Engineering spacer specificity of the Cre/loxP system.

Translational research on the Cre/loxP recombination system focuses on enhancing its specificity by modifying Cre/DNA interactions. Despite extensive efforts, the exact mechanisms governing Cre discrimination between substrates remains elusive. Cre recognizes 13 bp inverted repeats, initiating recombination in the 8 bp spacer region. While literature suggests that efficient recombination proceeds between lox sites with non-loxP spacer sequences when both lox sites have matching spacers, experimental validation for this assumption is lacking. To fill this gap, we investigated target site variations of identical pairs of the loxP 8 bp spacer region, screening 6000 unique loxP-like sequences. Approximately 84% of these sites exhibited efficient recombination, affirming the plasticity of spacer sequences for catalysis. However, certain spacers negatively impacted recombination, emphasizing sequence dependence. Directed evolution of Cre on inefficiently recombined spacers not only yielded recombinases with enhanced activity but also mutants with reprogrammed selective activity. Mutations altering spacer specificity were identified, and molecular modelling and dynamics simulations were used to investigate the possible mechanisms behind the specificity switch. Our findings highlight the potential to fine-tune site-specific recombinases for spacer sequence specificity, offering a novel concept to enhance the applied properties of designer-recombinases for genome engineering applications.

Pairing of single mutations yields obligate Cre-type site-specific recombinases.

Tyrosine site-specific recombinases (SSRs) represent a versatile genome editing tool with considerable therapeutic potential. Recent developments to engineer and evolve SSRs into heterotetramers to improve target site flexibility signified a critical step towards their broad utility in genome editing. However, SSR monomers can form combinations of different homo- and heterotetramers in cells, increasing their off-target potential. Here, we discover that two paired mutations targeting residues implicated in catalysis lead to simple obligate tyrosine SSR systems, where the presence of all distinct subunits to bind as a heterotetramer is obligatory for catalysis. Therefore, only when the paired mutations are applied as single mutations on each recombinase subunit, the engineered SSRs can efficiently recombine the intended target sequence, while the subunits carrying the point mutations expressed in isolation are inactive. We demonstrate the utility of the obligate SSR system to improve recombination specificity of a designer-recombinase for a therapeutic target in human cells. Furthermore, we show that the mutations render the naturally occurring SSRs, Cre and Vika, obligately heteromeric for catalytic proficiency, providing a straight-forward approach to improve their applied properties. These results facilitate the development of safe and effective therapeutic designer-recombinases and advance our mechanistic understanding of SSR catalysis.

Food insecurity and sociodemographic factors in Latin America during the COVID-19 pandemic.

Objective: To understand the association of food insecurity with sociodemographic factors in a sample population in Latin America during the COVID-19 pandemic. Methods: This was a multicenter cross-sectional study conducted in 10 countries in Latin America using an online survey through various digital platforms from October 14, 2020 to February 15, 2021. Statistical analysis of data was performed by applying descriptive statistics, chi-square test, and logistic regression analysis. Results: Of a total of 6 357 surveys, 58.2% of respondents experienced food security, 29.3% were slightly food insecure, 9.2% were moderately food insecure, and 3.3% were severely food insecure. Concerning the association food insecurity and sociodemographic variables, there is a significant association in the variables studied, including area of residence, education level, occupation, number of persons in the household, household with children younger than 10 years of age, and socioeconomic level. Conclusions: These findings indicate that sociodemographic factors associated with food insecurity during the COVID-19 pandemic in Latin America were rural residence; complete and incomplete basic and secondary schooling; occupation (homemaker, unemployed, and self-employed); low, medium-low, and medium socioeconomic level; household with more than four persons; and household with children younger than 10 years of age.

Efficacy and Safety of Erenumab, Galcanezumab, and Fremanezumab in the Treatment of Drug-Resistant Chronic Migraine: Experience in Real Clinical Practice.

