Use of ultraviolet-visible spectrophotometry associated with artificial neural networks as an alternative for determining the water quality index.

The water quality index (WQI) is an important tool for water resource management and planning. However, it has major disadvantages: the generation of chemical waste, is costly, and time-consuming. In order to overcome these drawbacks, we propose to simplify this index determination by replacing traditional analytical methods with ultraviolet-visible (UV-Vis) spectrophotometry associated with artificial neural network (ANN). A total of 100 water samples were collected from two rivers located in Assis, SP, Brazil and calculated the WQI by the conventional method. UV-Vis spectral analyses between 190 and 800 nm were also performed for each sample followed by principal component analysis (PCA) aiming to reduce the number of variables. The scores of the principal components were used as input to calibrate a three-layer feed-forward neural network. Output layer was defined by the WQI values. The modeling efforts showed that the optimal ANN architecture was 19-16-1, trainlm as training function, root-mean-square error (RMSE) 0.5813, determination coefficient between observed and predicted values (R2) of 0.9857 (p < 0.0001), and mean absolute percentage error (MAPE) of 0.57% ± 0.51%. The implications of this work's results open up the possibility to use a portable UV-Vis spectrophotometer connected to a computer to predict the WQI in places where there is no required infrastructure to determine the WQI by the conventional method as well as to monitor water body's in real time.

Sf9 Cell Metabolism Throughout the Recombinant Baculovirus and Rabies Virus-Like Particles Production in Two Culture Systems.

This work aimed to assess the Sf9 cell metabolism during growth, and infection steps with recombinant baculovirus bearing rabies virus proteins, to finally obtain rabies VLP in two culture systems: Schott flask (SF) and stirred tank reactor (STR). Eight assays were performed in SF and STR (four assays in each system) using serum-free SF900 III culture medium. Two non-infection growth kinetics assays and six recombinant baculovirus infection assays. The infection runs were carried out at 0.1 pfu/cell multiplicity of infection (MOI) for single baculovirus bearing rabies glycoprotein (BVG) and matrix protein (BVM) and a coinfection with both baculoviruses at MOI of 3 and 2 pfu/cell for BVG and BVM, respectively. The SF assays were done in triplicate. The glucose, glutamine, glutamate, lactate, and ammonium uptake or release specific rates were quantified over the exponential growth phase and infection stage. The highest uptake specific rate was observed for glucose (42.5 × 10-12 mmol cell/h) in SF and for glutamine (30.8 × 10-12 mmol/cell/h) in STR, in the exponential growth phases. A wave pattern was observed for assessed analytes throughout the infection phase and the glucose had the highest wave amplitude within the 10-10 mmol cell/h order. This alternative uptake and release behavior is in harmony with the lytic cycle of baculovirus in insect cells. The virus propagation and VLP generation were not limited by glucose, glutamine, and glutamate, neither by the toxicity of lactate nor ammonium under the conditions appraised in this work. The findings from this work can be useful to set baculovirus infection processes at high cell density to improve rabies VLP yield, purity, and productivity.

Sf9 Cells Metabolism and Viability When Coinfected with Two Monocistronic Baculoviruses to Produce Rabies Virus-like Particles.

This work aimed to describe the dynamics of the Sf9 insect cells death and primary metabolism when this host is infected simultaneously by two recombinant baculoviruses (BV) expressing rabies glycoprotein (BVG) and matrix protein (BVM) genes to produce rabies virus-like particles (VLP) at different multiplicities of infection (MOI). Schott flasks essays covering a wide range of MOI for both BV were performed. Viable cell density, cell viability, glucose, glutamine, glutamate, lactate, ammonium, and rabies proteins concentrations were monitored over the infection phase. The expression of both recombinant proteins was not limited by glucose, glutamine, and glutamate in a broad MOI (pfu/cell) range of BVG (0.15-12.5) and BVM (0.1-5.0) using SF900 III serum free culture medium. Death phase initiation and the specific death rate depend on BV MOI. The wave pattern of nutrient/metabolite profiles throughout the viral infection phase is related to the baculovirus lytic cycle. The optimal MOIs ratio between BVG (2.5-4.5) and BVM (1.0-3.0) for maximum protein expression was defined. The produced rabies VLP sizes are close to 78 nm. In general, these work outputs bring a better understanding of the metabolic performance of Sf9 cells when infected by BV for producing VLP, and specifically, for progressing in a rabies VLP vaccine development.

Zika virus-like particles (VLPs) produced in insect cells.

