Molecular Pharmacology of Gelsemium Alkaloids on Inhibitory Receptors.

Indole alkaloids are the main bioactive molecules of the Gelsemium genus plants. Diverse reports have shown the beneficial actions of Gelsemium alkaloids on the pathological states of the central nervous system (CNS). Nevertheless, Gelsemium alkaloids are toxic for mammals. To date, the molecular targets underlying the biological actions of Gelsemium alkaloids at the CNS remain poorly defined. Functional studies have determined that gelsemine is a modulator of glycine receptors (GlyRs) and GABAA receptors (GABAARs), which are ligand-gated ion channels of the CNS. The molecular and physicochemical determinants involved in the interactions between Gelsemium alkaloids and these channels are still undefined. We used electrophysiological recordings and bioinformatic approaches to determine the pharmacological profile and the molecular interactions between koumine, gelsemine, gelsevirine, and humantenmine and these ion channels. GlyRs composed of α1 subunits were inhibited by koumine and gelsevirine (IC50 of 31.5 ± 1.7 and 40.6 ± 8.2 µM, respectively), while humantenmine did not display any detectable activity. The examination of GlyRs composed of α2 and α3 subunits showed similar results. Likewise, GABAARs were inhibited by koumine and were insensitive to humantenmine. Further assays with chimeric and mutated GlyRs showed that the extracellular domain and residues within the orthosteric site were critical for the alkaloid effects, while the pharmacophore modeling revealed the physicochemical features of the alkaloids for the functional modulation. Our study provides novel information about the molecular determinants and functional actions of four major Gelsemium indole alkaloids on inhibitory receptors, expanding our knowledge regarding the interaction of these types of compounds with protein targets of the CNS.

Leukocyte- and Platelet-Rich Fibrin (L-PRF) Obtained from Smokers and Nonsmokers Shows a Similar Uniaxial Tensile Response In Vitro.

We evaluated and compared the biomechanical properties of Leukocyte-and Platelet Rich Fibrin L-PRF clots and membranes derived from smoker and nonsmoker donors. Twenty venous-blood donors (aged 18 to 50 years) were included after signing informed consent forms. L-PRF clots were analyzed and then compressed to obtain L-PRF membranes. L-PRF clot and membrane samples were tested in quasi-static uniaxial tension and the stress-stretch response was registered and characterized. Furthermore, scanning electron microscope representative images were taken to see the fibrin structure from both groups. The analysis of stress-stretch curves allowed us to evaluate the statistical significance in differences between smoker and nonsmoker groups. L-PRF membranes showed a stiffer response and higher tensile strength when compared to L-PRF clots. However, no statistically significant differences were found between samples from smokers and nonsmokers. With the limitations of our in vitro study, we can suggest that the tensile properties of L-PRF clots and membranes from the blood of smokers and nonsmokers are similar. More studies are necessary to fully characterize the effect of smoking on the biomechanical behavior of this platelet concentrate, to further encourage its use as an alternative to promote wound healing in smokers.

Amniotic fluid pulmonary embolism and COVID-19: Case report.

Amniotic fluid embolism is a rare complication of peripartum. It is caused by the entry of fetal components into the maternal systemic circulation. There are 2 main types: typical; it presents with the triad of hemodynamic collapse, respiratory distress and disseminated intravascular coagulation type coagulopathy, while atypical; disseminated intravascular coagulation does not occur. SARS CoV-2 infection causes coagulopathy due to the alteration of Virchow's triad and coagulation factors. We present the case of a 21-year-old pregnant woman who consulted for premature rupture of membranes, with an indication for cesarean section, and during surgery presented bradycardia, hypotension, and desaturation until cardiorespiratory arrest. An angiotomography showed amniotic fluid embolism associated with pulmonary edema, ruling out differential diagnoses associated with the disease, leaving as the only cause of the infection confirmed by COVID-19, which, it was inferred, was closely related to the immunological disorder suffered by the patient.

