Biophysical Characterization of Met-G-CSF: Effects of Different Site-Specific Mono-Pegylations on Protein Stability and Aggregation

The limited stability of proteins in vitro and in vivo reduces their conversion into effective biopharmaceuticals. To overcome this problem several strategies can be exploited, as the conjugation of the protein of interest with polyethylene glycol, in most cases, improves its stability and pharmacokinetics. In this work, we report a biophysical characterization of the non-pegylated and of two different site-specific mono-pegylated forms of recombinant human methionyl-granulocyte colony stimulating factor (Met-G-CSF), a protein used in chemotherapy and bone marrow transplantation. In particular, we found that the two mono-pegylations of Met-G-CSF at the N-terminal methionine and at glutamine 135 increase the protein thermal stability, reduce the aggregation propensity, preventing also protein precipitation, as revealed by circular dichroism (CD), Fourier transform infrared (FTIR), intrinsic fluorescence spectroscopies and dynamic light scattering (DLS). Interestingly, the two pegylation strategies were found to drastically reduce the polydispersity of Met-G-CSF, when incubated under conditions favouring protein aggregation, as indicated by DLS measurements. Our in vitro results are in agreement with preclinical studies, underlining that preliminary biophysical analyses, performed in the early stages of the development of new biopharmaceutical variants, might offer a useful tool for the identification of protein variants with improved therapeutic values.     

SorCS2 Controls Functional Expression of Amino Acid Transporter EAAT3 and Protects Neurons from Oxidative Stress and Epilepsy-Induced Pathology

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A molecular beacon, bead-based assay for the detection of nucleic acids by flow cytometry

Molecular beacons are dual-labelled probes that are typically used in real-time PCR assays, but have also been conjugated with solid matrices for use in microarrays or biosensors. We have developed a fluid array system using microsphere-conjugated molecular beacons and the flow cytometer for the specific, multiplexed detection of unlabelled nucleic acids in solution. For this array system, molecular beacons were conjugated with microspheres using a biotin-streptavidin linkage. A bridged conjugation method using streptavidin increased the signal-to-noise ratio, allowing for further discrimination of target quantitation. Using beads of different sizes and molecular beacons in two fluorophore colours, synthetic nucleic acid control sequences were specifically detected for three respiratory pathogens, including the SARS coronavirus in proof-of-concept experiments. Considering that routine flow cytometers are able to detect up to four fluorescent channels, this novel assay may allow for the specific multiplex detection of a nucleic acid panel in a single tube.     

Grossly Defective nef Gene Sequences in a Human Immunodeficiency Virus Type 1-Seropositive Long-Term Nonprogressor

We have been investigating a long-term nonprogressor who was found to be human immunodeficiency virus type 1 (HIV-1) seropositive in 1985 and has survived with stable CD4⁺ T-cell counts (>1,000 CD4 cells/μl) without any AIDS-related illness. We have previously reported that repeated attempts to measure HIV-1 RNA in the peripheral mononuclear cells obtained from this subject have invariably failed. In the present study, we have analyzed the molecular nature of the HIV-1 quasispecies infecting this patient by PCR amplification of two proviral regions, the 5′ long terminal repeat (5′LTR)/gag leader and the nef gene, directly from fresh uncultured peripheral mononuclear cells, followed by length polymorphism analysis (with 1994, 1995, and 1996 samples) and sequencing (with a 1996 sample). Only proviral forms with nef deletions were revealed by length polymorphism analysis in samples from all three time points. Sequence analysis of the nef gene from the 1996 sample confirmed the presence of similar proviral quasispecies characterized by the presence of several deletions located in the nef-alone and the nef/U3 overlapping regions. Length polymorphism analysis of the 5′LTR/gag leader region suggested the existence of two major quasispecies populations, one characterized by the presence of forms carrying deletions in the U3 region and the other showing a completely intact, full-length 5′LTR. Evidence of the role of nef gene defects in long-term survival of HIV-1-infected patients has been provided so far in two independent investigations involving patients infected with HIV through blood transfusion. Here we show the existence of a similar condition in a subject who acquired HIV-1 seropositivity through the sexual route.     

Growth and biomass productivity of Scenedesmus vacuolatus on a twin layer system and a comparison with other types of cultivations

Scenedesmus is a genus of microalgae employed for several industrial uses. Industrial cultivations are performed in open ponds or in closed photobioreactors (PBRs). In the last years, a novel type of PBR based on immobilized microalgae has been developed termed porous substrate photobioreactors (PSBR) to achieve significant higher biomass density during cultivation in comparison to classical PBRs. This work presents a study of the growth of Scenedesmus vacuolatus in a Twin Layer System PSBR at different light intensities (600 μmol photons m⁻² s⁻¹ or 1000 μmol photons m⁻² s⁻¹), different types and concentrations of the nitrogen sources (nitrate or urea), and at two CO₂ levels in the gas phase (2% or 0.04% v/v). The microalgal growth was followed by monitoring the attached biomass density as dry weight, the specific growth rate and pigment accumulation. The highest productivity (29 g m⁻² d⁻¹) was observed at a light intensity of 600 μmol photons m⁻² s⁻¹ and 2% CO₂. The types and concentrations of nitrogen sources did not influence the biomass productivity. Instead, the higher light intensity of 1000 μmol photons m⁻² s⁻¹ and an ambient CO₂ concentration (0.04%) resulted in a significant decrease of productivity to 18 and 10–12 g m⁻² d⁻¹, respectively. When compared to the performance of similar cultivation systems (15–30 g m⁻² d⁻¹), these results indicate that the Twin Layer cultivation System is a competitive technique for intensified microalgal cultivation in terms of productivity and, at the same time, biomass density.