Introdutcion: The Zika virus (ZIKV) infections are a healthcare concern mostly in the Americas, Africa, and Asia but have increased its endemicity area beyond these geographical regions. Due to the advances in infections by Zika virus, it is imperative to develop diagnostic and preventive tools against this viral agent. Virus-like particles (VLPs) appear as a suitable approach for use as antiviral vaccines. Methods: In this work, a methodology was established to produce virus-like particles containing the structural proteins, C, prM, and E of Zika virus produced in insect cells using the gene expression system derived from baculovirus. The vector pFast- CprME -ZIKV was constructed containing the gene sequences of Zika virus structural proteins and it was used to generate the recombinant bacmids (Bac- CprME -ZIKV) through transformation into DH10BacTM cells. The Bac- CprME -ZIKV was transfected in Spodoptera frugiperda (Sf9) insect cells and batches of BV- CprME -ZIKV were obtained by infection assays using a multiplicity of infection of 2. The Sf9 cells were infected, and the supernatant was collected 96 h post-infection. The expression of the CprME -ZIKV protein on the cell surface could be observed by immunochemical assays. To concentrate and purify virus-like particles, the sucrose and iodixanol gradients were evaluated, and the correct CprME -ZIKV proteins' conformation was evaluated by the Western blot assay. The virus-like particles were also analyzed and characterized by transmission electron microscopy. Results and discussion: Spherical structures like the native Zika virus from 50 to 65 nm containing the CprME -ZIKV proteins on their surface were observed in micrographs. The results obtained can be useful in the development path for a vaccine candidate against Zika virus.

Size and specificity of radiopharmaceuticals for sentinel lymph node detection.

BACKGROUND: Biological performance of radiotracers for sentinel node detection analyzed in the light of molecular design and dimension is not widely available. PURPOSE: To evaluate the effect of dextran molecular size and the presence of tissue-binding units (mannose) within the model of (99m)Tc-carbonyl conjugate for sentinel lymph node detection. MATERIAL AND METHODS: Four dextran conjugates with and without mannose in the chemical backbone were included. All polymers were radiolabeled using the precursor [(99m)Tc(OH(2))(3)(CO)(3)](+). Radiolabeling conditions targeted the best radiochemical purity and specific activity for each radiopharmaceutical, and partition coefficients were also defined. Lymphoscintigraphy and ex-vivo biodistribution in popliteal lymph node, liver and kidneys were performed in Wistar rats. The effects of molecular weight and mannose presence were assessed by a two-level factorial design. RESULTS: Radiochemical purity was indirectly related to molecular weight and presence of mannose in the polymer structure. All products were able to detect popliteal lymph node, however, uptake was strongly influenced by use of mannose (4-fold higher). Excretion was similarly modulated by differences in molecular weight. Mannose-enhanced lymph node uptake and higher molecule size in the range under study benefitted lymphoscintigraphic performance. CONCLUSION: Screening of radiopharmaceuticals for lymphoscintigraphy might improve with attention to the mentioned physico-chemical features of the molecule.

Production of Rabies VLPs in Insect Cells by Two Monocistronic Baculoviruses Approach.

Rabies is an ancient zoonotic disease that still causes the death of over 59,000 people worldwide each year. The rabies lyssavirus encodes five proteins, including the envelope glycoprotein and the matrix protein. RVGP is the only protein exposed on the surface of viral particle, and it can induce immune response with neutralizing antibody formation. RVM has the ability to assist with production process of virus-like particles. VLPs were produced in recombinant baculovirus system. In this work, two recombinant baculoviruses carrying the RVGP and RVM genes were constructed. From the infection and coinfection assays, we standardized the best multiplicity of infection and the best harvest time. Cell supernatants were collected, concentrated, and purified by sucrose gradient. Each step was used for protein detection through immunoassays. Sucrose gradient analysis enabled to verify the separation of VLPs from rBV. Through the negative contrast technique, we visualized structures resembling rabies VLPs produced in insect cells and rBV in the different fractions of the sucrose gradient. Using ELISA to measure total RVGP, the recovery efficiency of VLPs at each stage of the purification process was verified. Thus, these results encourage further studies to confirm whether rabies VLPs are a promising candidate for a veterinary rabies vaccine.

Optimization of Eugenia punicifolia (Kunth) D. C. leaf extraction using a simplex centroid design focused on extracting phenolics with antioxidant and antiproliferative activities.