Complete genome sequences of two Bacillus thuringiensis serovar kurstaki strains isolated from Lebanon and Tunisia, highly toxic against lepidopteran larvae.

Bacillus thuringiensis-based products are key in the biopesticides market. Bacillus thuringiensis kurstaki strains Lip and BLB1 were isolated from Lebanese and Tunisian soils, respectively. These strains are highly toxic against lepidopteran larvae, Ephestia kuehniella. Here, we report Lip and BLB1 complete genomes, including their plasmid and toxin contents.

Production of recombinant scorpion antivenoms in E. coli: current state and perspectives.

Scorpion envenomation is a serious health problem in tropical and subtropical zones. The access to scorpion antivenom is sometimes limited in availability and specificity. The classical production process is cumbersome, from the hyper-immunization of the horses to the IgG digestion and purification of the F(ab)'2 antibody fragments. The production of recombinant antibody fragments in Escherichia coli is a popular trend due to the ability of this microbial host to produce correctly folded proteins. Small recombinant antibody fragments, such as single-chain variable fragments (scFv) and nanobodies (VHH), have been constructed to recognize and neutralize the neurotoxins responsible for the envenomation symptoms in humans. They are the focus of interest of the most recent studies and are proposed as potentially new generation of pharmaceuticals for their use in immunotherapy against scorpion stings of the Buthidae family. This literature review comprises the current status on the scorpion antivenom market and the analyses of cross-reactivity of commercial scorpion anti-serum against non-specific scorpion venoms. Recent studies on the production of new recombinant scFv and nanobodies will be presented, with a focus on the Androctonus and Centruroides scorpion species. Protein engineering-based technology could be the key to obtaining the next generation of therapeutics capable of neutralizing and cross-reacting against several types of scorpion venoms. KEY POINTS: • Commercial antivenoms consist of predominantly purified equine F(ab)'2fragments. • Nanobody-based antivenom can neutralize Androctonus venoms and have a low immunogenicity. • Affinity maturation and directed evolution are used to obtain potent scFv families against Centruroides scorpions.

An updated examination of the perception of barriers for pharmacogenomics implementation and the usefulness of drug/gene pairs in Latin America and the Caribbean.

Pharmacogenomics (PGx) is considered an emergent field in developing countries. Research on PGx in the Latin American and the Caribbean (LAC) region remains scarce, with limited information in some populations. Thus, extrapolations are complicated, especially in mixed populations. In this paper, we reviewed and analyzed pharmacogenomic knowledge among the LAC scientific and clinical community and examined barriers to clinical application. We performed a search for publications and clinical trials in the field worldwide and evaluated the contribution of LAC. Next, we conducted a regional structured survey that evaluated a list of 14 potential barriers to the clinical implementation of biomarkers based on their importance. In addition, a paired list of 54 genes/drugs was analyzed to determine an association between biomarkers and response to genomic medicine. This survey was compared to a previous survey performed in 2014 to assess progress in the region. The search results indicated that Latin American and Caribbean countries have contributed 3.44% of the total publications and 2.45% of the PGx-related clinical trials worldwide thus far. A total of 106 professionals from 17 countries answered the survey. Six major groups of barriers were identified. Despite the region's continuous efforts in the last decade, the primary barrier to PGx implementation in LAC remains the same, the "need for guidelines, processes, and protocols for the clinical application of pharmacogenetics/pharmacogenomics". Cost-effectiveness issues are considered critical factors in the region. Items related to the reluctance of clinicians are currently less relevant. Based on the survey results, the highest ranked (96%-99%) gene/drug pairs perceived as important were CYP2D6/tamoxifen, CYP3A5/tacrolimus, CYP2D6/opioids, DPYD/fluoropyrimidines, TMPT/thiopurines, CYP2D6/tricyclic antidepressants, CYP2C19/tricyclic antidepressants, NUDT15/thiopurines, CYP2B6/efavirenz, and CYP2C19/clopidogrel. In conclusion, although the global contribution of LAC countries remains low in the PGx field, a relevant improvement has been observed in the region. The perception of the usefulness of PGx tests in biomedical community has drastically changed, raising awareness among physicians, which suggests a promising future in the clinical applications of PGx in LAC.

