Simultaneous monitoring of amoxicillin and paracetamol in synthetic biological fluids using a 3D printed disposable electrode with a lab-made conductive filament.

In this work, we are pleased to present for the first time a 3D-printed electrochemical device using a lab-made conductive filament based on graphite (Gr) and polylactic acid (PLA) polymer matrix for the simultaneous detection of amoxicillin (AMX) and paracetamol (PAR). The sensor was properly characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). Compared to the commercial glassy carbon electrode (GCE), the superior performance of the 3D-Gr/PLA electrode was verified with a 3.8-fold more favored charge transfer. A differential pulse voltammetry (DPV) method was proposed providing a linear working range of 4 to 12 µmol L-1 for both analytes and a limit of detection (LOD) of 0.80 and 0.51 µmol L-1 for AMX and PAR, respectively. Additionally, repeatability studies (n = 5, RSD < 5.7%) indicated excellent precision, and recovery percentages ranging from 89 to 109% when applied to synthetic human urine, saliva, and plasma samples, attested to the accuracy of the method. The studies also indicate that the sensor does not suffer significant interference from common substances (antibiotics and biomarkers) present in the biological fluids, which makes it a promising analytical tool considering its low-cost, ease of manufacturing, robustness, and electrochemical performance.

Study of the DNA damage and cell death in human peripheral blood mononuclear and HepG2/C3A cells exposed to the synthetic 3-(3-hydroxyphenyl)-7-hydroxycoumarin.

Hydroxycoumarins are an important source of biologically active compounds. Previous studies have shown that the number and position of the hydroxyl substituents in the scaffold play an important role for the observed biological activity. In the present study, 3-(3-hydroxyphenyl)-7-hydroxycoumarin was synthesized, and potential cytogenotoxic effects determined in human HepG2/C3A cells displaying phase 1 and phase 2 enzymes (metabolizing cell ability) and compared to human peripheral blood mononuclear cells (PBMC) without xenobiotics metabolizing capacity. Cell viability was determined with concentrations between 0.01 and 10 µg/ml of 3-(3-hydroxyphenyl)-7-hydroxycoumarin using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and trypan blue tests. Genotoxicity was determined utilizing the comet assay, and the clastogenic/aneugenic potential employing the micronucleus (MN) test. The results of the in vitro cytotoxicity assays showed a significant decrease in cell viability of PBMC following exposure to 10 µg/ml concentration of the studied compound after 48 and 72 hr. Comet assay observations noted significant DNA damage in PBMC after 4 hr treatment. No marked cytogenotoxic effects were found in HepG2/C3A cells. No chromosomal mutations were observed in both cell lines. It is important to note that 3-(3-hydroxyphenyl)-7-hydroxycoumarin may exert beneficial pharmacological actions at the low micromolar range and with half-life less than 24 hr. Therefore, the results obtained encourage the continuation of studies on this new molecule for medicinal purposes, but its potential toxicity at higher concentrations and longer exposure times needs to be investigated in further studies.

Communication and swallowing training of stroke-specialized health professionals using transdisciplinary knowledge in a patient-actor scenario: A case report.

