Multipronged approach to managing beta‐glucan contaminants in the downstream process: Control of raw materials and filtration with charge‐modified nylon 6,6 membrane filters

(1→3)‐β‐d ‐Glucans (beta‐glucans) have been found in raw materials used in the manufacture of recombinant therapeutics. Because of their biological activity, beta‐glucans are considered process contaminants and consequently their level in the product needs to be controlled. Although beta‐glucans introduced into the cell culture process can readily be removed by bind‐and‐elute chromatography process steps, beta‐glucans can also be introduced into the purification process through raw materials containing beta‐glucans as well as leachables from filters made from cellulose. This article reports a multipronged approach to managing the beta‐glucan contamination in the downstream process. Raw material screening and selection can be used to effectively limit the level of beta‐glucan introduced into the downstream process. Placement of a cellulosic filter upstream of the last bind‐and‐elute column step or effective preuse flushing can also limit the level of contaminant introduced. More importantly, this article reports the active removal of beta‐glucan from the downstream process when necessary. It was discovered that the Posidyne^® filter, a charge‐modified nylon 6,6 membrane filter, was able to effectively remove beta‐glucans from buffers at relatively low pH and salt concentrations. An approach of using low beta‐glucan buffer components combined with filtration of the buffer with a Posidyne membrane has been successfully demonstrated at preparative scale. Additionally, the feasibility of active removal of beta‐glucan from in‐process product pools by Posidyne membrane filtration has also been demonstrated. Based on the data presented, a mechanism for binding is proposed, as well as a systematic approach for sizing of the Posidyne filter. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:672–680, 2013     

Neocortex and Allocortex Respond Differentially to Cellular Stress In Vitro and Aging In Vivo

In Parkinson’s and Alzheimer’s diseases, the allocortex accumulates aggregated proteins such as synuclein and tau well before neocortex. We present a new high-throughput model of this topographic difference by microdissecting neocortex and allocortex from the postnatal rat and treating them in parallel fashion with toxins. Allocortical cultures were more vulnerable to low concentrations of the proteasome inhibitors MG132 and PSI but not the oxidative poison H₂O₂. The proteasome appeared to be more impaired in allocortex because MG132 raised ubiquitin-conjugated proteins and lowered proteasome activity in allocortex more than neocortex. Allocortex cultures were more vulnerable to MG132 despite greater MG132-induced rises in heat shock protein 70, heme oxygenase 1, and catalase. Proteasome subunits PA700 and PA28 were also higher in allocortex cultures, suggesting compensatory adaptations to greater proteasome impairment. Glutathione and ceruloplasmin were not robustly MG132-responsive and were basally higher in neocortical cultures. Notably, neocortex cultures became as vulnerable to MG132 as allocortex when glutathione synthesis or autophagic defenses were inhibited. Conversely, the glutathione precursor N-acetyl cysteine rendered allocortex resilient to MG132. Glutathione and ceruloplasmin levels were then examined in vivo as a function of age because aging is a natural model of proteasome inhibition and oxidative stress. Allocortical glutathione levels rose linearly with age but were similar to neocortex in whole tissue lysates. In contrast, ceruloplasmin levels were strikingly higher in neocortex at all ages and rose linearly until middle age. PA28 levels rose with age and were higher in allocortex in vivo, also paralleling in vitro data. These neo- and allocortical differences have implications for the many studies that treat the telencephalic mantle as a single unit. Our observations suggest that the topographic progression of protein aggregations through the cerebrum may reflect differential responses to low level protein-misfolding stress but also reveal impressive compensatory adaptations in allocortex.     

Linkage of Presumptive Multidrug Resistant Tuberculosis (MDR-TB) Patients to Diagnostic and Treatment Services in Cambodia

Setting

National Tuberculosis Programme, Cambodia.

Objective

In a cohort of TB patients, to ascertain the proportion of patients who fulfil the criteria for presumptive MDR-TB, assess whether they underwent investigation for MDR-TB, and the results of the culture and drug susceptibility testing (DST).

Methods

A cross sectional record review of TB patients registered for treatment between July-December 2011.

Results

Of 19,236 TB patients registered, 409 (2%) fulfilled the criteria of presumptive MDR-TB; of these, 187 (46%) were examined for culture. This proportion was higher among relapse, failure, return after default (RAD) and non-converters at 3 months of new smear positive TB patients (>60%) as compared to non-converters at 2 months of new TB cases (<20%). Nearly two thirds (n = 113) of the samples were culture positive; of these, three-fourth (n = 85) grew Mycobacterium tuberculosis complex (MTBc) and one-fourth (n = 28) grew non-tuberculous Mycobacteria. DST results were available for 96% of the MTBc isolates. Overall, 21 patients were diagnosed as MDR-TB (all diagnosed among retreatment TB cases and none from non-converters) and all of them were initiated on MDR-TB treatment.