Modelling the effects of assimilable nitrogen addition on fermentation in oenological conditions.

Alcoholic fermentation in oenological conditions is a biological process carried out under significant physiological constraints: deficiency of nitrogen and other nutriments (vitamins, lipids …) and different stresses (pH and osmotic). In literature, few models have been proposed to describe oenological fermentations. They focused on the initial conditions and did not integrate nitrogen addition during the fermentation process which is a widespread practice. In this work, two dynamic models of oenological fermentation are proposed to predict the effects of nitrogen addition at two different timings: at the beginning of the process and during the fermentation experiment. They were validated and compared against existing models showing an accurate fit to experimental data for CO2 release and CO2 production rate.

Biochemical limitations of Bacillus thuringiensis based biopesticides production in a wheat bran culture medium.

Bacillus thuringiensis, a gram-positive sporulating bacteria found in the environment, produces, during its sporulation phase, crystals responsible for its insecticidal activity, constituted of an assembly of pore-forming δ-endotoxins. This has led to its use as a biopesticide, an eco-friendly alternative to harmful chemical pesticides. To minimize production cost, one endemic Bacillus thuringiensis sv. kurstaki (Btk) strain Lip, isolated from Lebanese soil, was cultivated in a wheat bran (WB) based medium (IPM-4-Citrus project EC n° 734921). With the aim of studying the biochemical limitations of Btk biopesticide production in a wheat bran based medium, the WB was sieved into different granulometries, heat treated, inoculated with Btk Lip at flask scale, then filtered and separated into an insoluble and a permeate fractions. Several biochemical analyses, ie. bio performances, starch, elemental composition, total nitrogen and ashes, were then conducted on both fractions before and after culture. On a morphological level, two populations were distinguished, the fine starch granules and the coarse lignocellulosic particles. The biochemical analyses showed that both the raw and sieved WB have a similar proteins content (0.115 g/gdm WB), water content (0.116 g/gdm WB) and elemental composition (carbon: 45%, oxygen: 37%, nitrogen: 3%, hydrogen: 6%, ashes: 5%). The starch content was 17%, 14% and 34% and the fermentable fraction was estimated to 32.1%, 36.1% and 51.1% respectively for classes 2, 3 and 4. Both the elemental composition and Kjeldahl analyses showed that the nitrogen is the limiting nutrient of the culture.

Effects of Physical Exercise on the Incidence of Delirium and Cognitive Function in Acutely Hospitalized Older Adults: A Systematic Review with Meta-Analysis.

BACKGROUND: Acute care hospitalization increases the likelihood of developing cognitive impairment and delirium in older adults. OBJECTIVE: To summarize evidence about the effectiveness of exercise and physical rehabilitation interventions on the incidence of delirium and cognitive impairment in acutely hospitalized older patients. METHODS: Relevant articles were systematically searched (PubMed, Web of Science, and CINHAL databases) until 26 August 2021. Randomized and nonrandomized controlled trials of in-hospital physical exercise interventions and rehabilitation programs compared to usual care performed for older patients (> 65 years) hospitalized for an acute medical condition were selected. The primary endpoints were changes in the incidence of delirium and cognition during acute hospitalization. The secondary endpoints included functional independence, psychological measures, well-being status, length of hospital stay, transfer after discharge, fall occurrence, hospital readmissions, and mortality rate. The endpoints were evaluated at different time points (at admission, at discharge, and after discharge). RESULTS: Eleven studies from 8 trials (n = 3,646) were included. The methodological quality of the studies was mostly high. None of the studies reported any adverse events related to the intervention. Early rehabilitation improved cognitive function at 3 months postdischarge (Hedge's g = 0.33, 95% confidence interval [CI] 0.19 to 0.46, p < 0.001). No between-group differences were found for incident delirium and cognitive impairment during hospitalization (all p > 0.05). CONCLUSION: In-hospital physical exercise and early rehabilitation programs seem to be safe and effective interventions for enhancing cognitive function after discharge in older patients hospitalized for an acute medical condition. However, no potential benefits were obtained over usual hospital care for the incidence of delirium.