BACKGROUND: Most people with stroke exhibit a variety of impairments that need to be addressed by a multidisciplinary team. Communication and swallowing disorders are common and should be screened very early. To guarantee a patient-centred approach, all patients, even those with speech and language disorders, must be actively engaged in the healthcare process. Effective communication is essential to success in many of the needed interventions. However, healthcare professionals often do not receive formal training in communicating with these patients, thus increasing the risk of preventable adverse events. AIMS: To describe the design, implementation and evaluation of a post-acute stroke multidisciplinary team training using patient actors in a simulation approach. METHODS & PROCEDURES: A 2-day course focused on the transdisciplinary knowledge related to communication and swallowing that all members of the multidisciplinary stroke team should acquire was implemented. A case-based learning methodology used simulation and resorting to patients' actors. Learning outcomes were evaluated by comparing the results obtained in two knowledge tests, one for each topic, which participants performed before and after each day course. Reaction to the training was gathered concerning the content, teacher quality and course organization. The follow-up was performed 6 months later to assess training skills transfer to the workplace environment. OUTCOMES & RESULTS: All the participants considered that the programme objectives were relevant or truly relevant and revealed that the programme's dynamic, rhythm and scenarios set were excellent. After the end of the programme, both communication and swallowing knowledge increased. Most participants had the opportunity to employ the acquired training skills in their work environment. The main barriers identified to implementing these skills were the 'need for additional training', the 'lack of time' or 'the lack of opportunities'. CONCLUSIONS & IMPLICATIONS: Simulation is a central method to increase and improve health professionals' skills when intervening with stroke patients. Using simulation with patient actors allows flexibility and diversification of clinical situations under analysis, which can provide a multiplier effect of reflection and learning. The implemented training achieved its objectives. WHAT THIS PAPER ADDS: What is already known on this subject Simulation in the training of health professionals is increasingly used as a good practice, allowing the recreation of scenarios identical to those in the context of professional practice. This strategy is used not only in initial training but also for the development of advanced skills. What this study adds to the existing knowledge This study reports the use of simulation using actor patients for the development of transdisciplinary skills in the topics of communication and swallowing in people with stroke What are the clinical and practical implications of this work? The study demonstrates that in a short period of training, the use of simulation with actor patients favours the development of transdisciplinary skills in the topics of communication and swallowing in people with stroke. At the same time, the skills developed are transferable to professional practice.

Environmental vulnerability applied to the territorial planning of a tropical semiarid basin.

Tropical semiarid regions are naturally prone to environmental damage. Human activity can worsen this situation. To understand how human actions affect the ecosystem, plan land use effectively, and establish targeted management practices, assessing environmental vulnerability is crucial. This study focuses on a sub-basin receiving water transfers from the São Francisco River in Brazil's semiarid region. Here, we map and evaluate how land use and occupation alter natural vulnerability. We also propose zoning strategies to support water resource management and implement sustainable development policies in the region. To achieve this, we conducted an integrated analysis of physical factors (soil types, geology, climate, vegetation, and landforms) and spatial land-use data using geographic information systems (GIS) and map algebra techniques. Map algebra allowed us to combine these various datasets within the GIS environment, enabling the creation of maps that synthesize both natural and environmental vulnerability across the study area. Following analysis of these vulnerability maps, our findings reveal a high level of vulnerability. The areas with high to very high degrees of natural vulnerability coincide with the places that have high slopes, high altitudes, Lithic Neosols, and thick vegetation. Furthermore, the interaction between environmental factors and human activity exacerbates vulnerability. Based on the environmental vulnerability assessment, we defined four environmental management zones. These zones require distinct protection measures and management approaches. As a method to potentially improve the basin's vulnerability scenario, soil conservation measures are recommended. This approach is highly relevant for managing land in tropical semiarid regions and, with adaptations to specific regional factors, can be applied globally.

Lipoprotein Z, a hepatotoxic lipoprotein, predicts outcome in alcohol-associated hepatitis.

BACKGROUND AND AIMS: Lipoprotein Z (LP-Z) is an abnormal free cholesterol (FC)-enriched LDL-like particle discovered from patients with cholestatic liver disease. This study aims to define the diagnostic value of LP-Z in alcohol-associated hepatitis (AH) and interrogate the biology behind its formation. APPROACH AND RESULTS: We measured serum levels of LP-Z using nuclear magnetic resonance spectroscopy, a well-established clinical assay. Serum levels of LP-Z were significantly elevated in four AH cohorts compared with control groups, including heavy drinkers and patients with cirrhosis. We defined a Z-index, calculated by the ratio of LP-Z to total apolipoprotein B-containing lipoproteins, representing the degree of deviation from normal VLDL metabolism. A high Z-index was associated with 90-day mortality independent from the Model for End-Stage Liver Disease (MELD) and provided added prognosticative value. Both a Z-index ≤ 0.6 and a decline of Z-index by ≥0.1 in 2 weeks predicted 90-day survival. RNA-sequencing analyses of liver tissues demonstrated an inverse association in the expression of enzymes responsible for the extrahepatic conversion of VLDL to LDL and AH disease severity, which was further confirmed by the measurement of serum enzyme activity. To evaluate whether the FC in LP-Z could contribute to the pathogenesis of AH, we found significantly altered FC levels in liver explant of patients with AH. Furthermore, FC in reconstituted LP-Z particles caused direct toxicity to human hepatocytes in a concentration-dependent manner, supporting a pathogenic role of FC in LP-Z. CONCLUSIONS: Impaired lipoprotein metabolism in AH leads to the accumulation of LP-Z in the circulation, which is hepatotoxic from excessive FC. A Z-index ≤ 0.6 predicts 90-day survival independent from conventional biomarkers for disease prognostication.