BACKGROUND: Due to the recent introduction of new biologic drugs for chronic migraine, a global evaluation in real clinical practice is necessary. OBJECTIVE: The objective was to evaluate the effectiveness and safety in real clinical practice of drugs targeting the calcitonin gene-related peptide receptor (CGRPr) in patients with chronic migraine. METHODS: Single-center, restrospective study (2019-2022), including patients with chronic migraine treated with erenumab, galcanezumab, or fremanezumab. Effectiveness variables were recorded, namely, number of migraine headache days per month (MHD), Migraine Disability Assessment Scale (MIDAS) score, and Headache Impact Test-6 (HIT-6) score, assessing changes at week 12, 24 from baseline. Toxicity was recorded following the Common Terminology Criteria for Adverse Events (CTCAE) v5.0 criteria. RESULTS: In all, 104 patients were included (46.2% erenumab, 41.3% galcanezumab, 12.5% fremanezumab). A reduction in MHD, MIDAS, and HIT-6 was achieved at weeks 12 and 24 with erenumab (p75% at week 24 than those intensified; P = 0.041). There was no difference in efficacy (P = 0.154) or improvement in quality of life (P = 0.783, P = 0.150), but there was greater toxicity (P < 0.001) among nonresponders with erenumab 70 mg versus erenumab 140 mg. CONCLUSIONS: The results confirm the effectiveness and safety of anticalcitonin gene-related peptide (CGRP) drugs in real clinical practice. However, the study shows little benefit from erenumab intensification, with similar effectiveness and worse tolerability than the standard dose.

Chronic kidney disease acquired knowledge in a diabetic and hypertensive population using a translated and validated questionnaire.

Chronic kidney disease (CKD) is the progressive and irreversible functional and/or structural impairment of the kidney; its main etiologies include hypertension and diabetes. Mexico has the second highest prevalence of CKD worldwide with a high economic burden affecting public and private health systems. Patients with higher knowledge about CKD increase their adherence to preventive treatment. In this study we aim to describe the knowledge of CKD in a sample of Mexican high-risk population, comparing it with general Mexican population, medical students and nephrologists. A cross-sectional, observational study was performed divided in two phases: translation and validation of the knowledge questionnaire to Spanish, and cross-sectional survey to evaluate the knowledge of CKD in patients with diagnosis of diabetes and/or hypertension. We interviewed medical students, general population, and nephrologists to attain validation of the questionnaire in Spanish. The questionnaire was answered by 1,061 participants within the high-risk population. The results of the questionnaire were: 22/24, 18/24, 13.8/24, and 13.4/24 in nephrologists, medical students, normal subjects, and high-risk population, respectively. The questions with least correct answers were related to kidney functions and CKD risk factors. To our knowledge this is the first time a questionnaire for CKD knowledge is applied in Mexican population. These findings suggest poor understanding of kidney functions, risk factors, and symptoms of CKD. It is important not only to provide medical treatment to chronic illness but also awareness of the consequences of not achieving goals of treatment.

Determinants of COVID-19 vaccine hesitancy among pregnant persons.

BACKGROUND: Vaccines reduce the risk of contracting and developing complications from coronavirus disease 2019 (COVID-19). Pregnant people are at increased risk of disease-related complications but have a higher prevalence of vaccine hesitancy (VH) than their nonpregnant counterparts. OBJECTIVE: This study aimed to describe risk factors and COVID-19- and vaccine-related perspectives that lead to VH among pregnant people in Mexico to target strategies to increase vaccine acceptance in this population. METHODS: A cross-sectional survey-based study to evaluate risk factors and COVID-19- and vaccine-related perspectives associated with VH among pregnant people was conducted. Respondents were pregnant people of all ages attending a regular follow-up visit or admitted to labor and delivery in a third-level maternity hospital in Mexico. VH was defined as not having received a COVID-19 vaccine and either declining or being undecided to accept a vaccine during their pregnancy. We used bivariate and multivariable logistic regression models to estimate assess the relationship among demographic factors, COVID-19- and vaccine-related perspectives, and VH. RESULTS: A total of 1475 respondents completed the questionnaire; 216 (18%) were under the age of 18 years, and 860 (58%) had received at least one dose of a COVID-19 vaccine. In this sample, 264 (18%) were classified as vaccine hesitant. Key factors associated with VH were adolescence, having family as a primary source of information, first pregnancy, and history of vaccines in previous pregnancies. COVID-19 perspectives were also strongly associated with VH. CONCLUSIONS: Among pregnant people in Mexico, VH is associated with demographic factors, vaccination history, sources of information, and perceived risks to the fetus. This information is relevant to policy makers and health care professionals to identify those more likely to be hesitant and to inform strategies to increase vaccine uptake among pregnant people.