Reply to Inzitari et al. Comment on "Blancafort Alias et al. A Multi-Domain Group-Based Intervention to Promote Physical Activity, Healthy Nutrition, and Psychological Wellbeing in Older People with Losses in Intrinsic Capacity: AMICOPE Development Study. Int. J. Environ. Res. Public Health 2021, 18, 5979".

This is a reply to the comment by Inzitari et al [...].

Effects of game-based interventions on functional capacity in acutely hospitalised older adults: results of an open-label non-randomised clinical trial.

BACKGROUND: Hospitalisation-associated disability due to reduced physical activity levels and prolonged bedrest episodes are highly prevalent in older adults. OBJECTIVE: To assess the effect of gamified interventions on functional capacity in hospitalised older adults. METHODS: A three-armed non-randomised controlled trial with two experimental intervention groups and a control group was conducted in a tertiary public hospital in Navarre, Spain. Participants were allocated to a simple gamification group (SGG) (n = 21), a technology-based gamification group (TGG) (n = 23) or a control group (CG) (n = 26). The end points were changes in functional capacity, muscle strength, cognition, mood status and quality of life. RESULTS: Seventy patients (mean age 86.01 ± 4.27 years old) were included in the study; 29 (41.4%) were women. At discharge, compared to CG, a mean increase of 1.47 points (95%CI, 0.15-2.80 points) and 2.69 points (95%CI, 1.32-4.06 points) was observed (SGG and TGG, respectively) in the SPPB test; as well as an increase of 5.28 points (95%CI, 0.70-9.76 points) in the Barthel Index and 2.03 kg (95%CI, 0.33-3.72 kg) in handgrip strength in the TGG. Regression mediation analyses demonstrated that muscle strength changes (ß = 1.30; 95%CI, 0.45-2.14; indirect effect 0.864; 95%CI, 0.09-1.90) significantly mediated the TGG effect on the SPPB score. CONCLUSIONS: The TGG intervention programme may provide significant benefits in physical and muscle function over usual care and seems to reverse the functional decline frequently associated with acute hospitalisation in older adults.

Modulation of GABAA receptors and of GABAergic synapses by the natural alkaloid gelsemine.

The Gelsemium elegans plant preparations have shown beneficial activity against common diseases, including chronic pain and anxiety. Nevertheless, their clinical uses are limited by their toxicity. Gelsemine, one of the most abundant alkaloids in the Gelsemium plants, have replicated these therapeutic and toxic actions in experimental behavioral models. However, the molecular targets underlying these biological effects remain unclear. The behavioral activity profile of gelsemine suggests the involvement of GABAA receptors (GABAARs), which are the main biological targets of benzodiazepines (BDZs), a group of drugs with anxiolytic, hypnotic, and analgesic properties. Here, we aim to define the modulation of GABAARs by gelsemine, with a special focus on the subtypes involved in the BDZ actions. The gelsemine actions were determined by electrophysiological recordings of recombinant GABAARs expressed in HEK293 cells, and of native receptors in cortical neurons. Gelsemine inhibited the agonist-evoked currents of recombinant and native receptors. The functional inhibition was not associated with the BDZ binding site. We determined in addition that gelsemine diminished the frequency of GABAergic synaptic events, likely through a presynaptic modulation. Our findings establish gelsemine as a negative modulator of GABAARs and of GABAergic synaptic function. These pharmacological features discard direct anxiolytic or analgesic actions of gelsemine through GABAARs but support a role of GABAARs on the alkaloid induced toxicity. On the other hand, the presynaptic effects of the alkaloid provide an additional mechanism to explain their beneficial effects. Collectively, our results contribute novel information to improve understanding of gelsemine actions in the mammalian nervous system.