Oxidation-labile linkers for controlled drug delivery.

The continuous symbiosis throughout chemical biology and drug discovery has led to the design of innovative bifunctional molecules for targeted and controlled drug delivery. Among the different tools, protein-drug and peptide-drug conjugates are trend approaches to achieve targeted delivery, selectivity and efficacy. To meet the main goals of these bioconjugates, the selection of the appropriate payloads and linkers is crucial, as they must provide in vivo stability, while they may also help to achieve the therapeutic target and action. In neurodegenerative diseases or some cancer types, where oxidative stress plays an important role, linkers sensitive to oxidative conditions may be able to release the drug once the conjugate achieves the target. Considering specially this specific application, this mini-review covers the most relevant publications on oxidation-labile linkers.

Development of a 3D disposable device for the electrochemical determination of diclofenac in different matrices.

In this work, the development of a disposable electrochemical device (US$ 0.02 per electrode) using a 3D printed support (3Ds) of acrylonitrile butadiene styrene (ABS) insulating filament with a composite material (CM) based on graphite and nail polish, immobilized on the support surface, was described for the electrochemical determination of diclofenac (DCF). The device was compared to the commercial glassy carbon electrode (GCE) and showed superior electroanalytical performance with approximately 1.8-fold higher current density. Additionally, an amperometric method for DCF determination in tap water, synthetic urine, and pharmaceutical formulation samples with the proposed electrode, using a flow injection analysis (FIA-AD) system, was developed. The optimized method presented excellent detectability (LOD = 0.47 µmol L-1), with excellent precision and accuracy (relative standard deviation < 5.6%) and percent recovery from spiked samples ranging from 89 to 106%. In addition, the sensor showed optimal analytical frequency with approximately 108 injections per hour, which demonstrates the potential of this system using the proposed disposable electrode for implementation in routine analysis and quality control with good selectivity and sensitivity.

Evaluation of in vitro cytotoxic and genotoxic effects of the 3-(3,4-dihydroxyphenyl)-8-hydroxycoumarin.

A wide variety of natural and synthetic coumarins present therapeutic potential. Therefore, the assessment of their safety for humans is essential. 3-(3,4-Dihydroxyphenyl)-8-hydroxycoumarin is a coumarin derivative with antioxidant properties, among other biological activities. The aim of this study is to evaluate the cytotoxic and genotoxic potential of this molecule on peripheral blood mononuclear cells (PBMC) and human hepatocellular carcinoma cells (HepG2/C3A). The results obtained for the cytotoxicity assays, evaluated by the trypan blue staining assay, using concentrations between 0.1 and 20 µg/mL, showed that there is no decrease in cell viability for both cell lines. The MTT assay showed a significant decrease in the viability of HepG2/C3A cells at the highest concentrations tested, after 48 h, for all the tested concentrations, after 72 h of exposure. Regarding the genotoxic assays, the data obtained by the comet assay and the micronucleus test, up to the tested concentration of 10 µg/mL, do not show significant DNA damage and/or chromosomal mutations, for both cell lines. However, at the highest tested concentration of 20 µg/mL, a small but significant genotoxic effect was observed in PBMC. In view of the observed results, it can be concluded that the 3-(3,4-dihydroxyphenyl)-8-hydroxycoumarin, up to a concentration of 10 µg/mL, does not present genotoxic effects in human cells with and without liver enzymes metabolism. Additional studies with higher concentrations of this molecule need to be performed to address its complete biosafety.

Cytogenotoxicity assessment of 3-(3,4-dihydroxyphenyl)-7,8-dihydroxycoumarin on HepG2/C3A cells and leukocytes.

