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The Solid-state cooling based on caloric effects is considered a potential alternative to conventional refrigeration technology, which uses ozone-depleting gases. Several shape memory alloys have attracted attention for solid-state cooling since they present a high reversibility of the caloric effect, which depends mainly on thermal hysteresis and sensitivity to the applied field. In the present work, a study substitution of Mn by Ga in the Ni50Mn34Ti16 alloy led to diminished thermal hysteresis in the martensitic transformation by 9 K. The elastocaloric effect, thermal and microstructure properties of a polycrystalline Ni50Mn32Ti16Ga2 alloy have been characterized. The elastocaloric effect was obtained indirectly from the length change as a function of temperature at constant stress. An isothermal entropy change (ΔSISO) of 23.0 J kg−1 K−1 during heating and 22.0 J kg−1 K−1 during cooling was observed for applied stress of 160 MPa. In addition, the ΔSISO is reversible for a temperature span between 287 and 319 K, reaching a maximum of 20.5 J kg−1 K−1 at 299 K. The thermal hysteresis changed slightly while the applied stress increased up to 160 MPa since the sensitivity of the martensitic transformation temperatures to stress was 0.150 K/MPa during cooling and 0.160 K/MPa during heating. The X-ray diffraction analysis revealed a mixture of B2-type cubic austenite, 5M modulated martensite, and a second intermetallic phase identified as Ni3Ti. All these results were obtained around room temperature.

In the present work, the structural, mechanical and shape memory properties of Ni55Fe16Ga29 (at.%) melt-spun ribbons have been studied with a prospective of application in the elastocaloric devises. Particularly, a special thermo-mechanical treatment, consisting in the thermal aging under constant external stress, was elaborated to control the residual internal stress generated due to the melt-spinning processing and improve thermomechanical and superelastic responses of the ribbon. The stress-induced entropy change was evaluated for the ribbon with improved thermomechanical properties. Keywords: Melt spun ribbons, NiFeGa, Mechanical properties, Elastocaloric effect

Diabetes mellitus (DM) is a well-known risk factor for tuberculosis (TB). However, it is not known to what extent DM affects the outcome in patients with multidrug-resistant (MDR-TB) and extensively drug-resistant TB (XDR-TB) treated with second-line anti-TB drugs.The objective of this study was to compare the microbiological evolution (sputum smear and culture conversion) and final outcomes of MDR/XDR-TB patients with and without DM, managed at the national TB reference centre in Mexico City. Results: Ninety patients were enrolled between 2010 and 2015: 73 with MDR-TB (81.1%), 11 with pre-XDR-TB (e.g. MDR-TB with additional resistance to one injectable drug or a fluoroquinolone, 12.2%) and 6 (6.7%) with XDR-TB. Out of these, 49 (54.4%) had DM and 42 (86%) were undergoing insulin treatment.No statistically significant differences were found in treatment outcomes comparing DM vs. non-DM MDR-TB cases: 18/32 (56.3%) of DM cases and 19/24 (79.2%) non DM patients achieved treatment success (p = 0.07). The time to sputum smear and culture conversion was longer (although not statistically) in patients without DM, as follows: the mean (±SD) time to sputum smear conversion was 53.9 (±31.4) days in DM patients and 65.2 (±34.8) days in non-DM ones (p = 0.15), while the time to culture conversion was 66.2 (±27.6) days for DM and 81.4 (±37.7) days for non-DM MDR-TB cases (p = 0.06). Conclusions: The study results support the Mexican National TB programme to strengthen its collaboration with the DM programme, as an entry point for TB (and latent TB infection) screening and management. Keywords: Diabetes mellitus, Delay, Sputum and culture conversion, MDR-TB, High treatment adherence

Abstract Background A comprehensive non-invasive evaluation of bioprosthetic mitral valve (BMV) function can be challenging. We describe a novel method to assess BMV effective orifice area (EOA) based on phase contrast (PC) cardiovascular magnetic resonance (CMR) data. We compare the performance of this new method to Doppler and in vitro reference standards. Methods Four sizes of normal BMVs (27, 29, 31, 33 mm) and 4 stenotic BMVs (27 mm and 29 mm, with mild or severe leaflet obstruction) were evaluated using a CMR- compatible flow loop. BMVs were evaluated with PC-CMR and Doppler methods under flow conditions of; 70 mL, 90 mL and 110 mL/beat (n = 24). PC-EOA was calculated as PC-CMR flow volume divided by the PC- time velocity integral (TVI). Results PC-CMR measurements of the diastolic peak velocity and TVI correlated strongly with Doppler values (r = 0.99, P

Ectopic pregnancy (EP) is a common diagnosis, frequently misdiagnosed early in its presentation and a leading cause of first trimester mortality. Ultrasound (US) is a key component of evidence-based diagnostic algorithms. We present a systematic review on the frequency of the use of US in the diagnosis of EP in Sub-Saharan Africa. Methods: A librarian-assisted search of PUBMED, EMBASE, Cochrane, Web of Science, and POPLINE databases was performed. Inclusion criteria were original research studies that reported the proportion of patients receiving US as part of a workup for EP in a Sub-Saharan African country. Abstracts were reviewed and those potentially meeting criteria had a formal survey of the manuscript. Results: The initial search revealed 784 original publications. Manual review of abstracts narrowed this to 91 papers with potential relevance, and 12 studies were included in the final analysis. A total of 6055 patients diagnosed with EP were included. 8.7% received a pregnancy test. 92.3% were ruptured at the time of presentation. 42.9% were in shock and 75.8% received red blood cell transfusion. 73.7% were unaware of the pregnancy and 24.9% were seen by a healthcare worker prior to presentation, 1.1% of patients died. Overall, 12.6% received US to aid in the diagnosis. Conclusion: In this study, overall utilisation of US in the diagnosis of EP was found to be low. In this population, patients presented late and critically ill, obviating the need for US in many cases. However, studies in Sub-Saharan Africa in populations of patients with similar rates of late presentations have shown a substantial increase in the diagnosis of unruptured EP with the routine use of US.