A Multi-Domain Group-Based Intervention to Promote Physical Activity, Healthy Nutrition, and Psychological Wellbeing in Older People with Losses in Intrinsic Capacity: AMICOPE Development Study.

The World Health Organization has developed the Integrated Care of Older People (ICOPE) strategy, a program based on the measurement of intrinsic capacity (IC) as "the composite of all physical and mental attributes on which an individual can draw". Multicomponent interventions appear to be the most effective approach to enhance IC and to prevent frailty and disability since adapted physical activity is the preventive intervention that has shown the most evidence in the treatment of frailty and risk of falls. Our paper describes the development of a multi-domain group-based intervention addressed to older people living in the community, aimed at improving and/or maintaining intrinsic capacity by means of promoting physical activity, healthy nutrition, and psychological wellbeing in older people. The process of intervention development is described following the Guidance for reporting intervention development studies in health research (GUIDED). The result of this study is the AMICOPE intervention (Aptitude Multi-domain group-based intervention to improve and/or maintain IC in Older PEople) built upon the ICOPE framework and described following the Template for Intervention Description and Replication (TIDieR) guidelines. The intervention consists of 12 face-to-face sessions held weekly for 2.5 h over three months and facilitated by a pair of health and social care professionals. This study represents the first stage of the UK Medical Research Council framework for developing and evaluating a complex intervention. The next step should be carrying out a feasibility study for the AMICOPE intervention and, at a later stage, assessing the effectiveness in a randomized controlled trial.

A clinically viable vendor-independent and device-agnostic solution for accelerated cardiac MRI reconstruction.

BACKGROUND AND OBJECTIVE: Recent research has reported methods that reconstruct cardiac MR images acquired with acceleration factors as high as 15 in Cartesian coordinates. However, the computational cost of these techniques is quite high, taking about 40 min of CPU time in a typical current machine. This delay between acquisition and final result can completely rule out the use of MRI in clinical environments in favor of other techniques, such as CT. In spite of this, reconstruction methods reported elsewhere can be parallelized to a high degree, a fact that makes them suitable for GPU-type computing devices. This paper contributes a vendor-independent, device-agnostic implementation of such a method to reconstruct 2D motion-compensated, compressed-sensing MRI sequences in clinically viable times. METHODS: By leveraging our OpenCLIPER framework, the proposed system works in any computing device (CPU, GPU, DSP, FPGA, etc.), as long as an OpenCL implementation is available, and development is significantly simplified versus a pure OpenCL implementation. In OpenCLIPER, the problem is partitioned in independent black boxes which may be connected as needed, while device initialization and maintenance is handled automatically. Parallel implementations of both a groupwise FFD-based registration method, as well as a multicoil extension of the NESTA algorithm have been carried out as processes of OpenCLIPER. Our platform also includes significant development and debugging aids. HIP code and precompiled libraries can be integrated seamlessly as well since OpenCLIPER makes data objects shareable between OpenCL and HIP. This also opens an opportunity to include CUDA source code (via HIP) in prospective developments. RESULTS: The proposed solution can reconstruct a whole 12-14 slice CINE volume acquired in 19-32 coils and 20 phases, with an acceleration factor of ranging 4-8, in a few seconds, with results comparable to another popular platform (BART). If motion compensation is included, reconstruction time is in the order of one minute. CONCLUSIONS: We have obtained clinically-viable times in GPUs from different vendors, with delays in some platforms that do not have correspondence with its price in the market. We also contribute a parallel groupwise registration subsystem for motion estimation/compensation and a parallel multicoil NESTA subsystem for l1-l2-norm problem solving.

Effect of temperature on the production of a recombinant antivenom in fed-batch mode.