Eugenia punicifolia (Kunth) D. C. (Myrtaceae) has been showing interesting biological activities in the literature which was correlated to its phenolic compounds. In the sense of a better recovering of phenolics with the best antioxidant and antiproliferative activities, an extraction, based on multivariate analytical approach, was developed from E. punicifolia leaves. The different extractor solvents (ethanol, methanol and water) and their binary and ternary combinations were evaluated using a simplex-centroid mixture design and surface response methodology. The optimized crude extracts were investigated for phenol and flavonoid content and compared to their antioxidant (EC50) and antiproliferative properties against HEp-2 (cell line derived from the oropharyngeal carcinoma) and mononuclear viability cells. Ethanolic extracts showed the best phenolic content with the highest antioxidant activity and moderated activity antiproliferative to HEp-2. ESI-QTOF-MS revealed the presence of quercetin and myricetin derivatives, which was correlated to activities tested. Then, simplex-centroid design allowed us to correlate the Eugenia punicifolia biological activities with the extracts obtained from solvent different polarity mixtures.

Semliki Forest Virus replicon particles production in serum-free medium BHK-21 cell cultures and their use to express different proteins.

The production of biopharmaceuticals as vaccines in serum-free media results in reduced risk of contamination and simpler downstream processing. The production of enveloped viruses and viral vectors such as Semliki Forest Virus (SFV) typically requires lipids that are provided by supplementation with animal serum, so production under serum-free conditions is challenging. In this work, the capacity to deliver genetic material of SFV-viral replicon particles (SFV-VRPs) produced in BHK-21 cells adapted to serum-free medium (BHK/SFM) was evaluated. Three transgenes were evaluated: GFP used as a model protein, while hepatitis C virus nonstructural protein 3 protease domain (HCV-NS3p) and rabies virus glycoprotein (RVGP) were selected based on their distinct nature (enzyme and glycoprotein, respectively). BHK/SFM cells produced a sevenfold higher number of SFV-VRPs, as determined by qRT-PCR. These particles showed similar capacities of infecting BHK/FBS or BHK/SFM cells. GFP expression was evaluated by flow cytometry, HCV-NS3p activity by enzymatic assay, and RVGP expression by ELISA and Western Blot. Expression analysis revealed higher levels of GFP and HCV-NS3p in BHK/SFM, while the levels of RVGP were similar for BHK/SFM and BHK/FBS. In conclusion, the BHK/SFM cells showed increased SFV-VRP production yields, without affecting vector infectivity or heterologous gene expression, hence validating the use of BHK/SFM for industrial applications.

Influence of aeration-homogenization system in stirred tank bioreactors, dissolved oxygen concentration and pH control mode on BHK-21 cell growth and metabolism.

This work focused on determining the effect of dissolved oxygen concentration (DO) on growth and metabolism of BHK-21 cell line (host cell for recombinant proteins manufacturing and viral vaccines) cultured in two stirred tank bioreactors with different aeration-homogenization systems, as well as pH control mode. BHK-21 cell line adapted to single-cell suspension was cultured in Celligen without aeration cage (rotating gas-sparger) and Bioflo 110, at 10, 30 and 50 % air saturation (impeller for gas dispersion from sparger-ring). The pH was controlled at 7.2 as far as it was possible with gas mixtures. In other runs, at 30 and 50 % (DO) in Bioflo 110, the cells grew at pH controlled with CO2 and NaHCO3 solution. Glucose, lactate, glutamine, and ammonium were quantified by enzymatic methods. Cell concentration, size and specific oxygen consumption were also determined. When NaHCO3 solution was not used, the optimal DOs were 10 and 50 % air saturation for Celligen and Bioflo 110, respectively. In this condition maximum cell concentrations were higher than 4 × 10(6) cell/mL. An increase in maximum cell concentration of 36 % was observed in batch carried out at 30 % air saturation in a classical stirred tank bioreactor (Bioflo 110) with base solution addition. The optimal parameters defined in this work allow for bioprocess developing of viral vaccines, transient protein expression and viral vector for gene therapy based on BHK-21 cell line in two stirred tank bioreactors with different agitation-aeration systems.

Combining dose and injection volume for good performance of a specific radiopharmaceutical for sentinel node detection.

INTRODUCTION: The aim of this work was to quantify the effects of injection volume at different technetium-99m specific radiotracer doses on its lymphatic movement in animal model. PROCEDURES: Effects of injection volume (50, 100 µl) at different doses (0.05, 0.135, 0.22 nmol) on popliteal node (PN) detection were studied in rats. The radiotracer under study was (99m)Technetium-cysteine-mannose-dextran conjugate (30 kDa). RESULTS: At 0.05 nmol dose, higher PN uptake was observed at 50 µl injection volume (2.6 fold increase). Conversely, at 0.135 nmol dose, an increase of radiotracer retention in PN was achieved at 100 µl volume, 78% higher than 50 µl. However, at 0.22 nmol dose, the injection volume changes did not influence on the PN uptake. Considering as suitable radiotracer performance: high PN uptake and extraction, better combinations were 0.05 nmol/50 µl, 0.135 nmol/100 µl, 0.22/50 µl. CONCLUSION: Suitable performances could be reached by proper combinations of dose, injection volume and concentration for a specific radiotracer used in sentinel lymph node detection.