Background: The Mexican Ministry of Health has advocated for increased skilled birth attendance in hospital facilities to decrease maternal and newborn morbidity and mortality. Partners In Health (PIH) works in a rural region of Mexico where the home birth rate is 60%. The primary purpose of this project is to improve the quality of care, including adherence to evidence-based practices and promotion of respectful practices during childbirth, in a rural hospital in Chiapas, Mexico. Additionally, we hope to strengthen the role of professional midwives and obstetric nurses as competent clinicians in managing women with low-risk pregnancies during childbirth. Methods: PIH collaborated with Ariadne Labs to adapt and implement the WHO Safe Childbirth Checklist in a rural hospital in Ángel Albino Corzo, Chiapas. Four pause points (at admission, just before delivery, within 1h of birth, and before discharge) were adapted according to the local context. Additional items were added to capture respectful practices during childbirth. We provided coaching training to the professional midwife and perinatal and obstetric nurses to make optimal use of the checklist for ongoing quality improvement in a team-based environment. The checklist and coaching tools have been developed into a mobile application using CommCare for instant data capture and continuous monitoring and evaluation. We used a mixed-methods study to capture adherence to evidence-based practices and integrated these findings with patient and provider experiences during labour and delivery. We conducted post-discharge surveys and post-partum interviews to explore how patients perceive the care they received during childbirth. Findings: By August, 2016, five obstetric nurses, one perinatal nurse, and one professional midwife were using the adapted Safe Childbirth Checklist in the study hospital. Data collection using CommCare began in September, 2016, and will continue until June, 2017. By March, 2017, we had captured data from about 350 birthing women using the WHO Safe Childbirth Checklist and 160 women who had completed the discharge survey. We have conducted interviews with 25 postpartum women and some of their birth companions. Data collection is ongoing but preliminary data from September to December, 2016, show that 64% (171/266) of women were asked at least two questions about their preferences in labour, 13% (28/216) of women received episiotomies without indication, 68% (147/216) initiated skin-to-skin within 1h of delivery, and 56% (122/216) of women initiated breastfeeding after delivery. Preliminary analysis of the discharge surveys reveal high satisfaction with the care women received at the hospital. Qualitative interviews indicate some variation in birth experiences. Interpretation: There is some variation in adherence with evidence-based practices and perceptions of good quality of care at the rural hospital since implementing the adapted Safe Childbirth Checklist along with other PIH-driven community initiatives. While we do not have a baseline for these indicators, our intervention represents the first attempt at any quality improvement initiative for childbirth care in this region, and adoption will require continued buy-in from hospital staff. Funding: The MacArthur Foundation, and Brigham and Women's Hospital Division of Women's Health.