     

Evolución del microprolactinoma a largo plazo con tratamiento médico

IntroductionProlactinoma is the most frequent functioning pituitary adenoma. Most commonly occurs as microprolactinoma (less than 1 cm in size), which may be cured with medical therapy, but few long-term studies are available about optimal duration of treatment with dopamine agonists to ensure cure after drug discontinuation and its withdrawal without recurrence are do not report consistent results.ObjectiveTo establish criteria for cure of microprolactinoma with medical treatment and to analyze the potential predictors involved.PatientsA retrospective study was conducted on 47 adult women with microprolactinoma followed up between 1975 and 2010; none of them had undergone prior surgery or radiotherapy, and all of them received treatment with a dopamine agonist for at least 4 years. They were divided into two groups for analysis: cured patients with at least 4 years with normal prolactin levels after drug discontinuation, and not cured patients.ResultsCure was achieved in 57.4% of patients. Only age at diagnosis was a significant predictor: there were more young patients in the cured group and youngest patients needed less time to cure. Development of empty sella turcica or normal MRI were similar regarding time to cure.ConclusionsMicroprolactinoma may be cured with dopamine agonists, and life-long treatment is not required, although more than 10 years may be required to achieve cure, 11,6 ± 5,3 years in our experience.     

Is disruption of sleep quality a consequence of severe Covid-19 infection? A case-series examination

ABSTRACT

The Covid-19 outbreak put enormous stress on the health system worldwide, and objective data to handle the emergency are still needed. We aimed to objectively assess the consequence of severe symptoms of Covid-19 infection on sleep quality through wrist actigraphy monitoring of four patients during the sub-acute recovery stage of the disease. The sleep of those patients who had experienced the most severe respiratory symptoms and who had needed prolonged intensive care unit (ICU) stay showed lower Sleep Efficiency and Immobility Time and higher Fragmentation Index compared to those patients who had experienced only mild respiratory symptoms and not requiring ICU stay. Wrist actigraphy assessment provided important clinical information about the sleep and activity levels of Covid-19 patients during the post-acute rehabilitation management.     

Three Distinct Types of Monoclonal Antibodies After Long-Term Immunization of Rats with Mouse Nerve Growth Factor

This article reports the results of a systematic investigation of the different types of antibodies produced in the course of a long-term immunization of rats with mouse nerve growth factor (NGF). We have characterized three types of monoclonal antibodies, namely: (1) antibodies that bind to NGF and inhibit its binding to target cells and its biological activity in culture (type A); (2) antibodies that bind to and precipitate NGF but do not inhibit its binding to target cells or its biological activity (type B); (3) antibodies that fail to recognize NGF itself, but inhibit nonetheless its binding to target cells (type C). These antibodies bind to an antigen present on NGF target cells and not on rat fibroblasts lacking NGF receptor. They appear thus to be antiidiotypic antibodies directed against the NGF receptor, developed as a consequence of the long-term immunization with NGF.     

Nutrient removal from high strength nitrate containing industrial wastewater using <Emphasis Type="Italic">Chlorella</Emphasis> sp. strain ACUF_802

the research aim of this study was to characterize an isolated native strain of Chlorella sp. ACUF_802, well adapted to a high nitrate concentration environment and to investigate its potential to nitrate and phosphate removal from industrial wastewaters with the minimal addition of chemical reagents and energy. The isolated strain was identified and evaluated for its capability to support biomass growth and nutrient removal from synthetic wastewater in batch tests using different concentrations of carbon and nitrogen, different carbon sources and N:P ratios. The strain was isolated via the plating method from the settler of a pilot scale moving bed biofilm reactor performing a nitrification process. The strain was identified using molecular analysis with rDNA primers. Using sodium bicarbonate as carbon source, the batch productivity (71.43 mg L^?1 day^?1) of the strain Chlorella sp. ACUF_802 was calculated with a logistic model and compared to the values reported in the literature. Assays on the effect of the N:P ratio indicated that the productivity was increased 36% when the N:P ratio was close to 1 (111.96 mg L^?1 day^?1), but for a complete phosphorus removal a 5:1 N:P ratio with nitrate concentrations ≤125 mg?L^?1 is recommended. The isolated microalgae strain Chlorella sp. ACUF_802 showed versatility to grow in the synthetic industrial wastewaters tested and can be considered as an appropriate organism for nitrogen removal from industrial wastewaters in the presence of an organic or inorganic carbon source.     

A novel functional crosstalk between DDR1 and the IGF axis and its relevance for breast cancer

ABSTRACT

In the last decades increasing importance has been attributed to the Insulin/Insulin-like Growth Factor signaling (IIGFs) in cancer development, progression and resistance to therapy. In fact, IIGFs is often deregulated in cancer. In particular, the mitogenic insulin receptor isoform A (IR-A) and the insulin-like growth factor receptor (IGF-1R) are frequently overexpressed in cancer together with their cognate ligands IGF-1 and IGF-2. Recently, we identified discoidin domain receptor 1 (DDR1) as a new IR-A interacting protein. DDR1, a non-integrin collagen tyrosine kinase receptor, is overexpressed in several malignancies and plays a role in cancer progression and metastasis.

Herein, we review recent findings indicating that DDR1 is as a novel modulator of IR and IGF-1R expression and function. DDR1 functionally interacts with IR and IGF-1R and enhances the biological actions of insulin, IGF-1 and IGF-2. Conversely, DDR1 is upregulated by IGF-1, IGF-2 and insulin through the PI3K/AKT/miR-199a-5p circuit. Furthermore, we discuss the role of the non-canonical estrogen receptor GPER1 in the DDR1-IIGFs crosstalk. These data suggest a wider role of DDR1 as a regulator of cell response to hormones, growth factors, and signals coming from the extracellular matrix.