3-(3,4-Dihydroxyphenyl)-7,8-dihydroxycoumarin is a newly synthesized coumarin derivative with a potent antioxidant effect. The aim of the present study is to investigate the safety of this compound, determining the in vitro cytotoxic and genotoxic in human peripheral blood mononuclear cells (PBMC) and in HepG2/C3A cells. Cell viability has been investigated by the trypan blue staining test and MTT assay and the genotoxicity by the comet assay and micronucleus test, using concentrations between 0.01 and 10 µg/ml. The compound proved to be noncytotoxic in both cell lines, at all tested concentrations, protecting the cells from the DNA damage. In addition, this molecule does not show clastogenic/aneugenic effects when performing the micronucleus test with cytokinesis blockade. Based on the obtained data, and the conditions of the experiments, we can conclude that the 3-(3,4-dihydroxyphenyl)-7,8-dihydroxycoumarin is a safe molecule up to a concentration of 10 µg/ml, which encourages further studies aiming to explore its potential as a drug candidate.

Effect of Lactobacillus spp. cell-free supernatant against planktonic growth and biofilm formation of foodborne Escherichia coli isolates.

The aim of this work was to evaluate the anti-Escherichia coli effect of cell-free supernatant (CFS) of Lactobacillus spp. against planktonic and biofilm forms of foodborne isolates. Escherichiacoli strains (P12, P25, P35 and P36), previously isolated from fresh filets of fish, were subjected to antimicrobial susceptibility determination by the disc-diffusion agar method. Subsequently, the antagonistic effect between probiotic and pathogenic strains was determined by spot overlay assay. Finally, the CFS activity against pre-established (12 h) biofilms was demonstrated through biomass quantification by crystal violet staining and scanning electron microscopy (SEM). All isolates presented some pattern of resistance, primarily to ampicillin and tetracycline. Probiotic strains presented high antagonistic effects against all E. coli strains, presenting inhibition zones (R) ranging from 15.60 to 20.67 mm. Additionally, the residual biomass of pre-established (12 h) biofilm was drastically reduced about 50% after CFS treatment (P < 0.01). What can be noted by SEM images, which show less surface-attached cells of CFS-treated biofilms of E. coli (P12). Thus, cell-free preparations produced from Lactobacillus spp. may represent a tool in the battle against planktonic cells and biofilm forms of antibiotic-resistant E. coli.

When body dysmorphia symptomatology meets disordered eating: The role of shame and self-criticism.

Body Dysmorphic Disorder (BDD) is a serious psychiatric condition that has long been identified as an important risk factor for the development of eating-related difficulties. However, little is known about the mechanisms that might explain this association. Therefore, the current study aimed to explore the link between body dysmorphic symptomatology and disordered eating, and test whether this relationship is mediated by higher levels of shame and self-criticism. This cross-sectional study included 291 women from the community, aged between 18 and 62 years old, who completed self-report measures. Path analysis revealed that BDD symptomatology has not only a direct effect on disordered eating, but also an indirect effect, mediated by shame and self-criticism. The path model revealed a very good fit, accounting for 38% and 31% of internal and external shames' variances, respectively, for 69% of self-criticism variance, and 58% of the variance of disordered eating. These findings seem to suggest that in women with BDD symptomatology, disordered eating may emerge as a compensatory strategy to cope with general feelings of inferiority/defectiveness, particularly in the presence of shame experiences and self-critical attitudes/behaviours. Moreover, this study emphasizes the importance to invest in innovative treatment and prevention approaches for BDD that specifically target shame and self-criticism, such as compassion-based therapies. LEVEL OF EVIDENCE: IV, cross-sectional study.

Rapid and sensitive detection of E. coli O157:H7 by lateral flow immunoassay and silver enhancement.

The aim of this study was to develop a sensitive lateral flow immunoassay (LFIA) for the rapid detection of Escherichia coli (E. coli) O157:H7, a pathogen contributor to diseases and fatalities worldwide. Au nanoparticles with high stability, uniform size, and shape were synthesized and coated with heterobifunctional PEG polymer with carboxyl groups, and they were bioconjugated to be used as label in sandwich-LFIA. Then, a silver enhancement strategy was developed as an accessible, rapid, and cost-effective approach for signal amplification to reduce the limit of detection (LOD). The optimal results were achieved when a solution of silver nitrate and hydroquinone/citrate buffer was added to the strips for 4 min. This led to a decrease in the visual LOD from 2 × 106 (CFU mL-1) to 2 × 103 (CFU mL-1), resulting in a threefold improvement in sensitivity compared to the conventional LFIA system. The specificity of the system was evaluated by using non-target bacteria (E. coli BL21 and E. coli T515) and its reliability was determined by testing commercial food samples (milk, tap water, and orange juice), demonstrating its effectiveness for quickly detecting pathogenic bacteria in food products.