Neurotensin(8-13) analogue: radiolabeling and biological evaluation using different chelators.

INTRODUCTION: Several strategies on the development of radiopharmaceuticals have been employed. Bifunctional chelators seem to be a promising approach since high radiochemical yields as well as good in vitro and in vivo stability have been achieved. To date, neurotensin analogs have been radiolabeled using the (99m)Tc-carbonyl approach and none was described employing the bifunctional chelating agent technique. AIM: The purpose of this study was to evaluate the radiochemical and biological behaviour of NT(8-13) analogue radiolabeled with (99m)Tc, using HYNIC and NHS-S-acetyl-MAG(3) as chelator agents. METHODS: Radiolabeling, in vitro stability toward cysteine and glutathione, partition coefficient and plasma protein binding were assessed for both radioconjugates. Biodistribution in healthy Swiss mice were carried out in order to evaluate the biological behaviour of the radiocomplexes. RESULTS: Radiochemical yields were higher than 97% and no apparent instability toward transchelant agents was observed for both radioconjugates. A higher lipophilic character was observed for the radioconjugate labeled via MAG(3). The chelators seem to have no effect on the percentage of the radioconjugate bound to plasma proteins. A similar biological pattern was observed for both radioconjugates. Total blood, bone and muscle values revealed a slightly slower clearance for the radiocomplex labeled via MAG(3). Moreover, a remarkable liver and intestinal uptake was observed for the radiocomplex labeled via MAG(3) even at the later time points studied. CONCLUSION: The high radiochemical yields achieved and the similar in vivo pattern found for both radioconjugates make them potential candidates for imaging tumors using nuclear medicine techniques.

Neovascularization after ischemic injury: evaluation with 99(m)Tc-HYNIC-RGD.

PURPOSE: Angiogenesis involves many mediators including integrins, and the tripeptide RGD is a target amino acid recognition sequence for many of them. Hindlimb ischemia is a simple and convenient animal model however standardization of the injection procedures in the devascularized and control limb is lacking, thus rendering difficult the interpretation of results. The aim of this investigations was to evaluate neovascularization in a hindlimb murine model by means of 99(m)Tc-HYNIC-ß-Ala-RGD. METHODS: 99(m)Tc-HYNIC-RGD analog was prepared using coligands. Ischemia was induced in Wistar rats by double- ligation of the common femoral artery. Radiolabeled RGD was injected after 2h, as well as 1, 3, 5, 7, 10 and 14 days. Uptake was evaluated by planar imaging and biodistribution studies. RESULTS: The highest ratio between ischemia and control was achieved at the 7th day (2.62 ± 0.95), with substantial decrease by the 14th day. For pertechnetate the 7th day ratio was 0.87 ± 0.23. Scintigraphic image confirmed different uptakes. CONCLUSION: 99(m)Tc-HYNIC-RGD analog concentrated in ischemic tissue by the time of widespread angiogenesis and pertechnetate confirmed reduction in blood flow. In this sense, the protocol can be recommended for ischemic models.

Parameters optimization defined by statistical analysis for cysteine-dextran radiolabeling with technetium tricarbonyl core.

The objective of this study was the development of a statistical approach for radiolabeling optimization of cysteine-dextran conjugates with Tc-99m tricarbonyl core. This strategy has been applied to the labeling of 2-propylene-S-cysteine-dextran in the attempt to prepare a new class of tracers for sentinel lymph node detection, and can be extended to other radiopharmaceuticals for different targets. The statistical routine was based on three-level factorial design. Best labeling conditions were achieved. The specific activity reached was 5 MBq/µg.

Influence of colloid particle profile on sentinel lymph node uptake.

