Extension of human GCSF serum half-life by the fusion of albumin binding domain

Granulocyte colony stimulating factor (GCSF) can decrease mortality of patients undergo chemotherapy through increasing neutrophil counts. Many strategies have been developed to improve its blood circulating time. Albumin binding domain (ABD) was genetically fused to N-terminal end of GCSF encoding sequence and expressed as cytoplasmic inclusion bodies within Escherichia coli. Biological activity of ABD-GCSF protein was assessed by proliferation assay on NFS-60 cells. Physicochemical properties were analyzed through size exclusion chromatography, circular dichroism, intrinsic fluorescence spectroscopy and dynamic light scattering. Pharmacodynamics and pharmacokinetic properties were also investigated in a neutropenic rat model. CD and IFS spectra revealed that ABD fusion to GCSF did not significantly affect the secondary and tertiary structures of the molecule. DLS and SEC results indicated the absence of aggregation formation. EC50 value of the ABD-GCSF in proliferation of NFS-60 cells was 75.76 pg/ml after 72 h in comparison with control GCSF molecules (Filgrastim: 73.1 pg/ml and PEG-Filgrastim: 44.6 pg/ml). Animal studies of ABD-GCSF represented improved serum half-life (9.3 ± 0.7 h) and consequently reduced renal clearance (16.1 ± 1.4 ml/h.kg) in comparison with Filgrastim (1.7 ± 0.1 h). Enhanced neutrophils count following administration of ABD-GCSF was comparable with Filgrastim and weaker than PEG-Filgrastim treated rats. In vitro and in vivo results suggested the ABD fusion as a potential approach for improving GCSF properties.

Contributions of Escherichia coli and its motility to the formation of dual-species biofilms with Vibrio cholerae

Biofilm formation is important in both the environmental and intestinal phases of the Vibrio cholerae life cycle. Nevertheless, most studies of V. cholerae biofilm formation focus on mono-species cultures, whereas nearly all biofilm communities found in nature consist of a variety of microorganisms. Multi-species biofilms formed between V. cholerae and other bacteria in the environment and the interactions that exist between these species are still poorly understood. In this study, the influence of Escherichia coli on the biofilm formation of V. cholerae was studied in the context of both in vitro coculture and in vivo coinfection. To understand the underlying synergistic mechanisms between these two species and to investigate the role of E. coli in V. cholerae biofilm formation, different pathotypes of E. coli and corresponding deletion mutants lacking genes that influence flagella motility, curli fibers, or type I pili were cocultured with V. cholerae . Our findings demonstrate that the presence of commensal E. coli increases biofilm formation at the air-liquid interface in vitro and the generation of biofilm-like multicellular clumps in the mice feces. Examination of laboratory E. coli flagellar-motility mutants Δ fliC and Δ motA in the dual-species biofilm formation suggests that flagellar motility plays an important role in the synergistic interaction and co-aggregation formation between V. cholerae and E. coli . This study facilitates a better understanding of how V. cholerae resides in harsh environments and colonizes the intestine. IMPORTANCE Biofilms play an important role in the V. cholerae life cycle. Until now, mono-species biofilm formation of V. cholerae has been well studied. However, in nature, bacteria live in complex microbial communities, where biofilm is mostly composed of multiple microbial species that interact to cooperate with or compete against each other. Uncovering how V. cholerae forms multi-species biofilm is critical for furthering our understanding of how V. cholerae survives in the environment and transitions to infecting the human host. In this work, the dual-species biofilm between V. cholerae and E. coli was investigated. We demonstrate that the presence of commensal E. coli increased overall biofilm formation. Furthermore, we demonstrate that the motility of E. coli flagella is important for V. cholerae and E. coli to form co-aggregation clumps in dual-species biofilm. These results shed light on a new mechanism for understanding the survival and pathogenesis of V. cholerae .