Cost-effective protocol to produce 3D-printed electrochemical devices using a 3D pen and lab-made filaments to ciprofloxacin sensing.

A novel conductive filament based on graphite (Gr) dispersed in polylactic acid polymer matrix (PLA) is described to produce 3D-electrochemical devices (Gr/PLA). This conductive filament was used to additively manufacture electrochemical sensors using the 3D pen. Thermogravimetric analysis confirmed that Gr was successfully incorporated into PLA, achieving a composite material (40:60% w/w, Gr and PLA, respectively), while Raman and scanning electron microscopy revealed the presence of defects and a high porosity on the electrode surface, which contributes to improved electrochemical performance. The 3D-printed Gr/PLA electrode provided a more favorable charge transfer (335 Ω) than the conventional glassy carbon (1277 Ω) and 3D-printed Proto-pasta® (3750 Ω) electrodes. As a proof of concept, the ciprofloxacin antibiotic, a species of multiple interest, was selected as a model molecule. Thus, a square wave voltammetry (SWV) method was proposed in the potential range + 0.9 to + 1.3 V (vs Ag|AgCl|KCl(sat)), which provided a wide linear working range (2 to 32 µmol L-1), 1.79 µmol L-1 limit of detection (LOD), suitable precision (RSD < 7.9%), and recovery values from 94 to 109% when applied to pharmaceutical and milk samples. Additionally, the sensor is free from the interference of other antibiotics routinely employed in veterinary practices. This device is disposable, cost-effective, feasibly produced in financially limited laboratories, and consequently promising for evaluation of other antibiotic species in routine applications.

Recommendations of good practice to prevent aspiration pneumonia in older adults at risk of oropharyngeal dysphagia living in nursing homes: A modified e-Delphi study protocol.

BACKGROUND: Aspiration pneumonia (AP) is a subset of pneumonia caused by the aspiration of food and fluids to the lungs and is highly prevalent in the older population. Oropharyngeal dysphagia (OD) is one of the risk factors for AP and it is also associated with malnutrition, dehydration and poor functional outcomes. As pneumonia is the second most common infection in nursing homes (NHs) and OD represents a major concern to NH staff, good practices for the prevention of AP in older adults at risk of OD are needed. PURPOSE: The aim of this modified e-Delphi study is to build consensus among a panel of experts regarding a set of recommendations for NH staff on good practices to prevent AP in older adults at risk of OD living in NHs. The objective of this paper is to establish the methodology inherent to the Delphi study. METHODS: An online modified Delphi study will be developed in three rounds. Criteria for the Delphi panel participants include holding a master's or doctoral degree in OD or speech and language therapy; or having 10 or more years of experience in OD; or having at least one scientific publication related to OD. A previously described modified Delphi methodology will be used to achieve consensus (75% agreement). An additional round will be performed to collect the experts' perspectives regarding the priority for application of each recommendation previously validated. DISCUSSION: This protocol aimed to describe the methodology of a future Delphi study on the prevention of AP, seeking to fulfil the gap in the literature regarding this topic. The modified Delphi technique is a widely used method for collecting experts' opinion in health sciences, but the absence of standardised guidelines allows some heterogeneity between studies with the same aim. WHAT THIS PAPER ADDS: What is already known on the subject Aspiration pneumonia (AP) is related to three main risk factors: impaired safety of swallow, impaired nutritional status and poor oral health. It is known that being dependent for feeding is one of the main risk factors for AP and around 50% of nursing home (NH) residents need feeding assistance. Thus, it is important to promote specialised intervention and care by the NH staff for preventing AP. What this paper adds to existing knowledge It is hypothesised that increasing the knowledge of NH staff regarding the best practices for preventing AP in older adults at risk of oropharyngeal dysphagia (OD) will improve outcomes such as quality of life, incidence of AP and mortality. What are the potential or actual clinical implications of this work? The recommendations resulting from this study will address a current gap in healthcare practice of NH staff regarding older adults at increased risk for OD and, consequently, for AP.

Evidence for Extensive Duplication and Subfunctionalization of FCRL6 in Armadillo (Dasypus novemcinctus).