This study aimed to determine the prevalence of V. parahaemolyticus in oysters from the northwestern coast of Mexico and to identify the serotypes, virulence factors, and antibiotic resistance of the strains. Oyster samples were collected from 2012 to 2020 from the northwest coast of Mexico; biochemical and molecular methods were used to identify V. parahaemolyticus from oysters; antiserum reaction to determine V. parahaemolyticus serotypes, and PCR assays were performed to identify pathogenic (tdh and/or trh) or pandemic (toxRS/new, and/or orf8) strains and antibiotic resistance testing. A total of 441 oyster samples were collected and tested for V. parahaemolyticus. Forty-seven percent of oyster samples were positive for V. parahaemolyticus. Ten different O serogroups and 72 serovars were identified, predominantly serotype O1:KUT with 22.2% and OUT:KUT with 17.3%. Twenty new serotypes that had not been previously reported in our region were identified. We detected 4.3% of pathogenic clones but no pandemic strains. About 73.5% of strains were resistant to at least one antibiotic, mainly ampicillin and ciprofloxacin; 25% were multi-drug resistant. In conclusion, the pathogenic strains in oysters and antibiotic resistance are of public health concern, as the potential for outbreaks throughout northwestern Mexico is well established., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Assessing the umbilical artery pulsatility index via Doppler measurements plays a crucial role in evaluating fetal growth impairment., Objective: This study aimed to investigate perinatal outcomes associated with discordant pulsatility indices of umbilical arteries in fetuses with growth restriction., Study Design: In this retrospective cohort study, all singleton pregnancies were included if their estimated fetal weight and/or abdominal circumference fell below the 10th percentile for gestational age (2017-2022). Eligible cases included singleton pregnancies with concurrent sampling of both umbilical arteries within 14 days of birth at the ultrasound evaluation closest to delivery. The exclusion criteria included births before 22 weeks of gestation, evidence of absent or reverse end-diastolic flow in either umbilical artery, and known fetal genetic or structural anomalies. The study compared cases with discordant umbilical artery pulsatility index values (defined as 1 umbilical artery pulsatility index at ≤95th percentile and the other umbilical artery pulsatility index at >95th percentile for gestational age) to pregnancies where both umbilical artery pulsatility indices had normal pulsatility index values and those with both umbilical arteries displaying abnormal pulsatility index values. The primary outcome assessed was the occurrence of composite adverse neonatal outcomes. Multivariable logistic regressions were performed, adjusting for relevant covariates., Results: The study encompassed 1014 patients, including 194 patients (19.1%) with discordant umbilical artery pulsatility index values among those who had both umbilical arteries sampled close to delivery, 671 patients (66.2%) with both umbilical arteries having normal pulsatility index values, and 149 patients (14.7%) with both umbilical arteries exhibiting abnormal values. Pregnancies with discordant umbilical artery pulsatility index values displayed compromised sonographic parameters compared with those with both umbilical arteries showing normal pulsatility index values. Similarly, the number of abnormal umbilical artery pulsatility index values was associated with adverse perinatal outcomes in a dose-response manner. Cases with 1 abnormal (discordant) umbilical artery pulsatility index value showed favorable sonographic parameters and perinatal outcomes compared with cases with both abnormal umbilical artery pulsatility index values, and cases with both abnormal umbilical artery pulsatility index values showed worse sonographic parameters and perinatal outcomes compared with cases with discordant UA PI values. Multivariate analysis revealed that discordant umbilical artery pulsatility indices were significantly and independently associated with composite adverse perinatal outcomes, with an adjusted odds ratio of 1.75 (95% confidence interval, 1.24-2.47; P = .002)., Conclusion: Evaluating the resistance indices of both umbilical arteries may provide useful data and assist in assessing adverse perinatal outcomes among fetuses with growth restriction., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Our study aimed to establish reference values for nesting females and compare them with those previously reported to understand olive ridley turtles' health status and contribute to long-term health assessment and monitoring in foraging and nesting areas from the state of Sinaloa, Mexico. In August and September 2018, morphometric data and biochemical profiles were collected from 33 nesting olive ridley turtles from Ceuta Beach Sanctuary (CBS) and 14 foraging female turtles captured at the foraging site, Navachiste Marine Area (NMA). Nesting turtles sampled had greater CCL (65.86 ± 1.70 cm) than those from the foraging area (61.54 ± 1.22) (p < 0.05). Regarding biochemical profiles, post-nesting turtles had higher packed cell volume (PCV), albumin, blood urea nitrogen (BUN), cholesterol, triglycerides, and calcium than turtles from the foraging area (p < 0.05). Phosphorus levels were higher in foraging turtles than in nesting turtles (p = 0.001), while the remaining parameters showed no significant differences. The present study describes for the first time the blood biochemical values of nesting turtles from the Ceuta Beach Sanctuary in southern Sinaloa, Mexico, similar to those of foraging turtles from the north of the state. The significant differences observed between the two analysis groups may be due to the energy reserves and reproductive and nesting activity of the nesting turtles, so the blood biochemistry values described in this study can be used as a standard reference blood value for the olive ridley turtle population of Sinaloa, Mexico., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction: OnabotulinumtoxinA is used as treatment for refractory idiopathic and neurogenic detrusor overactivity in children. Many patients perform intermittent self-catheterization and therefore have higher rates of asymptomatic bacteriuria, which may increase their risk of symptomatic urinary tract infection (UTI) following treatment. Multiple injections are often needed due to the short-term efficacy of onabotulinumtoxinA treatment, which may also increase the risk of UTI., Objective: We aim to evaluate whether a sterile urinary tract is necessary to decrease the risk of postoperative UTI in pediatric patients treated with onabotulinumtoxinA., Study Design: A retrospective review of patients undergoing intradetrusor onabotulinumtoxinA injection from 2014 to 2021 was performed. Demographic data, clinical characteristics, antibiotic treatment and culture results were collected. A positive urine culture was defined as ≥ 10 3  CFU/ml of uropathogenic bacteria. Our primary outcome was symptomatic UTI within 14 days of the procedure., Results: 103 patients underwent 158 treatments with onabotulinumtoxinA. The incidence of postoperative UTI was 3.2%. The incidence of symptomatic postoperative UTI in patients with asymptomatic bacteriuria compared to those with sterile urine was not significantly different (3.8% vs 0%, p = 0.57). Obtaining a preoperative urinalysis or urine culture did not affect the incidence of postoperative UTI (p = 0.54). The number needed to treat with antibiotics to prevent one postoperative UTI was 27. The incidence of postoperative UTI was highest in patients with low-risk bladders (p = 0.043). Prior history of multi-drug resistant UTI was a risk factor for postoperative UTI (p = 0.048)., Discussion: For children undergoing onabotulinumtoxinA injection, there are no evidence-based recommendations regarding antibiotic prophylaxis and the need to screen for and treat asymptomatic bacteruria prior to treatment. Our study addresses this important clinical question, and shows no difference in the rate of postoperative UTI between patients with asymptomatic bacteriuria and those with sterile urine. Patients with a history of multi-drug resistant UTI are at increased risk of symptomatic postoperative UTI and may benefit from preoperative urine testing and treatment. Limitations of our retrospective study include its small sample size in the face of such a low incidence of our primary outcome., Conclusions: The risk of UTI following onabotulinumtoxinA injection in children is low. The presence of sterile urine at the time of surgery does not significantly decrease the risk of postoperative UTI. Routine treatment of asymptomatic bacteriuria prior to surgery results in a large number of patients receiving unnecessary antibiotics. As a result, we recommend against preoperative urine testing for most asymptomatic patients., Competing Interests: Conflicts of interest CK has served as a consultant and meeting planning director for Intuitive Surgical (Sunnyvale, California). The other authors declare that they have no conflicts of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Introduction: Chorioamnionitis is an adverse condition in human pregnancy caused by many bacterial pathogens including Escherichia coli (E. coli); which has been associated with higher risk of preterm birth. We recently reported that human maternal decidua (MDec) tissue responds to E. coli infection by secreting extracellular heat-shock proteins (eHsp)-60, -70 and interlukin-1β (IL-1β). Previous studies have shown that progesterone (P4) regulates the immune response, but it is unknown whether P4 inhibits the secretion of eHsp. The aim of this investigation was to determine the role of P4 on the secretion of eHsp-27, -60, -70 and IL-1β in MDec after 3, 6, and 24 h of E. coli infection., Methods: Nine human feto-maternal interface (HFMi) tissues were included and mounted in the Transwell culture system. Only the maternal decidua (MDec) was stimulated for 3, 6 and 24 h with E. coli alone or in combination with progesterone and RU486. After each treatment, the HFMi tissue was recovered to determine histological changes and the culture medium recovered to evaluate the levels of eHsp-27, -60, -70 and IL-1β by ELISA and mRNA expression by RT-PCR., Results: No structural changes were observed in the HFMi tissue treated with P4 and RU486. However, stimulation with E. coli produces diffuse inflammation and ischemic necrosis. E. coli induced infection decreases, in time- and dose-dependent manner, eHsp-27 and increases eHsp-60, eHsp-70 and IL-1β levels. In contrast, incubation of HFMi tissue with E. coli + P4 reversed eHsp and IL-1β secretion levels relative to E. coli stimulation group but not relative to the control group. The same profile was observed on the expression of eHsp-27 and eHsp-60., Discussion: we found that progesterone modulates the anti-inflammatory (eHsp-27) and pro-inflammatory (eHsp-60 and eHsp-70) levels of eHsp induced by E. coli infection in human choriodecidual tissue. eHsp-60 and eHsp-70 levels were not completely reversed; maintaining the secretion of IL-1β, which has been associated with adverse events during pregnancy., Competing Interests: Declaration of competing interest All authors declare that they have no conflict or financial interest. All authors carefully read the final version of the manuscript and gave their consent for journal submission., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: To assess the quality of harms reporting in systematic reviews (SRs) regarding minimally invasive hysterectomies (MIHs)., Data Sources: We performed a cross-sectional analysis on SRs addressing MIHs to assess completeness of harms reporting. Hysterectomies are common gynecologic procedures and are associated with complications. Such adverse events can directly affect physician decision making and patient outcomes. Thus, it is important that SRs equally weigh the harms and benefits surrounding MIHs., Methods of Study Selection: On May 15, 2022, we searched MEDLINE (PubMed and Ovid), Embase, Epistemonikos, and the Cochrane Database of Systematic Reviews for SRs with or without a meta-analysis on MIH for any indication. Eligible studies underwent full-text screening, data extraction, harms reporting assessment, and A Measurement Tool to Assess Systematic Reviews-2 quality assessment in a masked, duplicate fashion. The corrected covered area was calculated to indicate any overlap between SR dyads., Tabulation, Integration, and Results: A total of 52 SRs met the inclusion criteria for data extraction. We found that >44 of included SRs (of 52; 84.6%) reported >50% of the harms items. Completeness of harms reporting was significantly associated with harms specification as a primary outcome (p <.05). The corrected covered area was 0.60%., Conclusion: The harms reporting was more complete than hypothesized, but still had deficiencies throughout, such as inconsistent use of severity scales to classify harms. Even though MIH harms reporting in sample SRs was more complete than other fields of medicine, it was still suboptimal overall and requires refinement., (Copyright © 2022 AAGL. Published by Elsevier Inc. All rights reserved.)