The Small Ones Matter—sHsps in the Bacterial Chaperone Network

Small heat shock proteins (sHsps) are an evolutionarily conserved class of ATP-independent chaperones that form the first line of defence during proteotoxic stress. sHsps are defined not only by their relatively low molecular weight, but also by the presence of a conserved α-crystallin domain, which is flanked by less conserved, mostly unstructured, N- and C-terminal domains. sHsps form oligomers of different sizes which deoligomerize upon stress conditions into smaller active forms. Activated sHsps bind to aggregation-prone protein substrates to form assemblies that keep substrates from irreversible aggregation. Formation of these assemblies facilitates subsequent Hsp70 and Hsp100 chaperone-dependent disaggregation and substrate refolding into native species. This mini review discusses what is known about the role and place of bacterial sHsps in the chaperone network.

Fertilization and Tree Species Influence on Stable Aggregates in Forest Soil

Background and objectives: aggregation and structure play key roles in the water-holding capacity and stability of soils and are important for the physical protection and storage of soil carbon (C). Forest soils are an important sink of ecosystem C, though the capacity to store C may be disrupted by the elevated atmospheric deposition of nitrogen (N) and sulfur (S) compounds by dispersion of soil aggregates via acidification or altered microbial activity. Furthermore, dominant tree species and the lability of litter they produce can influence aggregation processes. Materials and methods: we measured water-stable aggregate size distribution and aggregate-associated organic matter (OM) content in soils from two watersheds and beneath four hardwood species at the USDA Forest Service Fernow Experimental Forest in West Virginia, USA, where one watershed has received (NH4)2SO4 fertilizer since 1989 and one is a reference/control of similar stand age. Bulk soil OM, pH, and permanganate oxidizable carbon (POXC) were also measured. Research highlights: fertilized soil exhibited decreased macro-aggregate formation and a greater proportion of smaller micro-aggregates or unassociated clay minerals, particularly in the B-horizon. This shift in aggregation to soil more dominated by the smallest (<53 µm) fraction is associated with both acidification (soil pH) and increased microbially processed C (POXC) in fertilized soil. Intra-aggregate OM was also depleted in the fertilized soil (52% less OM in the 53–2000 µm fractions), most strongly in subsurface B-horizon soil. We also document that tree species can influence soil aggregation, as soil beneath species with more labile litter contained more OM in the micro-aggregate size class (<250 µm), especially in the fertilized watershed, while species with more recalcitrant litter promoted more OM in the macro-aggregate size classes (500–2000 µm) in the reference watershed. Conclusions: long-term fertilization, and likely historic atmospheric deposition, of forest soils has weakened macro-aggregation formation, with implications for soil stability, hydrology, and storage of belowground C.

Pathogenic and protective roles of extracellular vesicles in neurodegenerative diseases

Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and polyglutamine diseases are caused by aggregation and abnormal accumulation of the disease-causative proteins in brain and spinal cord. Recent studies have suggested that proteins associated with neurodegenerative diseases are secreted and transmitted intercellularly via extracellular vesicles (EVs), which may be involved in propagation of abnormal protein accumulation and progressive degeneration in patient brains. On the other hand, it has been also reported that EVs have neuroprotective roles in these diseases, which potentially contribute to preventing aggregation formation and aberrant accumulation of the disease-associated proteins. In this review, I summarize the current understanding of the roles of EVs in neurodegenerative diseases, especially focussing on the pathogenic and neuroprotective aspects. Elucidation of these two aspects of EVs would provide insight into not only potential therapeutic targets for treatment of neurodegenerative diseases but also development of EV-based biomarkers for disease diagnostics

Perfusion reduces bispecific antibody aggregation via mitigating mitochondrial dysfunction-induced glutathione oxidation and ER stress in CHO cells

