On‐Line Control of Glucose Concentration in High‐Yielding Mammalian Cell Cultures Enabled Through Oxygen Transfer Rate Measurements

Glucose control is vital to ensure consistent growth and protein production in mammalian cell cultures. The typical fed‐batch glucose control strategy involving bolus glucose additions based on infrequent off‐line daily samples results in cells experiencing significant glucose concentration fluctuations that can influence product quality and growth. This study proposes an on‐line method to control and manipulate glucose utilizing readily available process measurements. The method generates a correlation between the cumulative oxygen transfer rate and the cumulative glucose consumed. This correlation generates an on‐line prediction of glucose that has been successfully incorporated into a control algorithm manipulating the glucose feed‐rate. This advanced process control (APC) strategy enables the glucose concentration to be maintained at an adjustable set‐point and has been found to significantly reduce the deviation in glucose concentration in comparison to conventional operation. This method has been validated to produce various therapeutic proteins across cell lines with different glucose consumption demands and is successfully demonstrated on micro (15 mL), laboratory (7 L), and pilot (50 L) scale systems. This novel APC strategy is simple to implement and offers the potential to significantly enhance the glucose control strategy for scales spanning micro‐scale systems through to full scale industrial bioreactors.     

Brain uptake of multivalent and multi-specific DVD-Ig proteins after systemic administration

Therapeutic monoclonal antibodies and endogenous IgG antibodies show limited uptake into the central nervous system (CNS) due to the blood-brain barrier (BBB), which regulates and controls the selective and specific transport of both exogenous and endogenous materials to the brain. The use of natural transport mechanisms, such as receptor-mediated transcytosis (RMT), to deliver antibody therapeutics into the brain have been studied in rodents and monkeys. Recent successful examples include monovalent bispecific antibodies and mono- or bivalent fusion proteins; however, these formats do not have the capability to bind to both the CNS target and the BBB transport receptor in a bivalent fashion as a canonical antibody would. Dual-variable-domain immunoglobulin (DVD-Ig) proteins offer a bispecific format where monoclonal antibody-like bivalency to both the BBB receptor and the therapeutic target is preserved, enabling independent engineering of binding affinity, potency, valency, epitope and conformation, essential for successful generation of clinical candidates for CNS applications with desired drug-like properties. Each of these parameters can affect the binding and transcytosis ability mediated by different receptors on the brain endothelium differentially, allowing exploration of diverse properties. Here, we describe generation and characterization of several different DVD-Ig proteins, specific for four different CNS targets, capable of crossing the BBB through transcytosis mediated by the transferrin receptor 1 (TfR1). After systemic administration of each DVD-Ig, we used two independent methods in parallel to observe specific uptake into the brain. An electrochemiluminescent-based sensitive quantitative assay and a semi-quantitative immunohistochemistry technique were used for brain concentration determination and biodistribution/localization in brain, respectively. Significantly enhanced brain uptake and retention was observed for all TfR1 DVD-Ig proteins regardless of the CNS target or the systemic administration route selected.     

Trimethoprim-sulphamethoxazole in Acute Brucellosis

Eight patients with proved brucella infection were treated with trimethoprim-sulphamethoxazole. The dose varied from two to four tablets given twice daily for three weeks. Clinical response was rapid and all patients were asymptomatic and afebrile within two to seven days of starting therapy. Three patients relapsed clinically and bacteriologically within three weeks of ending treatment. There were no side effects of the treatment. It is suggested that the treatment be continued for at least six weeks to prevent relapses.     

Number of Polypeptide Components in Bacteriophage T2L Contractile Sheaths

Isolated purified contractile tail sheaths of bacteriophage T2L were analyzed for their carboxyl terminal amino acids by carboxypeptidase treatment and hydrazinolysis. Glycine and serine were identified as the only two carboxyl end groups. Using corrections for the yields of these two amino acids upon hydrazinolysis, we calculated that there are 154 (+/-30) moles of C-terminal glycine and 130 (+/-45) moles of C-terminal serine per mole of sheath. It appears likely that sheaths contain two types of polypeptide chains in equal numbers, probably 144 of each. The relation of these two components to the mechanism of sheath contraction is discussed.     