In the pharmaceutical industry, nanobodies show promising properties for its application in serotherapy targeting the highly diffusible scorpion toxins. The production of recombinant nanobodies in Escherichia coli has been widely studied in shake flask cultures in rich medium. However, there are no upstream bioprocess studies of nanobody production in defined minimal medium and the effect of the induction temperature on the production kinetics. In this work, the effect of the temperature during the expression of the chimeric bispecific nanobody CH10-12 form, showing high scorpion antivenom potential, was studied in bioreactor cultures of E. coli. High biomass concentrations (25 g cdw/L) were achieved in fed-batch mode, and the expression of the CH10-12 nanobody was induced at temperatures 28, 29, 30, 33, and 37°C with a constant glucose feed. For the bispecific form NbF12-10, the induction was performed at 29°C. Biomass and carbon dioxide yields were reported for each culture phase, and the maintenance coefficient was obtained for each strain. Nanobody production in the CH10-12 strain was higher at low temperatures (lower than 30°C) and declined with the increase of the temperature. At 29°C, the CH10-12, NbF12-10, and WK6 strains were compared. Strains CH10-12 and NbF12-10 had a productivity of 0.052 and 0.021 mg/L/h of nanobody, respectively, after 13 h of induction. The specific productivity of the nanobodies was modeled as a function of the induction temperature and the specific growth rates. Experimental results confirm that low temperatures increase the productivity of the nanobody.Key points• Nanobodies with scorpion antivenom activity produced using two recombinant strains.• Nanobodies production was achieved in fed-batch cultures at different induction temperatures.• Low induction temperatures result in high volumetric productivities of the nanobody CH10-12.

Impact of Game-Based Interventions on Health-Related Outcomes in Hospitalized Older Patients: A Systematic Review.

OBJECTIVES: To examine the effectiveness of game-based interventions compared with usual care on health-related outcomes for acutely hospitalized older patients. DESIGN: Systematic review of randomized controlled trials (RCT) and nonrandomized trials. SETTING AND PARTICIPANTS: Adults aged 65 years or older admitted to an Acute Care for Elderly unit were selected. MEASURES: Health-related outcomes (eg, functional capacity, quality of life, adherence to treatment). RESULTS: Four RCTs were included in the review. The interventions were based on the implementation of serious-game programs using Nintendo Wii in acute medical patients. Across the included studies, no significant differences were observed between groups on functional capacity and health-related quality of life. Significant differences were found between groups on the adherence to treatment (in favor of the control group), but no differences were obtained in other outcomes such as enjoyment and motivation. CONCLUSIONS AND IMPLICATIONS: In general, there is very limited evidence for the efficacy to reach conclusions about the effects of game-based interventions on health-related outcomes in acutely hospitalized older patients. Future studies are needed to improve our knowledge in the field; however, we consider that these strategies should be considered in the future complementary to usual care.

A Feasibility Study for Implementation "Health Arcade": A Study Protocol for Prototype of Multidomain Intervention Based on Gamification Technologies in Acutely Hospitalized Older Patients.

The aim of this article is to present the research protocol for a study that will evaluate the feasibility of implementation of Health Arcade prototype multidomain intervention based on physical and cognitive training using gamification technologies at improving care for older people hospitalized with an acute illness. A total of 40 older people will be recruited in a tertiary public hospital at Pamplona, Spain. The intervention duration will be four to nine consecutive days. Additionally, the patients will receive encouragement for maintaining active during hospital stay and for reducing sedentary time. Primary implementation-related outcomes will be the adherence to treatment (i.e., number of games and days completed during the intervention period), reaction or response time, and number of success and failures in each game per day. Secondary implementation-related outcomes will be self-perceived grade of difficulty, satisfaction, enjoyment per game and session, and self-perceived difficulties in handling the prototype hardware. Other health-related outcomes will also be assessed such as functional capacity in activities of daily living, mood status, quality of life, handgrip strength, physical activity levels, and mobility. The current study will provide additional evidence to support the implementation of multidomain interventions designed to target older persons with an acute illness based on friendly technology. The proposed intervention will increase accessibility of in-clinical geriatrics services, improve function, promote recovery of the health, and reduce economic costs.