Low-income, rural frontline communities of California's Central Valley experience environmental and socioeconomic injustice, water insecurity, extremely poor air quality, and lack of fundamental infrastructure (sewage, green areas, health services), which makes them less resilient. Many communities depend financially on agriculture, while water scarcity and associated policy may trigger farmland retirement further hindering socioeconomic opportunities. Here we propose a multi-benefit framework to repurpose cropland in buffers inside and around (400-m and 1600-m buffers) 154 rural disadvantaged communities of the Central Valley to promote socioeconomic opportunities, environmental benefits, and business diversification. We estimate the potential for (1) reductions in water and pesticide use, nitrogen leaching, and nitrogen gas emissions, (2) managed aquifer recharge, and (3) economic and employment impacts associated with clean industries and solar energy. Retiring cropland within 1600-m buffers can result in reductions in water use of 2.18 km 3 /year, nitrate leaching into local aquifers of 105,500 t/year, greenhouse gas emissions of 2,232,000 t CO 2 -equivalent/year, and 5388 t pesticides/year, with accompanying losses in agricultural revenue of US$4213 million/year and employment of 25,682 positions. Buffer repurposing investments of US$27 million/year per community for ten years show potential to generate US$101 million/year per community (total US$15,578 million/year) for 30 years and 407 new jobs/year (total 62,697 jobs/year) paying 67 % more than prior farmworker jobs. In the San Joaquin Valley (southern Central Valley), where groundwater overdraft averages 2.3 km 3 /year, potential water use reduction is 1.8 km 3 /year. We have identified 99 communities with surficial soils adequate for aquifer recharge and canals/rivers within 1600 m. This demonstrates the potential of managed aquifer recharge in buffered zones to substantially reduce overdraft. The buffers framework shows that well-planned land repurposing near disadvantaged communities can create multiple benefits for farmers and industry stakeholders, while improving quality of life in disadvantaged communities and producing positive externalities for society., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Owing to the frequent perioperative use of ketorolac tromethamine and its ability to minimise postoperative opioid requirements, it is important to continually reassess harms associated with its use. Our primary objective was to investigate the extent of harms reporting in systematic reviews (SRs) on ketorolac for perioperative pain., Methods: In May 2022, we conducted a search of major databases, MEDLINE (PubMed and Ovid), Embase, Epistemonikos, and the Cochrane Database of Systematic Reviews to identify eligible SRs on ketorolac for perioperative pain. Screening and data extraction were performed in masked, duplicate fashion. A MeaSurement Tool to Assess systematic Reviews-2 (AMSTAR-2) was used to appraise the methodological quality of included SRs. Corrected covered area (CCA) was calculated to determine overlap of primary studies between SR dyads., Results: A total of 28 SRs evaluating 630 primary studies met the inclusion criteria. Seven SRs (7/28, 25%) reported no harms and 17 SRs (17/28, 60.7%) reported ≤50% of harms items. A significant association was found between completeness of harms reporting and whether harms were specified as a primary outcome (P<0.001). No other associations were statistically significant. Regarding methodological quality, 22 SRs were appraised as 'critically low' (22/28, 78.6%), 5 as 'low' (5/28, 17.9%), and 1 as 'high' (1/28, 3.6%). One SR dyad had a CCA >50% but neither reported harms., Conclusions: The extent of harms reporting in systematic reviews was inadequate. Given the importance that systematic reviews have on guiding perioperative decision-making, it is essential to improve the completeness of harms reporting., (Copyright © 2022 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

Early screening for placenta accreta spectrum (PAS) is not established in Canada, yet has been demonstrated with a 2-stage strategy in patients classified as at risk at the nuchal translucency examination. We performed a retrospective analysis of women with singleton pregnancies who had first-trimester screening and subsequently had a pathology-confirmed PAS. We sought those who met high-risk clinical criteria for PAS: previous uterine surgeries and an anterior low-lying placenta. Over 80% of women met both high-risk criteria at the nuchal translucency examination. Lack of early recognition and risk stratification resulted in a late median gestational age at suspicion and diagnosis of PAS., (Copyright © 2022 The Society of Obstetricians and Gynaecologists of Canada/La Société des obstétriciens et gynécologues du Canada. Published by Elsevier Inc. All rights reserved.)