Geographical context outweighs habitat disturbance effects in explaining mistletoe population genetic differentiation at a regional scale.

Genetic differentiation depends on ecological and evolutionary processes that operate at different spatial and temporal scales. While the geographical context is likely to determine large-scale genetic variation patterns, habitat disturbance events will probably influence small-scale genetic diversity and gene flow patterns. Therefore, the genetic diversity patterns that we observe today result from the combination of both processes, but they are rarely assessed simultaneously. We determined the population structure and genetic diversity of a hemiparasitic mistletoe (Tristerix corymbosus) from the temperate rainforests of southern Chile to determine the effects of geographical context and habitat disturbance at a regional scale and if it is affected by the abundance and occurrence of its seed disperser mutualist (the arboreal marsupial Dromiciops gliroides). We genotyped 359 individuals from 12 populations using single nucleotide polymorphisms, across three different geographical contexts and four disturbance conditions. We also used camera traps to estimate the abundance and occurrence of the seed disperser. Our results suggest that genetic differences among populations are related more to geographical context than to habitat disturbance. However, as disturbance increased, D. gliroides abundance and occurrence decreased, and mistletoe inbreeding index (FIS ) increased. We also found highly uneven gene flow among study sites. Despite the high levels of disturbance that these temperate rainforests are facing, our results suggest that mistletoe genetic differentiation at a regional scale was more influenced by historical events. However, habitat disturbance can indirectly affect mistletoe population genetic differentiation via the seed dispersal process, which may increase levels of inbreeding.

Lasting metabolic effect of a high-fructose diet on global cerebral ischemia.

Introduction: Obesity is a public health problem that is associated with cerebrovascular diseases, such as ischemic stroke. The coexistence of obesity with cerebral ischemia has been suggested to be considerably detrimental to the neurological system. Objective: Hence, in this study, we evaluated the long-term effects of a 20% high fructose diet (HFD) and global cerebral ischemia on neurological, cognitive and emotional performance in three-month-old male Wistar rats. Results: Our results demonstrated that fructose intake led to increases in body weight and blood glucose, as well as reduced insulin sensitivity. The co-morbidity of fructose intake and cerebral ischemia resulted to hyperlipidemia, as well as increases in liver and adipocyte damage, which worsened neurological performance and resulted in alterations in learning and emotional skills at two weeks post-ischemia. No significant biochemical changes in autophagy and plasticity markers at the late stage of ischemia were observed. Conclusion: These results suggested that obesity causes a lasting effect on metabolic disorders that can contribute to increased neurological impairment after cerebral ischemia.

Performance of sewage treatment technologies for the removal of Cryptosporidium sp. and Giardia sp.: Toward water circularity.

Cryptosporidium sp. and Giardia sp. are parasites that cause diseases in the population. Most of parasite diseases regarding the consumption of drinking water polluted with sewage are caused by Cryptosporidium sp. or Giardia sp. it is because of the incomplete disinfection of the wastewater treatment. Therefore, in this work the removal or inactivation efficiency of different treatment technologies presented by around 40 scientific studies was evaluated, with a view to water circularity. For Cryptosporidium sp., we conclude that the most efficient secondary technologies are aerobic technologies, which remove between 0.00 and 2.17 log units (Ulog), with activated sludge presenting the greatest efficiency, and that the tertiary technologies with the greatest removal are those that use ultrasound, which reach removal values of 3.17 Ulog. In the case of Giardia sp., the secondary technologies with the greatest removal are anaerobic technologies, with values between 0.00 and 3.80 Ulog, and the tertiary technologies with the greatest removal are those that combine filtration with UV or a chemical disinfection agent. Despite the removal values obtained, the greatest concern remains detecting and quantifying the infectious forms of both parasites in effluents; therefore, although the technologies perform adequately, discharge effluents must be monitored with more sensitive techniques, above all aiming for circularity of the treated water in a context of the water scarcity that affects some parts of the world.