Inhibition of the Glycine Receptor alpha 3 Function by Colchicine.

Colchicine is a plant alkaloid that is widely used as a therapeutic agent. It is widely accepted that colchicine reduces the production of inflammatory mediators mainly by altering cytoskeleton dynamics due to its microtubule polymerization inhibitory activity. However, other lines of evidence have shown that colchicine exerts direct actions on the function of ion channels, which are independent of cytoskeleton alterations. Colchicine is able to modify the function of several pentameric ligand-gated ion channels, including glycine receptors (GlyRs). Previous electrophysiological studies have shown that colchicine act as an antagonist of GlyRs composed by the α 1 subunit. In addition, it was recently demonstrated that colchicine directly bind to the α 3 subunit of GlyRs. Interestingly, other studies have shown a main role of α 3GlyRs on chronic inflammatory pain. Nevertheless, the functional effects of colchicine on the α 3GlyR function are still unknown. Here, by using electrophysiological techniques and bioinformatics, we show that colchicine inhibited the function of the α 3GlyRs. Colchicine elicited concentration-dependent inhibitory effects on α 3GlyRs at micromolar range and decreased the apparent affinity for glycine. Single-channel recordings show that the colchicine inhibition is associated with a decrease in the open probability of the ion channel. Molecular docking assays suggest that colchicine preferentially bind to the orthosteric site in the closed state of the ion channel. Altogether, our results suggest that colchicine is a competitive antagonist of the α 3GlyRs.

Clinical Effectiveness of Bulk-Fill and Conventional Resin Composite Restorations: Systematic Review and Meta-Analysis.

The objective of this systematic review and meta-analysis was to determine the clinical effectiveness of bulk-fill and conventional resin in composite restorations. A bibliographic search was carried out until May 2020, in the biomedical databases Pubmed/MEDLINE, EMBASE, Scopus, CENTRAL and Web of Science. The study selection criteria were: randomized clinical trials, in English, with no time limit, with a follow-up greater than or equal to 6 months and that reported the clinical effects (absence of fractures, absence of discoloration or marginal staining, adequate adaptation marginal, absence of post-operative sensitivity, absence of secondary caries, adequate color stability and translucency, proper surface texture, proper anatomical form, adequate tooth integrity without wear, adequate restoration integrity, proper occlusion, absence of inflammation and adequate point of contact) of restorations made with conventional and bulk resins. The risk of bias of the study was analyzed using the Cochrane Manual of Systematic Reviews of Interventions. Sixteen articles were eligible and included in the study. The results indicated that there is no difference between restorations with conventional and bulk resins for the type of restoration, type of tooth restored and restoration technique used. However, further properly designed clinical studies are required in order to reach a better conclusion.

A protocol for recombinant protein quantification by densitometry.

The protein purity is generally checked using SDS-PAGE, where densitometry could be used to quantify the protein bands. In literature, few studies have been reported using image analysis for the quantification of protein in SDS-PAGE: that is, imaged with Stain-Free™ technology. This study presents a protocol of image analysis for electrophoresis gels that allows the quantification of unknown proteins using the molecular weight markers as protein standards. Escherichia coli WK6/pHEN6 encoding the bispecific nanobody CH10-12 engineered by the Pasteur Institute of Tunisia was cultured in a bioreactor and induced with isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 28°C for 12 hr. Periplasmic proteins extracted by osmotic shock were purified by immobilized metal affinity chromatography (IMAC). Images of the SDS-PAGE gels were analyzed using ImageJ, and the lane profiles were obtained in grayscale and uncalibrated optical density. Protein load and peak area were linearly correlated, and optimal image processing was then performed by background subtraction using the rolling ball algorithm with radius size 250 pixels. No brightness and contrast adjustment was applied. The production of the nanobody CH10-12 was obtained through a fed-batch strategy and quantified using the band of 50 kDa in the marker as reference for 750 ng of recombinant protein. The molecular weight marker was used as a sole protein standard for protein quantification in SDS-PAGE gel images.