, was observed in all cases of supplementation and, in particular, significant 68 and 57 % increases were observed when adding 0.385 mM glucuronic acid plus galactosamine or 0.385 mM fructose, respectively. Increased expression levels of the gene kfoC, coding for a K4 polymerase, evaluated in different growth conditions, confirmed the results at the molecular level. Furthermore, batch fermentations, performed in lab-scale reactors (2 L), allowed to double the K4 capsular polysaccharide production values obtained in shake flask conditions, by means of a strict control of the growth parameters.     

Identification, Genomic Organization, and Analysis of the Group III Capsular Polysaccharide Genes kpsD, kpsM, kpsT, and kpsE from an Extraintestinal Isolate of Escherichia coli (CP9, O4/K54/H5)

Group III capsular polysaccharides (e.g., K54) of extraintestinal isolates of Escherichia coli, similar to group II capsules (e.g., K1), are important virulence traits that confer resistance to selected host defense components in vitro and potentiate systemic infection in vivo. The genomic organization of group II capsule gene clusters has been established as a serotype-specific region 2 flanked by regions 1 and 3, which contain transport genes that are highly homologous between serotypes. In contrast, the organization of group III capsule gene clusters is not well understood. However, they are defined in part by an absence of genes with significant nucleotide homology to group II capsule transport genes in regions 1 and 3. Evaluation of isogenic, TnphoA-generated, group III capsule-minus derivatives of a clinical blood isolate (CP9, O4/K54/H5) has led to the identification of homologs of the group II capsule transport genes kpsDMTE. These genes and their surrounding regions were sequenced and analyzed. The genomic organization of these genes is distinctly different from that of their group II counterparts. Although kps_K54DMTE are significantly divergent from their group II homologs at both the DNA and protein levels phoA fusions and computer-assisted analyses suggest that their structures and functions are similar. The putative proteins Kps_K54M and Kps_K54T appear to be the integral membrane component and the peripheral ATP-binding component of the ABC-2 transporter family, respectively. The putative Kps_K54E possesses features similar to those of the membrane fusion protein family that facilitates the passage of large molecules across the periplasm. At one boundary of the capsule gene cluster, a truncated kpsM (kpsM_truncated) and its 5′ noncoding regulatory sequence were identified. In contrast to the complete kps_K54M, this region was highly homologous to the group II kpsM. Fifty-three base pairs 3′ from the end of kpsM_truncated was a sequence 75% homologous to the 39-bp inverted repeat in the IS110 insertion element from Streptomyces coelicolor. Southern analysis established that two copies of this element are present in CP9. These findings are consistent with the hypothesis that CP9 previously possessed group II capsule genes and acquired group III capsule genes via IS110-mediated horizontal transfer.     

The role of alien fish (the centrarchid Micropterus salmoides) in lake food webs highlighted by stable isotope analysis

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Modulation of Alpha-Synuclein Aggregation by Dopamine Analogs

The action of dopamine on the aggregation of the unstructured alpha-synuclein (α-syn) protein may be linked to the pathogenesis of Parkinson''s disease. Dopamine and its oxidation derivatives may inhibit α-syn aggregation by non-covalent binding. Exploiting this fact, we applied an integrated computational and experimental approach to find alternative ligands that might modulate the fibrillization of α-syn. Ligands structurally and electrostatically similar to dopamine were screened from an established library. Five analogs were selected for in vitro experimentation from the similarity ranked list of analogs. Molecular dynamics simulations showed they were, like dopamine, binding non-covalently to α-syn and, although much weaker than dopamine, they shared some of its binding properties. In vitro fibrillization assays were performed on these five dopamine analogs. Consistent with our predictions, analyses by atomic force and transmission electron microscopy revealed that all of the selected ligands affected the aggregation process, albeit to a varying and lesser extent than dopamine, used as the control ligand. The in silico/in vitro approach presented here emerges as a possible strategy for identifying ligands interfering with such a complex process as the fibrillization of an unstructured protein.     

Effects of cooling and freezing on the motility of Ostrea edulis (L., 1758) spermatozoa after thawing

The aim of this work was to evaluate the effects of temperature, cryoprotectant agents and freezing curves on sperm motility of Ostrea edulis. All phases of cryopreservation were studied (evaluation of semen motility pattern, choice of cryoprotectants and freezing rates) to restore after thawing the motility characteristics distinctive of fresh semen.To assess the temperature effects on sperm motility, semen was activated using four different temperatures (25, 18, 10 and 3 °C). Sperm aliquots were maintained inactive at these temperatures for 1 and 3 h, then activated with FSW at same temperature of conservation. Sperm was activated and incubated to 3 °C with dimethylsulfoxide (Me₂SO), ethylene glycol (EG), 1–2 propylene glycol (PG) (5%, 7%, 10% and 15% final concentrations), glycerol (GlOH; 5%, 10% and 15% final concentrations) and methanol (MetOH; 4% and 10% final concentrations) for 10, 20 and 30 min. A first evaluation of freezing rates was made by testing four freezing curves: −1, −3, −6 and −10 °C/min. Then, an optimization was made by testing four freezing curves: −2.5, −3.0, −3.5 and −4 °C/min.The selected temperature for short term conservation has been 3 °C, because only this temperature has allowed good sperm motility conservation after 3 h of dry-storage; this is a time sufficient to conduct cryopreservation procedures. The sperm showed a particular sensitivity to GlOH and PG to all tested concentrations and to 15% Me₂SO. EG and MetOH to all concentrations and Me₂SO to concentrations lower than 15% have not shown significant toxic effects. The freezing rate −3 °C/min using 15% EG has shown an highest percentage of RVF (rapid, vigorous and forward) spermatozoa (class 3, about 75% of fresh semen) and an highest sperm motility duration.     

Improved fructosylated chondroitin production by kfoC overexpression in E. coli K4

Escherichia coli K4 is one of the bacteria expressing a surface polysaccharide, indicated as capsular polysaccharide (K-antigen), showing a chemical structure that resembles that of metabolites commonly used in pharmaceutical applications. In this study we provide evidence that homologous overexpression of the chondroitin polymerase, encoded by the kfoC gene, acts on a potential bottleneck for production of capsular polysaccharide, and increases productivity by 100%. However, we also demonstrate that genetic engineering and scale-up of the production process with E. coli K4 is not straight forward due to genetic instability of recombinant strains, partly overcome by multiple additions of antibiotic throughout fermentation that prove to increase plasmid maintenance inside the cells. A lower resistance to the antibiotic was nevertheless highlighted in the stationary phase suggesting other concomitant causes for plasmid instability. The latter might partly be related to a newly discovered endogenous mobile element that we indicate as pK4EC05. Sequencing and analysis of a 1900 bp fragment of pK4EC05 shows a high percentage of sequence similarity to large conjugative plasmids isolated from Shigella, Salmonella and E. coli strains.