EF-hand is a common motif in Ca 2+ -binding proteins, some of which present a conformational change upon Ca 2+ -binding, a relevant property for signal transduction. In the present work, we investigated the behavior of Calbindin D 9k , a modulator protein with a high affinity for Ca 2+ but structurally insensitive to its presence. Its non-canoncal N-terminal EF-hand was replaced by chimeric motifs, containing increasing structural elements from the sensor troponin C SCIII motif. We demonstrated that the loop and helix II were the necessary elements for a conformational change promoted by calcium in chimeric Calbindin D 9k . Fusion of the isolated chimeric motifs to an activity reporter gene showed the loop as the minimal element to promote a conformational change. The discrepancy between these results is discussed in the light of inter-motif interactions and helix I participation in modulating the Ca 2+ affinity and restricting motif conformation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The aim of this work was to evaluate the expression profile of genes involved in signaling, intracellular and extracellular Ca +2 concentration and apoptosis pathways of osteoblasts in contact with a scaffold made of a composite of BCN/MWCNTs. Osteoblasts were cultivated on BCN, MWCNTs and their mixtures. Osteoblast RNA was extracted for sintering cDNA to amplify genes of interest by PCR; intra- and extracellular calcium (Ca 2+) was also quantified. Regarding the genes that participate in the regulation paths (MAPK and NF-KB), it was found that only the expression of NF-KB was affected in all treatments. The expression of VEGFA increased, except in the treatment of high concentration of MWCNTs, where remained unchanged. The expression of genes Apaf-1 and Bcl-2/Bax and TP53 increased as compared to the control (except for TP53 in BC and C1/MWCNTs) indicating that cells are responding to the presence of BCN-MWCNTs composites scaffolds. The results suggest that osteoblast developed a modification in the expression profile of genes that actively participate in cellular processes such as proliferation, vasculogenesis and apoptosis, which may be modulated by the increase of intra- and extracellular Ca 2+ concentration., (Copyright © 2020. Published by Elsevier B.V.)

Lipids are fundamental to life as structural components of cellular membranes and for signaling. They are also key regulators of different cellular processes such as cell division, proliferation, and death. Regulated cell death (RCD) requires the engagement of lipids and lipid metabolism for the initiation and execution of its killing machinery. The permeabilization of lipid membranes is a hallmark of RCD that involves, for each kind of cell death, a unique lipid profile. While the permeabilization of the mitochondrial outer membrane allows the release of apoptotic factors to the cytosol during apoptosis, permeabilization of the plasma membrane facilitates the release of intracellular content in other nonapoptotic types of RCD like necroptosis and ferroptosis. Lipids and lipid membranes are important accessory molecules required for the activation of protein executors of cell death such as BAX in apoptosis and MLKL in necroptosis. Peroxidation of membrane phospholipids and the subsequent membrane destabilization is a prerequisite to ferroptosis. Here, we discuss how lipids are essential players in apoptosis, the most common form of RCD, and also their role in necroptosis and ferroptosis. Altogether, we aim to highlight the contribution of lipids and membrane dynamics in cell death regulation., (© 2020 Elsevier Inc. All rights reserved.)

Although investigation with human embryonic stem cells (HESC) is not decreasing, the derivation of new lines has been diminished. The preeminence of only a few HESC lines in research is accompanied by lack of universal applicability of results as well as by genetic under-representation. We previously reported the derivation of one line with male karyotype from Mexican population. Here, we derived one HESC line (Amicqui-2) with female karyotype from poor-quality embryos. These line comply the pluripotent requirements (normal karyotype, detection of pluripotency-associated markers, mycoplasma test and teratoma formation) and could be a valuable model for studying diseases specific to under-represented population., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

MicroRNAs are heterochronic molecules important during brain development, which could be altered by gestational diabetes mellitus (GDM). To explore these molecules in maternal serum, we performed an RT-qPCR analysis. Our results revealed the heterochronic character of some neural development-related microRNA in serum samples of pregnant women. In relation to the first trimester, higher levels of miR-183-5p, -200b-3p, and -125-5p in the second trimester, and higher levels of miR-137 in the third trimester, were found. Furthermore, an insult such as GDM led to higher levels of miR-183-5p, -200b-3p, -125-5p, and -1290 relative to the control in the first trimester, which might be related to changes in neurogenesis and cell proliferation. An in silico analysis suggested that increased microRNAs in the second trimester in the control contributed to cell proliferation and neuron differentiation and that the rise in miR-137 in the third trimester led to neuron maturation. In the diabetic, higher levels of the microRNAs in the first trimester suggested alterations in cell proliferation and neuron differentiation. In conclusion, we showed that fetal-related microRNAs can be detected in the serum of pregnant woman and exhibit temporary regulation during pregnancy and that microRNAs involved in cell proliferation and neuron differentiation are upregulated under GDM., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

In this paper we explore the use of native bacterial cellulose (BC) in combination with functionalized multi-walled carbon nanotubes (MWNTs) as an original biomaterial, suitable three-dimensional (3D) scaffold for osteoblastic cell culture. Functionalized MWNTs were mixed with native BC (secreted by Gluconacetobacter xylinus) with the aim of reinforcing the mechanical properties of BC. The results indicate that BC-MWNTs scaffolds support osteoblast viability, adhesion and proliferation at higher levels as compared to traditional culture substrates. Chemically functionalized MWNTs are also an excellent material to be used as scaffold because these did not affect cell viability and showed an enhanced osteoblast adhesion. These results suggest the potential for this combination of biomaterials, i.e. BC and carbon nanomaterials, as scaffolds for bone regeneration., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introduction: Deficits in cholinergic function have been found in the aged brain and in neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD). The vesicular acetylcholine transporter (VAChT) is a reliable biomarker for the cholinergic system. We previously reported the initial in vitro and ex vivo characterization of (-)-[(11)C]TZ659 as a VAChT specific ligand. Here, we report the in vivo specificity, tracer kinetics, and dose-occupancy studies in the nonhuman primate brain., Methods: MicroPET brain imaging of (-)-[(11)C]TZ659 was performed under baseline conditions in two male macaques. Tracer kinetic modeling was carried out using a two-tissue compartment model (2TCM) and Logan plot with arterial blood input function and using a simplified reference tissue model (SRTM) and Logan plot (LoganREF) without blood input. Specificity for VAChT was demonstrated by pretreatment with (+)-pentazocine, (-)-vesamicol, or S-(-)-eticlopride. Target occupancy (Occ) was calculated following pretreatment with escalating doses of (-)-vesamicol., Results: Baseline PET imaging revealed selective retention in the striatum with rapid clearance from the cerebellar hemispheres as a reference region. Total volume of distribution (VT) values derived from both 2TCM and Logan analysis with blood input revealed ~3-fold higher levels of (-)-[(11)C]TZ659 in the striatum than the cerebellar hemispheres. Injection of (-)-vesamicol either as a blocking or displacing agent significantly reduced striatal uptake of (-)-[(11)C]TZ659. In contrast, pretreatment with the sigma-1 ligand (+)-pentazocine had no impact. Pretreatment with the S-(-)-eticlopride, a dopamine D2-like receptor antagonist, increased striatal uptake of (-)-[(11)C]TZ659. Striatal binding potential (BPND, range of 0.33-1.6 with cerebellar hemispheres as the reference region) showed good correlation (r(2)=0.97) between SRTM and LoganREF. Occupancy studies found that ~0.0057 mg/kg of (-)-vesamicol produced 50% VAChT occupancy in the striatum., Conclusion: (-)-[(11)C]TZ659 demonstrated specific and reversible VAChT binding and favorable pharmacokinetic properties for assessing the density of VAChT in the living brain., (Copyright © 2015 Elsevier Inc. All rights reserved.)