Environmental risk assessment of heavy metals pollution in aquatic ecosystem—A case study: Sediment of Kor River,Iran

A comprehensive investigation of fractionation and environmental risk of nine heavy metals is carried out for 12 sediment samples collected from Kor River, Iran. For this purpose, the 5-stage sequential extraction method, along with individual contamination factor, global contamination factor, and Environmental Risk Index (ERI), is used. Total concentrations of Cr, Hg, Ni, and Zn were found to be beyond the threshold effect level. The results of fractionation patterns indicate that As, Cr, Ni, Pb, and Zn are mostly associated with Fe-Mn oxide fraction, organic fraction, and residual fraction, while Cd and Mo are predominantly associated with carbonate fraction. Cu and Hg are mostly associated with organic and exchangeable fractions. The results of ERI revealed High to Dangerous risks in 40% of Kor River stations. The applied approach in this study is beneficial to other environmental studies that require analysis of complex data.     

Leukocyte Gene Expression and Plasma Concentration in Multiple Sclerosis: Alteration of Transforming Growth Factor-βs,Claudin-11, and Matrix Metalloproteinase-2

Multiple sclerosis is a neurodegenerative disease characterized by the present of leukocytes in the brain tissue and subsequently the formation of sclerotic plaques. Leukocytes penetration into the blood–brain barrier is related to several factors, such as, the conversion of leukocyte gene expression or plasma characteristics. In this frame, we explore alteration of matrix metalloproteinase-2 (MMP-2), transforming growth factor beta (TGF-β) family, and Claudin-11 (as a main myelin structural protein) in leukocytes and blood plasma of multiple sclerosis patients compared to the normal group. Blood samples were collected from thirteen men affected by MS and fifteen healthy men. Leukocyte gene expression was measured using real-time PCR and plasma parameters were examined by ELISA. The results of this study showed that the gene expression of Claudin-11 was significantly higher in MS group compared with normal. Interestingly, the MMP-2 pattern was similar to Claudin-11 and correlated positively with it. It was observed that, although the expressions of TGF-β1 and TGF-β2 are down-regulated in the leukocytes of subjects with MS, they showed higher levels of these cytokines in blood plasma. The plasma level of TGF-β3 in MS patients was higher than normal and correlated with Claudin-11 concentration. In conclusion, the aberrant pattern of Claudin-11, TGF-βs family, and MMP-2 expression in leukocytes of the MS patients was observed in this study. Moreover, the plasma levels of TGF-βs family increased in the MS group. The findings of this study provide clues for further investigations to assay MS pathogenesis.     

Printing protein arrays from DNA arrays

We describe a method, DNA array to protein array (DAPA), which allows the 'printing' of replicate protein arrays directly from a DNA array template using cell-free protein synthesis. At least 20 copies of a protein array can be obtained from a single DNA array. DAPA eliminates the need for separate protein expression, purification and spotting, and also overcomes the problem of long-term functional storage of surface-bound proteins.     

MODELING THREE-DIMENSIONAL MORPHOLOGICAL STRUCTURES USING SPHERICAL HARMONICS

Quantifying morphological shape is a fundamental issue in evolutionary biology. Recent technological advances (e.g., confocal microscopy, laser scanning, computer tomography) have made the capture of detailed three-dimensional (3D) morphological structure easy and cost-effective. In this article, we develop a 3D analytic framework (SPHARM—spherical harmonics) for modeling the shapes of complex morphological structures from continuous surface maps that can be produced by these technologies. Because the traditional SPHARM methodology has limitations in several of its processing steps, we present new algorithms for two SPHARM processing steps: spherical parameterization and SPHARM registration. These new algorithms allow for the numerical characterization of a much larger class of 3D models. We demonstrate the effectiveness of the method by applying it to modeling the cerci of Enallagma damselflies.