INTRODUCTION: Particle size of colloids employed for sentinel lymph node (LN) detection is not well studied. This investigation aimed to correlate particle size and distribution of different products with LN uptake. METHODS: All agents (colloidal tin, dextran, phytate and colloidal rhenium sulfide) were labeled with (99m)Tc according to manufacturer's instructions. Sizing of particles was carried out on electron micrographs using Image Tool for Windows (Version 2.0). Biodistribution studies in main excretion organs as well as in popliteal LN were performed in male Wistar rats [30 and 90 min post injection (p.i.)]. The injected dose was 0.1 ml (37 MBq) in the footpad of the left posterior limb. Dynamic images (0-15 min p.i.) as well as static ones (30 and 90 min) were acquired in gamma camera. RESULTS: Popliteal LN was clearly reached by all products. Nevertheless, particle size remarkably influenced node uptake. Colloidal rhenium sulfide, with the smallest diameter (5.1 x 10(-3)+/-3.9 x 10(-3) microm), permitted the best result [2.72+/-0.64 percent injected dose (%ID) at 90 min]. Phytate displayed small particles (<15 microm) with favorable uptake (1.02+/-0.14%ID). Dextran (21.4+/-12.8 microm) and colloidal tin (39.0+/-8.3 microm) were less effective (0.55+/-0.14 and 0.06+/-0.03%ID respectively). Particle distribution also tended to influence results. When asymmetric, it was associated with biphasic uptake which increased over time; conversely, symmetric distribution (colloidal tin) was consistent with a constant pattern. CONCLUSION: The results are suggesting that particle size and symmetry may interfere with LN radiopharmaceutical uptake.

Semliki forest virus as a vector: pros and cons for its use in biopharmaceuticals production

The number of biopharmaceuticals for medical and veterinarian use produced in mammalian cells is increasing year after year. All of them are obtained by stable recombinant cell lines. However, it is recognized that transient gene expression produces high level expression in a short time. In that sense, viral vectors have been extensively used for producing recombinant proteins on lab-scale. Among them, Semliki Forest virus is commonly employed for this purpose. This review discusses the main aspects related to the use of Semliki Forest virus technology as well as its advantages and drawbacks which limit currently its utilization in biopharmaceutical industry on large-scale.

Neovascularization after ischemic injury: evaluation with 99mTc-HYNIC-RGD / Neovascularização após lesão isquêmica: avaliação com 99mTc-HYNIC-RGD

Purpose: Angiogenesis involves many mediators including integrins, and the tripeptide RGD is a target amino acid recognition sequence for many of them. Hindlimb ischemia is a simple and convenient animal model however standardization of the injection procedures in the devascularized and control limb is lacking, thus rendering difficult the interpretation of results. The aim of this investigations was to evaluate neovascularization in a hindlimb murine model by means of 99mTc-HYNIC-ß-Ala-RGD. Methods: 99mTc-HYNIC-RGD analog was prepared using coligands. Ischemia was induced in Wistar rats by double- ligation of the common femoral artery. Radiolabeled RGD was injected after 2h, as well as 1, 3, 5, 7, 10 and 14 days. Uptake was evaluated by planar imaging and biodistribution studies. Results: The highest ratio between ischemia and control was achieved at the 7th day (2.62 ± 0.95), with substantial decrease by the 14th day. For pertechnetate the 7th day ratio was 0.87 ± 0.23. Scintigraphic image confirmed different uptakes. Conclusion: 99mTc-HYNIC-RGD analog concentrated in ischemic tissue by the time of widespread angiogenesis and pertechnetate confirmed reduction in blood flow. In this sense, the protocol can be recommended for ischemic models.

Objetivo: A angiogênese em resposta a fenômenos isquêmicos envolve vários mediadores como as integrinas, sendo que o tripeptídeo RGD possui uma seqüência de aminoácidos com reconhecimento para este alvo. O modelo animal de isquemia de pata traseira é simples e conveniente, porém não há uma padronização do procedimento de injeção e controle radioisotópico em membro desvascularizado, dificultando, portanto a interpretação de resultados. O objetivo deste estudo foi avaliar a neovascularização em modelo murino de isquemia de pata traseira através do radiotraçador 99mTc-HYNIC-ß-Ala-RGD. Métodos: O análogo 99mTc-HYNIC-RGD foi preparado usando coligantes. A isquemia foi induzida em ratos Wistar por dupla-ligação da artéria femoral comum na prega inguinal. Peptídeo RGD radiomarcado foi injetado após 2h, assim como 1, 3, 5, 7, 10 e 14 dias. A captação foi avaliada por imagem planar e estudos de biodistribuição. Resultados: A maior diferença de captação entre isquemia e pata controle foi obtida no 7º dia (2,62 ± 0,95), com decréscimo acentuado no 14º dia. Para o pertecnetato a razão no 7º dia foi 0,87 ± 0,23. A imagem cintilográfica confirmou as diferentes captações. Conclusões: O análogo 99mTc-HYNIC-RGD concentrou-se no tecido isquêmico na etapa em que a angiogênese é mais acentuada, e o estudo do pertecnetato confirmou a redução no fluxo sanguíneo. Desta maneira, este protocolo diagnóstico pode ser recomendado para modelos isquêmicos.