The control of infections by the vertebrate adaptive immune system requires careful modulation to optimize defense and minimize harm to the host. The Fc receptor-like (FCRL) genes encode immunoregulatory molecules homologous to the receptors for the Fc portion of immunoglobulin (FCR). To date, nine different genes (FCRL1-6, FCRLA, FCRLB and FCRLS) have been identified in mammalian organisms. FCRL6 is located at a separate chromosomal position from the FCRL1-5 locus, has conserved synteny in mammals and is situated between the SLAMF8 and DUSP23 genes. Here, we show that this three gene block underwent repeated duplication in Dasypus novemcinctus (nine-banded armadillo) resulting in six FCRL6 copies, of which five appear functional. Among 21 mammalian genomes analyzed, this expansion was unique to D. novemcinctus. Ig-like domains that derive from the five clustered FCRL6 functional gene copies show high structural conservation and sequence identity. However, the presence of multiple non-synonymous amino acid changes that would diversify individual receptor function has led to the hypothesis that FCRL6 endured subfunctionalization during evolution in D. novemcinctus. Interestingly, D. novemcinctus is noteworthy for its natural resistance to the Mycobacterium leprae pathogen that causes leprosy. Because FCRL6 is chiefly expressed by cytotoxic T and NK cells, which are important in cellular defense responses against M. leprae, we speculate that FCRL6 subfunctionalization could be relevant for the adaptation of D. novemcinctus to leprosy. These findings highlight the species-specific diversification of FCRL family members and the genetic complexity underlying evolving multigene families critical for modulating adaptive immune protection.

Quality Standard for Rehabilitation of Young Deaf Children Receiving Cochlear Implants.

Background and objectives: More and more children with severe-to-profound hearing loss are receiving cochlear implants (CIs) at an early age to improve their hearing and listening abilities, speech recognition, speech intelligibility, and other aspects of spoken language development. Despite this, the rehabilitation outcomes can be very heterogeneous in this population, not only because of issues related to surgery and fitting or the specific characteristics of the child with his/her additional disabilities but also because of huge differences in the quality of the support and rehabilitation offered by the therapist and the family. These quality standards for the rehabilitation of young deaf children receiving CIs are developed within the European KA202 Erasmus+ project "VOICE"-vocational education and training for speech and language therapists and parents for the rehabilitation of children with CIs, Ref. No.: 2020-1-RO01-KA202-080059. Material and methods: To develop these quality standards, we used the input from the face-to-face interviews of 11 local rehabilitation experts in CIs from the four partner countries of the project and the outcomes of the bibliographic analysis of 848 publications retrieved from six databases: Pub Med, Psych Info, CINAHL, Scopus, Eric, and Cochrane. Based on all this information, we created a first set of 32 quality standards over four domains: general, fitting, rehabilitation, and for professionals. Further on, the Delphi method was used by 18 international rehabilitation experts to discuss and agree on these standards. Results: The results from the literature analysis and the interviews show us that more than 90% of the consulted international experts agreed on 29 quality standards. They focus on different aspects of rehabilitation: the multidisciplinary team, their expertise and knowledge, important rehabilitation topics to focus on, and programming issues related to rehabilitation. Conclusions: These quality standards aim to optimize the activity of speech rehabilitation specialists so that they reach the optimal level of expertise. Also presented is the necessary equipment for the IC team to carry out the rehabilitation sessions in good conditions. This set of quality standards can be useful to ensure the appropriate postoperative care of these children. As a result, the rehabilitation process will be more relaxed, and therapists will have the opportunity to focus more on the specific needs of each child, with the provision of quality services, which will result in better results. This theme is particularly complex and dependent on multifactorial aspects of medicine, education, speech therapy, social work, and psychology that are very intricate and interdependent.

Single Mutation on Trastuzumab Modulates the Stability of Antibody-Drug Conjugates Built Using Acetal-Based Linkers and Thiol-Maleimide Chemistry.