One major challenge observed for the expression of therapeutic bispecific antibodies (BisAbs) is high product aggregates. Aggregates increase the risk of immune responses in patients and therefore must be removed at the expense of purification yields. BisAbs contain engineered disulfide bonds, which have been demonstrated to form product aggregates, if mispaired. However, the underlying intracellular mechanisms leading to product aggregate formation remain unknown. We demonstrate that impaired glutathione regulation underlies BisAb aggregation formation in a CHO cell process. Aggregate formation was evaluated for the same clonal CHO cell line producing a BisAb using fed-batch and perfusion processes. The perfusion process produced significantly lower BisAb aggregates compared to the fed-batch process. Perfusion bioreactors attenuated mitochondrial dysfunction and ER stress resulting in a favorable intracellular redox environment as indicated by improved reduced to oxidized glutathione ratio. Conversely, mitochondrial dysfunction-induced glutathione oxidation and ER stress disrupted the intracellular redox homeostasis, leading to product aggregation in the fed-batch process. Combined, our results demonstrate that mitochondrial dysfunction and ER stress impaired glutathione regulation leading to higher product aggregates in the fed-batch process. This is the first study to utilize perfusion bioreactors as a tool to demonstrate the intracellular mechanisms underlying product aggregation formation.

The Role of FAT10 in Alcoholic Hepatitis Pathogenesis

FAT10 expression is highly up-regulated by pro-inflammatory cytokines IFNγ and TNFα in all cell types and tissues. Increased FAT10 expression may induce increasing mitotic non-disjunction and chromosome instability, leading to tumorigenesis. In this review, we summarized others’ and our work on FAT10 expression in liver biopsy samples from patients with alcoholic hepatitis (AH). FAT10 is essential to maintain the function of liver cell protein quality control and Mallory–Denk body (MDB) formation. FAT10 overexpression in AH leads to balloon degeneration and MDB aggregation formation, all of which is prevented in fat10-/- mice. FAT10 causes the proteins’ accumulation, overexpression, and forming MDBs through modulating 26s proteasome’s proteases. The pathway that increases FAT10 expression includes TNFα/IFNγ and the interferon sequence response element (ISRE), followed by NFκB and STAT3, which were all up-regulated in AH. FAT10 was only reported in human and mouse specimens but plays critical role for the development of alcoholic hepatitis. Flavanone derivatives of milk thistle inhibit TNFα/IFNγ, NFκB, and STAT3, then inhibit the expression of FAT10. NFκB is the key nodal hub of the IFNα/TNFα-response genes. Studies on Silibinin and other milk thistle derivatives to treat AH confirms that overexpressed FAT10 is the major key molecule in these networks.

Spectroscopic Studies of a Phosphonium Ionic Liquid in Supercritical CO2

Fluorescence spectroscopy was used to study a solution comprised of coumarin 153 (C153)+ trihexyltetradecylphosphonium bis(trifluoromethylsulfonyl)imide ([P6,6,6,14]+ [Tf2N]−)+ supercritical CO2 (scCO2). We compare the spectroscopy of C153 in neat scCO2 to that of C153/scCO2 with the addition of ionic liquid (IL). Excitation and emission peak frequencies of C153 in scCO2 and in IL/scCO2 diverged at reduced densities (ρr = ρ/ρc) below the CO2 critical density. At low fluid density, spectral changes in the IL/scCO2 solutions showed evidence that C153 experiences a very different microenvironment—one that is unlike neat scCO2. The data show that the presence of IL clearly influences the C153 excitation and emission profiles. Excitation was broadened and red shifted by >2000 cm−1 and the presence of an additional low-energy emission component that was red shifted by ~3000 cm−1 was clearly visible and not observed in neat scCO2. The solution heterogeneity was controlled by changing the scCO2 density and at high fluid density, both the excitation and emission spectra were more similar to those in neat scCO2. Steady-state anisotropy also showed that at low fluid density, the C153 emission was significantly polarized. Aggregation of C153 has been reported in the literature and this led us to hypothesize the possibility that C153 dimer (aggregation) formation may be occurring in scCO2. Another possible explanation is that dye–IL aggregates may dissolve into the scCO2 phase due to C153 acting as a “co-solvent” for the IL. Time-resolved intensity decay measurements yielded only slightly non-exponential decays with accompanying time constants of ~3–4 ns that were significantly shorter than the 5–6 ns time constants in neat scCO2, which are suggestive of C153–IL interactions. However, these data did not conclusively support dimer formation. Pre-exponential factors of the time constants showed that almost all of the emission was due to monomeric C153.