From 2010 to 2012, 35 ciguatera fish poisoning (CFP) events involving 87 individuals who consumed locally-caught fish were reported in Guadeloupe (French West Indies). For 12 of these events, the presence of ciguatoxins (CTXs) was indicated in meal remnants and in uncooked fish by the mouse bioassay (MBA). Caribbean ciguatoxins (C-CTXs) were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Using a cell-based assay (CBA), and the only available standard Pacific ciguatoxin-1 (P-CTX-1), the lowest toxins level detected in fish samples causing CFP was 0.022 µg P-CTX-1 equivalent (eq.)·kg(-1) fish. Epidemiological and consumption data were compiled for most of the individuals afflicted, and complete data for establishing the lowest observable adverse effects level (LOAEL) were obtained from 8 CFP events involving 21 individuals. Based on toxin intakes, the LOAEL was estimated at 4.2 ng P-CTX-1 eq./individual corresponding to 48. 4 pg P-CTX-1 eq.kg(-1) body weight (bw). Although based on limited data, these results are consistent with the conclusions of the European Food Safety Authority (EFSA) opinion which indicates that a level of 0.01 µg P-CTX-1 eq.kg(-1) fish, regardless of source, should not exert effects in sensitive individuals when consuming a single meal. The calculated LOAEL is also consistent with the U.S. Food and Drug Administration guidance levels for CTXs (0.1 µg C-CTX-1 eq.kg(-1) and 0.01 µg P-CTX-1 eq.kg(-1) fish)., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers., (Copyright © 2015. Published by Elsevier B.V.)

Introduction: Escherichia coli is recognized as an etiological bacteria associated with chorioamnionitis and the preterm premature rupture of fetal membranes. This pathological condition induces pro-inflammatory cytokines and degradative metalloproteinases, which are considered biological markers secreted in an acute stage of infection. Heat-shock proteins (HSPs) are an important component of the innate immunity response and are found in different pathological conditions. They have not been previously measured in human fetal membranes in response to infectious conditions. We hypothesized that the choriodecidual tissue and amniotic epithelium secreted temporal and differential Hsp-60, Hsp-70, and interleukin (IL)-1β mediated by E. coli infection., Methods: Fetal membranes were mounted in a two-compartment culture system and infected with two passes of live E. coli at different doses (10², 10⁴, 10⁵, and 10⁶ colony-forming units (CFU)/mL) and intervals of incubation (3, 6, and 24 h). The culture medium was collected, and Hsp-60, Hsp-70, and IL-1β were assessed using the enzyme-linked immunosorbent assay (ELISA) method., Results: After 3 and 6 h of infection, E. coli induced an increase in Hsp-70 secretion in the choriodecidual tissue. However, after 24 h of incubation, Hsp-70 was downregulated and we observed an increase in IL-1β secretion. By contrast, E. coli induced a lower Hsp-60 secretion in the amnion compared to Hsp-70., Discussion: Human fetal membranes responded actively to E. coli infection, with an increase in Hsp-70 during the first hours of infection. After 24 h, there was an increase in the liberation of IL-1β., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Seminal coagulum formation in spider monkeys (Ateles geoffroyi) interferes with the efficient recovery and evaluation of spermatozoa. The main objective was to assess the effect of increasing concentrations of trypsin on dissolution of seminal coagulum and spermatic parameters. Seminal coagulum was incubated at 37 °C without trypsin or in the presence of increasing trypsin concentrations (0.1%, 1.0%, and 5.0%). For each sample, coagulum dissolution time was measured, and sperm concentration, viability, motility, and morphology were evaluated using light microscopy and/or transmission electronic microscopy (TEM). Trypsin concentrations of 1.0% and 5.0% more rapidly liquefied seminal coagulum, averaging 32 and 21 min, respectively, compared with nontrypsinized controls, with maintenance of greater sperm viability (70.8% and 72.5%, respectively). Coagulum treated with 1.0% trypsin and the liquid ejaculate fraction averaged higher sperm motility (40.1% and 55.6%, respectively) than control samples, and both 1.0% and 5.0% trypsin treatment allowed recovery of increased numbers of motile spermatozoa. There was greater sperm fragmentation at the head and midpiece level after treatment with 1.0% and 5.0% trypsin (55.8% and 55.9%); however, the percentage of normal morphology in structurally intact spermatozoa did not differ relative to controls. With transmission electronic microscopy imaging, there were similar percentages of spermatozoa with plasma membrane swelling in the midpiece and acrosomal regions in trypsin-treated samples and controls. In conclusion, trypsin treatment of spider monkey seminal coagulum exerted a concentration-dependent effect on dissolution time and various spermatic parameters. Higher trypsin concentrations caused more rapid liquefaction of coagulum and recovery of greater numbers of motile spermatozoa, but may adversely affect fragmentation of spermatozoa and could compromise sperm function and cryopreservation potential., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Overall, 1-4% of all births in the US are complicated by choriamnionitis. Choriamnionitis is a polymicrobial infection most often due to ascending genital microbes which, in over 65% of positive amniotic fluid cultures, involves two or more organisms. In this study, we determine the cytokines expression (IL-1β, TNFα) and prometalloproteinase activation (proMMP-2 and proMMP-9) after double o single infection an in vitro model of human fetal membranes. Fetal membranes at term were mounted in the Transwell culture system and after 24 h of infection, choriodecidual, and amnion media was collected. IL-1β and TNFα were evaluated by ELISA, whereas proMMP-9 and proMMP-2 were determined by substrate gel zymography. The choriodecidual and amnion compartments actively respond to the infectious process, which induced the secretion of IL-1β, TNFα, and proMMP-9 after either mixed or single infection. The proMMP-2 secretion profile was the same after all experimental conditions. There was no synergy between Streptococcus agalactiae and Escherichia coli for inducing the secretion of inflammatory factors or degradative metalloproteinase. In conclusion, these two bacteria could initiate different pathways to induce chorioamnioitis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: In 1997 the US Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) reported high proportions of drug-resistant Mycobacterium tuberculosis in three Mexican states: Sinaloa, Baja California, and Oaxaca. In 2006, we showed that resistance to anti-tuberculosis drugs remained frequent in Sinaloa., Objectives: The objectives of this study were to describe drug-resistant tuberculosis (TB) trends and to investigate the probability that patients acquire resistance to first-line anti-TB drugs on recurrence after treatment in Sinaloa., Methods: Sputum specimens were collected from patients diagnosed with TB at all the health care institutions of Sinaloa during 1997-2005. Isolates were tested for susceptibility to first-line drugs., Results: Among 671 isolates tested from 1997 to 2002, the overall resistance rate was 34.9% (95% confidence interval (CI) 31.2-38.4) with a 1.2% increase per year (Chi-square=4.258, p=0.03906). The prevalence of multi-drug resistance (MDR) was 17.9% (95% CI 14.9-20.7) with a 1.2% increase per year (Chi-square=8.352, p=0.00385). Of 50 patients registered twice between 1997 and 2005, 15 were fully susceptible at first registration, of whom six (40%) acquired drug resistance. Of 35 cases with any drug resistance at first registration, 21 (60%) came to acquire resistance to at least one other drug., Conclusions: The proportion of drug-resistant TB increased during 1997-2005 in Sinaloa. Major efforts are needed to prevent the further rise and spread of drug-resistant and MDR TB., (Copyright © 2011 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Objective: The aim was to compare the clinical and radiographic efficacy of enamel matrix derivative and self-hardening calcium hydroxide as direct pulp capping materials on decayed primary molars, with observation periods of 1, 6, and 12 months., Study Design: A clinical, randomized, controlled trial was performed, following the "split-mouth" design. A total of 90 primary molars were treated. Assignation of materials and operative initial side were selected in a randomized manner. Five outcome variables were considered: internal dentin resorption, pain, gingival sinus tract, root external resorption, and pathologic mobility. The appearance of any of these signs or symptoms was considered to be a failure of treatment., Results: Significant statistical or clinical differences were not found between the study groups. Two treatments were judged as failures, 1 per study group; both occurred during the first postoperative month., Conclusions: The technique used for direct pulp capping on primary molars in this study is recommended on the basis of the obtained clinical and radiographic results.