Conclusion

There is a need to strengthen mechanisms for linking patients with presumptive MDR-TB to culture centers. The policy of testing non-converters for culture and DST needs to be reviewed.     

Antineoplastic Effects of α-Santalol on Estrogen Receptor-Positive and Estrogen Receptor-Negative Breast Cancer Cells through Cell Cycle Arrest at G2/M Phase and Induction of Apoptosis

Anticancer efficacy and the mechanism of action of α-santalol, a terpenoid isolated from sandalwood oil, were investigated in human breast cancer cells by using p53 wild-type MCF-7 cells as a model for estrogen receptor(ER)-positive and p53 mutated MDA-MB-231 cells as a model for ER-negative breast cancer. α-Santalol inhibited cell viability and proliferation in a concentration and time-dependent manner in both cells regardless of their ER and/or p53 status. However, α-santalol produced relatively less toxic effect on normal breast epithelial cell line, MCF-10A. It induced G2/M cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cells. Cell cycle arrest induced by α-santalol was associated with changes in the protein levels of BRCA1, Chk1, G2/M regulatory cyclins, Cyclin dependent kinases (CDKs), Cell division cycle 25B (Cdc25B), Cdc25C and Ser-216 phosphorylation of Cdc25C. An up-regulated expression of CDK inhibitor p21 along with suppressed expression of mutated p53 was observed in MDA-MB-231 cells treated with α-santalol. On the contrary, α-santalol did not increase the expression of wild-type p53 and p21 in MCF-7 cells. In addition, α-santalol induced extrinsic and intrinsic pathways of apoptosis in both cells with activation of caspase-8 and caspase-9. It led to the activation of the executioner caspase-6 and caspase-7 in α-santalol-treated MCF-7 cells and caspase-3 and caspase-6 in MDA-MB-231 cells along with strong cleavage of poly(ADP-ribose) polymerase (PARP) in both cells. Taken together, this study for the first time identified strong anti-neoplastic effects of α-santalol against both ER-positive and ER-negative breast cancer cells.     

Purification and Characterization of Neurotoxin Complex from a Dual Toxin Gene Containing Clostridium Botulinum Strain PS-5

Botulinum neurotoxins are produced as a toxin complex (TC) which consists of neurotoxin (NT) and neurotoxin associated proteins. The characterization of NT in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. The presence of NT genes was validated by PCR amplification of toxin specific fragments from genomic DNA of Clostridium botulinum strain PS-5 which indicated the presence of both serotype A and B genes on PS-5 genome. Further, TC was purified and characterized by Western blotting, Digoxin-enzyme linked immunosorbent assay, endopeptidase activity assay, and Liquid chromatography–Mass spectrometry. The data showed the presence of serotype A specific neurotoxin. Based on the analysis of neurotoxin genes and characterization of TC, PS-5 strain appears as a serotype A (B) strain of C. botulinum which produces only serotype A specific TC in the cell culture medium.     

Correlation between substituent constants and hyperpolarizabilities for di-substituted trans-azobenzenes

Nonlinear optical properties of a series of disubstituted trans-azobenzenes were studied. The structures were fully optimized by B3LYP/6-31+G* and both static polarizabilities and hyperpolarizabilities were then calculated by the derivative method. In order to show the relationships between dipole moments, (hyper)polarizabilities and the structures, three kinds of substituent constants were applied to correlate with both ground state dipole moment and hyperpolarizabilities. Both physical properties have a satisfactory correlation with substituent constants Σσ_+/? and bond length alternation. Overall, the electronic excitation contribution to the hyperpolarizabilities is rationalized in terms of the two-level model.     

Surface‐based protein binding pocket similarity

Protein similarity comparisons may be made on a local or global basis and may consider sequence information or differing levels of structural information. We present a local three‐dimensional method that compares protein binding site surfaces in full atomic detail. The approach is based on the morphological similarity method which has been widely applied for global comparison of small molecules. We apply the method to all‐by‐all comparisons two sets of human protein kinases, a very diverse set of ATP‐bound proteins from multiple species, and three heterogeneous benchmark protein binding site data sets. Cases of disagreement between sequence‐based similarity and binding site similarity yield informative examples. Where sequence similarity is very low, high pocket similarity can reliably identify important binding motifs. Where sequence similarity is very high, significant differences in pocket similarity are related to ligand binding specificity and similarity. Local protein binding pocket similarity provides qualitatively complementary information to other approaches, and it can yield quantitative information in support of functional annotation. Proteins 2011; © 2011 Wiley‐Liss, Inc.