Universal screening for SARS-CoV-2 in obstetric patients.

Not available.

COVID-19 vaccine hesitancy and acceptance in Mexico: a web-based nationwide survey.

OBJECTIVE: To identify factors associated with COVID-19 vaccine hesitancy and acceptance among the Mexican population. METHODS: In a web-based nationwide survey in early December 2020, respondents were inquired about their sociodemographic characteristics and their willingness to accept a hypothetical COVID-19 vaccine given a 50% or 90% effectiveness. A logistic regression model was used to identify the factors associated with hesitancy and acceptance. RESULTS: A total 3 768 responses were analyzed. A 90% effective vaccine was accepted by 85% of respondents, while only 46% would accept being vaccinated with a 50% effective vaccine. In univariate analysis, each age group (40-49, 50-59, and ≥60) was strongly associated with vaccine hesitancy for a 90% effective vaccine (OR 0.48, 95% CI 0.38, 0.63; OR 0.33, 95 CI 0.26, 0.41; and OR 0.28, 95 CI 0.21, 0.38, respectively) compared to the 18-39 age group. After multivariable adjustment, similar magnitudes of association were observed. Being female and higher socioeconomic status were also associated with higher vaccine hesitancy. CONCLUSIONS: Vaccine hesitancy represents a major public health problem in Mexico and is driven by multiple factors. Our study provides relevant insights for the development of effective policies and strategies to ensure widespread vaccination in Mexico.

Adsorption of Cr(VI) in Aqueous Solution Using a Surfactant-Modified Bentonite.

Clay minerals can be modified organically by a cationic surfactant resulting in materials known as organoclays. The organoclays have been used as adsorbents of most of the organic contaminants in the aqueous solution and oxyanions of the heavy metal. In this study, a Colombian bentonite was modified with hexadecyltrimethylammonium bromide to obtain an organobentonite, and its capacity to adsorb Cr(VI) oxyanions in the aqueous solution was evaluated. The effect of pH, stirring speed, adsorbent amount, contact time, and ionic strength were investigated at 25°C. Stirring speeds above 200 rpm, contact times greater than 120 min, and the addition of NaCl (0.1 to 2.0 mM) did not have a significant effect on Cr(VI) removal. The influence of the adsorbent amount and pH on Cr(VI) adsorption was studied by the response surface methodology (RSM) approach based on a complete factorial design 32. Results proved that the Cr(VI) adsorption follows a quadratic model with high values of coefficient of determination (R 2 = 95.1% and adjusted R 2 = 93.9%). The optimal conditions for removal of Cr(VI) from an aqueous solution of 50 mg/L were pH of 3.4 and 0.44 g amount of the adsorbent. The adsorption isotherm data were fitted to the Langmuir and Freundlich adsorption isotherm models, and the model parameters were evaluated. The maximum adsorption capacity of Cr(VI) onto organobentonite calculated from the Langmuir model equation was 10.04 ± 0.34 mg/g at 25°C. The results suggest that organobentonite is an effective adsorbent for Cr(VI) removal, with the advantage of being a low-cost material.

Molecular weight distribution of the recalcitrant organic matter contained in kraft mill effluents and the identification of microbial consortia responsible for an anaerobic biodegradable fraction.

The objective of this research was to evaluate the distribution of the molecular weights of the recalcitrant organic matter contained in kraft mill effluents and identify microbial consortia responsible for an anaerobic biodegradable fraction. As a result, the average removal efficiencies of chemical organic demand (COD) and biological oxygen demand (BOD5) during the entire period of operation were 28% and 53%, respectively. The non-biodegradable organic matter was detected at molecular weights less than 1000 Da. However, most of the organic matter was in the molecular weight fraction higher than 10000 Da with 32 ± 11.6% COD as well as color (42.3 ± 8.7%), total phenolic compounds (35.9 ± 7.9%) and adsorbable organic compounds (AOX) (13.0 ± 2.7%). Methanogenic acetoclastic archaea of the genera Methanomethylovorans and Methanosarcina were found in the surface and middle zones of the reactor. Moreover, Methanosaeta and Methanolinea were identified in the low zone of the reactor. In all zones of the reactor, Desulfomicrobium and Desulfovibrio were found to be the most dominant genera of sulfate-reducing bacteria (SRB).