Background: The contraceptive effect of the progestogen norethisterone (NET) and its main metabolites 5alpha-NET and 3beta,5alpha-NET has been demonstrated in several species, and most studies have focused on the effects of these compounds in the uterus. We previously reported that 5alpha-NET inhibits the progesterone (P(4))-induced acrosome reaction in pig and mouse spermatozoa and induces severe morphological damage in two-cell fertilized mouse oocytes., Study Design: The main goal of this study was to analyze the possible role of P(4) receptor (PR) in the effects of NET and 5alpha-NET on the oocyte fertilization process. Different steroid treatments were used with or without cumulus-enclosed oocytes., Results: It was demonstrated that NET increases the percentage of fertilized oocytes in the same manner as P(4) does, while 5alpha-NET reduces the percentage of fertilized oocytes. This effect was not reversed by P(4) in the same concentrations., Conclusion: A possible molecular mechanism for the effects of 5alpha-NET may be through a PR localized in the oocyte plasma membrane.

Contemporary enzymes are highly efficient and selective catalysts. However, due to the intrinsically very reactive nature of active sites, gratuitous secondary reactions are practically unavoidable. Consequently, even the smallest cell, with its limited enzymatic repertoire, has the potential to carry out numerous additional, very likely inefficient, secondary reactions. If selectively advantageous, secondary reactions could be the basis for the evolution of new fully functional enzymes. Here, we investigated if Escherichia coli has cryptic enzymatic activities related to thiamin biosynthesis. We selected this pathway because this vitamin is essential, but the cell's requirements are very small. Therefore, enzymes with very low activity could complement the auxotrophy of strains deleted of some bona fide thiamin biosynthetic genes. By overexpressing the E. coli's protein repertoire, we selected yjbQ, a gene that complemented a strain deleted of the thiamin phosphate synthase (TPS)-coding gene thiE. In vitro studies confirmed TPS activity, and by directed evolution experiments, this activity was enhanced. Structurally oriented mutagenesis allowed us to identify the putative active site. Remote orthologs of YjbQ from Thermotoga, Sulfolobus, and Pyrococcus were cloned and also showed thiamin auxotrophy complementation, indicating that the cryptic TPS activity is a property of this protein family. Interestingly, the thiE- and yjbQ-coded TPSs are analog enzymes with no structural similarity, reflecting distinct evolutionary origin. These results support the hypothesis that the enzymatic repertoire of a cell such as E. coli has the potential to perform vast amounts of alternative reactions, which could be exploited to evolve novel or more efficient catalysts.

HSCT provides the opportunity to replace a damaged tissue. It is the most important treatment for high risk hematologic malignant and non malignant disorders. An important challenge in the identification of matched donors/patients is the HLA diversity. The Mexican Bone Marrow Registry (DONORMO) has nowadays > 5000 donors. The prevalent alleles are Amerindian, Mediterranean (Semitic and Spanish genes) and African. In theory, it is possible to find 11% of 6/6 A-B-DR low resolution matches for 70% of patients with Mexican ancestry. We contributed with 39 unrelated, cord blood and autologous HSCT for patients with malignant, genetic and autoimmune disorders. Overall disease survival was 50% (2-7 years) depending on the initial diagnosis, conditioning, disease evolution or other factors. Clinical studies using autologous and unrelated HSC are performed on patients with refractory autoimmune diseases producing mixed results: mainly, T1D, RA, MS, SLE. Improvement has been observed in skin damage and quality of life in SLE and systemic sclerosis. Disease stabilization in 2/3 of MS patients. However, in RA and T1D, initial benefits have been followed by eventual relapse. With growing clinical experience and protocol improvement, treatment-related mortality is decreasing. Proof efficacy will be achieved by comparing HSCT with standard therapy in autoimmunity.