Antibody-drug conjugates (ADCs) are a class of targeted therapeutics used to selectively kill cancer cells. It is important that they remain intact in the bloodstream and release their payload in the target cancer cell for maximum efficacy and minimum toxicity. The development of effective ADCs requires the study of factors that can alter the stability of these therapeutics at the atomic level. Here, we present a general strategy that combines synthesis, bioconjugation, linker technology, site-directed mutagenesis, and modeling to investigate the influence of the site and microenvironment of the trastuzumab antibody on the stability of the conjugation and linkers. Trastuzumab is widely used to produce targeted ADCs because it can target with high specificity a receptor that is overexpressed in certain breast cancer cells (HER2). We show that the chemical environment of the conjugation site of trastuzumab plays a key role in the stability of linkers featuring acid-sensitive groups such as acetals. More specifically, Lys-207, located near the reactive Cys-205 of a thiomab variant of the antibody, may act as an acid catalyst and promote the hydrolysis of acetals. Mutation of Lys-207 into an alanine or using a longer linker that separates this residue from the acetal group stabilizes the conjugates. Analogously, Lys-207 promotes the beneficial hydrolysis of the succinimide ring when maleimide reagents are used for conjugation, thus stabilizing the subsequent ADCs by impairing the undesired retro-Michael reactions. This work provides new insights for the design of novel ADCs with improved stability properties.

Clinical Features and Neurodevelopmental Outcomes for Infants with Perinatal Vertical Transmission of Zika Virus, Colombia.

Transplacental transmission of Zika virus has been reported during all trimesters of pregnancy and might lead to central nervous system anomalies, including microcephaly. We report 3 cases of perinatal Zika infection identified during the epidemic in Colombia and provide detailed descriptions of clinical features, diagnosis, and neurodevelopmental outcome at 18 months of age (corrected).

Immunolocalization of melatonin receptor type 1 in the sheep ovary and involvement of the PI3K/Akt/FOXO3a signaling pathway in the effects of melatonin on survival and in vitro activation of primordial follicles.

This study characterized the expression of melatonin receptor type 1 (MT1 ) protein in sheep ovaries, evaluated melatonin effects on primordial follicle survival and development after in vitro culture of ovarian tissue and verified the possible involvement of the phosphatidylinositol-3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FOXO3a) pathway in the melatonin actions. Ovine ovarian fragments were cultured in α-modified minimum essential medium alone (α-MEM+ ) or supplemented with 100, 500, or 1000 pg/ml melatonin for 7 days. PI3K inhibition was performed through pretreatment of ovarian fragments with LY294002. Thereafter, immunohistochemistry was performed to evaluate the expression of cleaved caspase-3, Akt, phosphorylated-Akt, and phosphorylated-FOXO3a (p-FOXO3a). The immunohistochemical localization of the MT1 receptor protein was documented in sheep preantral and antral follicles. After in vitro culture, 100 pg/ml melatonin showed higher follicular survival and activation than α-MEM+ and other melatonin concentrations. After PI3K inhibition, there was an increase in cleaved caspase-3-positive follicles, and a decrease in the primordial follicle activation, Akt phosphorylation, and nuclear exclusion of p-FOXO3a. In conclusion, MT1 receptor protein is present in the sheep ovary. Furthermore, 100 pg/ml melatonin maintains survival and stimulates activation of primordial follicles through the PI3K/Akt/FOXO3a signaling pathway after in vitro culture of sheep ovarian tissue.

Batch injection analysis with amperometric detection for fluoroquinolone determination in urine, pharmaceutical formulations, and milk samples using a reduced graphene oxide-modified glassy carbon electrode.

In this work, the batch injection analysis system with amperometric detection using reduced graphene oxide as a modifier of glassy carbon electrode (GCE) was investigated for the simple, fast, and sensitive monitoring of levofloxacin (LEVO) and ciprofloxacin (CIPRO) in samples of pharmaceutical formulations, synthetic urine, and milk (low- and high-fat content). LEVO and CIPRO were quantified in seven samples using amperometric measurements at +1.10 V vs Ag/AgCl, KCl(sat). The developed methods showed excellent analytical performance with limits of detection of 0.30 and 0.16 µmol L-1, linear range from 3.0 to 50 µmol L-1 and 1.0 to 50 µmol L-1, relative standard deviation below 9.7 and 3.1%, and recovery ranges ranging from 80 to 107% and from 78 to 109% for LEVO and CIPRO, respectively. In addition, the minimum sample preparation (simple dilution) combined with a high analytical frequency (130 to 180 analyses per hour) can be highlighted. Thus, the methods are promising for implementation in routine analysis and quality control to different samples.