Tartrazine Removal from Aqueous Solution by HDTMA-Br-Modified Colombian Bentonite.

The effect of pH, ionic strength (NaCl added), agitation speed, adsorbent mass, and contact time on the removal of tartrazine from an aqueous solution, using an organobentonite, has been studied. A complete factorial design 32 with two replicates was used to evaluate the influence of the dye concentration (30, 40, and 50 mg/L) and amount of adsorbent (25, 35, and 45 mg) on decolorization of the solution. Experimental data were evaluated with Design Expert® software using a response surface methodology (RSM) in order to obtain the interaction between the processed variables and the response. pH values between 2 and 9, stirring speed above 200 rpm, and contact time of 60 min did not have a significant effect on decolorization. The optimum conditions for maximum removal of tartrazine from an aqueous solution of 30 mg/L were follows: pH = 6.0, NaCl concentration = 0.1 M, stirring speed = 230 rpm, temperature = 20°C, contact time = 60 min, and the organobentonite amount = 38.04 mg. The equilibrium isotherm at 20°C was analyzed by means of the Langmuir and Freundlich models, and the maximum adsorption capacity obtained was 40.79 ± 0.71 mg/g. This adsorption process was applied in a sample of industrial wastewater containing tartrazine and sunset yellow, having obtained a decolorization rate higher than 98% for both dyes. These results suggest that organobentonite is an effective adsorbent for the removal of anionic dyes from an aqueous solution.

Effects of biflavonoids from Garcinia madruno on a triple transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is pathologically characterized by the deposition of ß-amyloid (ßA) peptides in senile plaques and neurofibrillary tangles in the brain. Flavonoids have recently been used to prevent and treat a variety of neurodegenerative diseases, but little is known about bioflavonoids. In this study, we evaluate whether a biflavonoid fraction (BF) exerts neuroprotective effects on an aged triple transgenic mouse mode of AD (3xTg-AD). Then, 21-24-month-old 3xTg AD mice were i.p. injected with 25mg/kg of a BF from Garcinia madruno composed of morelloflavone (65%), volkensiflavone (12%), GB 2a (11%), fukugiside (6%) and amentoflavone (0.4%) every 48h for 3 months. The BF treatment reduced ßA deposition in different regions of the brain (the hippocampus, entorhinal cortex and amygdala), reduced ßA1-40 and ßA1-42 levels, BACE1-mediated cleavage of APP (CTFß), tau pathology, astrogliosis and microgliosis in the brains of aged 3xTg-AD mice. Although the BF treatment weakly improved learning, animals treated with BF spent more time in the open arms of the elevated plus maze test and displayed greater risk assessment behavior than the control groups. In summary, the BF reverses histopathological hallmarks and reduces emotional disorders in the 3xTg-AD mouse model, suggesting that the biflavonoids from G. madruno represent a potential natural therapeutic option for AD if its bioavailability is improved.

CDK5 downregulation enhances synaptic plasticity.

CDK5 is a serine/threonine kinase that is involved in the normal function of the adult brain and plays a role in neurotransmission and synaptic plasticity. However, its over-regulation has been associated with Tau hyperphosphorylation and cognitive deficits. Our previous studies have demonstrated that CDK5 targeting using shRNA-miR provides neuroprotection and prevents cognitive deficits. Dendritic spine morphogenesis and forms of long-term synaptic plasticity-such as long-term potentiation (LTP)-have been proposed as essential processes of neuroplasticity. However, whether CDK5 participates in these processes remains controversial and depends on the experimental model. Using wild-type mice that received injections of CDK5 shRNA-miR in CA1 showed an increased LTP and recovered the PPF in deficient LTP of APPswe/PS1Δ9 transgenic mice. On mature hippocampal neurons CDK5, shRNA-miR for 12 days induced increased dendritic protrusion morphogenesis, which was dependent on Rac activity. In addition, silencing of CDK5 increased BDNF expression, temporarily increased phosphorylation of CaMKII, ERK, and CREB; and facilitated calcium signaling in neurites. Together, our data suggest that CDK5 downregulation induces synaptic plasticity in mature neurons involving Ca2+ signaling and BDNF/CREB activation.

Characterization of Colombian Clay and Its Potential Use as Adsorbent.

This paper presents a mineralogical and physicochemical characterization of a Colombian clay found in an area with the greatest exploitation potential of smectites and possible use as an adsorbent for the removal of chromium. The clay was characterized by using X-ray diffraction (XRD), cation exchange capacity (CEC), X-ray fluorescence (XRF), Fourier transform infrared spectroscopy (FT-IR), thermal analysis (TGA/DSC), and nitrogen adsorption at 77 K. The homoionized clay was used as an adsorbent for the removal of Cr(III) in an aqueous solution. The homoionized clay was modified with hexadecyltrimethylammonium bromide (HDTMA-Br), and the organoclay obtained was evaluated for the adsorption of Cr(VI) in aqueous solution. The XRD analysis showed that the clay from Armero-Guayabal is primarily constituted by smectite (48 wt%) followed by quartz mineral (21 wt%). The chemical analysis of bulk clay showed that the predominant oxides are SiO2 (55.81 wt%), Al2O3 (16.25 wt%), and Fe2O3 (7.51 wt%), and the nitrogen adsorption indicated that the bulk clay has a specific surface area of 45.1 m2/g. Homoionized clay and organoclay achieved Cr(III) and Cr(VI) removals greater than 85.05 ± 2.04% (pH between 3 and 4) and 82.93 ± 1.03% (pH between 3 and 5), respectively, proving the potential of these materials for the removal of heavy metals in an aqueous solution.

[MLVA analysis and virulence profile of community-acquired methicillin-resistant Staphylococcus aureus causing infections in Paraguayan children]. / Análisis MLVA y perfil de virulencia de aislamientos de Staphylococcus aureus resistentes a la meticilina adquiridos en la comunidad causantes de infecciones pediátricas en Paraguay.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is one of the first causes of skin and soft tissue infections, and can also produce severe diseases such as osteomyelitis and pneumonia. The aim of this descriptive study was to determine the SCCmec type and virulence profile and to study the genetic diversity by MLVA analysis of 21 CA-MRSA isolates that infected Paraguayan children in 2010. The SCCmec type and virulence factors were performed by PCR and genetic diversity by MLVA (multiple locus variable analysis). All the isolates carried SCCmec cassette iv. hla, hlb and sea genes were detected in 28,6%, 9,5% and 4,8% respectively. The MLVA analysis showed high genetic diversity with congruent antibiotic resistance and virulence profiles. This study revealed the presence of CA-MRSA harbouring SCCmeciv with high genetic diversity, providing information not available in our country.

Sulfated hyaluronan attenuates inflammatory signaling pathways in macrophages involving induction of antioxidants.

It is well recognized that high molecular weight hyaluronan (H-HA) exerts potent anti-inflammatory effects while its fragmentation into low molecular weight HA (L-HA) is discussed to promote inflammation. Chemical modification of HA with sulfate groups has been shown to foster its anti-inflammatory activity which seems to be maintained in sulfated low molecular weight HA derivatives (sL-HA). However, the molecular mechanisms by which sL-HA produces its anti-inflammatory activity are not understood. In this study, we used global quantitative proteomics combined with targeted analysis of key proteins to characterize the effect of sL-HA on fully differentiated human inflammatory macrophages (iMФ). Culture of iMФ with sL-HA did not affect cell viability but resulted in a reduced pro-inflammatory cytokine response of iMФ after activation indicating a profound counter-regulation of their initial inflammatory phenotype. Rapid internalization of sL-HA involving CD44 and scavenger receptors was observed. Furthermore, an upregulation of the antioxidants SOD2 and SOD3 was found while no oxidative stress was induced. Consequently, activity of transcription factors for inflammatory gene expression was downregulated in iMФ with sL-HA after activation whereas anti-inflammatory proteins were induced. This study proves anti-inflammatory properties of sL-HA and provides information on its regulatory mode of action on iMФ.