Autoimmune diseases need to be considered at a genetic and mechanistic level. T1D is an autoimmune, chronic, multifactorial and polygenic disease characterized by the destruction of the pancreatic beta-cells associated with long term dysfunction of several organs and tissues. Mechanisms of susceptibility include epi-genetic and post-transcriptional effects that regulate transmission and expression of the inherited genes. The HLA complex, constitutes the most relevant region contributing 50% of the inherited risk for T1D. An additional 17 genes with variable but small effects have been described. In non-Caucasians, the presence of E-DRbeta1-74 and/or D-DRbeta1-57 are relevant in predisposition. The "Diabetogenic haplotypes" in Mexicans were DRB1*0301-DQA1*0501-DQB1*0201 (OR = 21.4); DRB1*0405-DQA1-*0301-DQB1*0302 (OR = 44.5) and the same DQA1/DQB1 with DRB1*0404/*0401 conferring lower risk, increasing (OR = 61.3) with an early age at onset and a heterozygote DR3/DR4 genotype. In most populations, the absence of D-57 and the presence of R-52 are important to the susceptibility, but in Hispanics, all DR4s (including the protective DRB1*0403/*0407/*0411) are in linkage disequilibrium with DQA1/DQB1 susceptibility alleles. Thus, susceptibility alleles in Latin American Mestizos are of Mediterranean ancestry whereas protective alleles are of Amerindian origin. In this review, we discuss the complexity of T1D and some aspects of prevention/intervention based on immunogenetics.

The aim of this study was to determine the role of certain bovine lymphocyte antigens (BoLA) regions in the resistance or susceptibility to Boophilus microplus tick infestation in two different breeds of cattle. The breeds were maintained, one in natural conditions and the second one in an experimental setting at the research station in Martinez de la Torre, Veracruz, Mexico. The study took place from June to August 2001 (natural infestation) using 33 crossbreed steers (crossbreed is here defined as 3/4 European = 1/2 Simmenthal x 1/4 Holstein x 1/4 Zebu, a cross resulting from F1 x Simmenthal), ranging from 15 to 20 months old. Fifty-nine F1 cows (1/2 Holstein x 1/2 Zebu) were included in the experimental setting, infested and followed during 25 days in November 2001 and 2002. Experiment A included thirty-one 2-7-year-old F1 cows, and experiment B included twenty-eight 18-24-month-old F1 heifers. Both groups were analysed separately and were not comparable because of the different infestation methods and genetic background. All ticks > or =4mm long were counted on the total body of F1 animals and on one side of the 3/4 European steers. In this case, susceptible animals were defined when having ticks = X + 1S.D. (29 +/- 16). In the experimental setting susceptibility was defined when the number of ticks was over the 75 percentile (> or =79). DNA was extracted from peripheral blood samples of all animals. The BoLA DRB3, DRBP1, RM185 and BM1815 microsatellite loci were amplified using a PCR method. Genescan software was used for analysis in an ABI sequencer. The SPSS statistical program was used and the comparisons were assessed using the Fisher's exact test. In the naturally infested animals, DRB3-184 was found positively associated with tick infestation (P = 0.018; Pc = NS; OR = 5; EF = 28%). DRBP1-128 was also found to be increased (P = 0.03; Pc = NS; OR = 6; EF = 42%). In the experimentally infested animals, two more loci were found to be associated, BM1815-152 (P = 0.01; Pc = NS; OR = 15; EF = 74%) and DRBP1-130 (P = 0.05; Pc = NS; OR = 4; EF = 77%). None of them remained significant after correction, indicating that a larger sample size is needed to confirm the results. This is the first study showing MHC genes associated with tick infestation based on class II microsatellite polymorphisms. Further studies are needed to confirm the susceptibility traits and to determine haplotype segregation in families.

The reporter enzyme beta-glucuronidase was mutagenized and evolved for thermostability. After four cycles of screening the best variant was more active than the wild-type enzyme, and retained function at 70 degrees C, whereas the wild-type enzyme lost function at 65 degrees C. Variants derived from sequential mutagenesis were shuffled together, and re-screened for thermostability. The best variants retained activities at even higher temperatures (80 degrees C), but had specific activities that were now less than that of the wild-type enzyme. The mutations clustered near the tetramer interface of the enzyme, and many of the evolved variants showed much greater resistance to quaternary structure disruption at high temperatures, which is also a characteristic of naturally thermostable enzymes. Together, these results suggest a pathway for the evolution of thermostability in which enzymes initially become stable at high temperatures without loss of activity at low temperatures, while further evolution leads to enzymes that have kinetic parameters that are optimized for high temperatures., (Copyright 2002 Academic Press.)

The EcoRI endonuclease is an important recombinant DNA tool and a paradigm of sequence-specific DNA-protein interactions. We have isolated temperature-sensitive (TS) EcoRI endonuclease mutants (R56Q, G78D, P90S, V97I, R105K, M157I, C218Y, A235E, M255I, T261I and L263F) and characterized activity in vivo and in vitro. Although the majority were TS for function in vivo, all of the mutant enzymes were stably expressed and largely soluble at both 30 degrees C and 42 degrees C in vivo and none of the mutants was found to be TS in vitro. These findings suggest that these mutations may affect folding of the enzyme at elevated temperature in vivo. Both non-conservative and conservative substitutions occurred but were not correlated with severity of the mutation. Of the 12 residues identified, 11 are conserved between EcoRI and the isoschizomer RsrI (which shares 50% identity), a further indication that these residues are critical for EcoRI structure and function. Inspection of the 2.8 A resolution X-ray crystal structure of the wild-type EcoRI endonuclease-DNA complex revealed that: (1) the TS mutations cluster in one half of the globular enzyme; (2) several of the substituted residues interact with each other; (3) most mutations would be predicted to disrupt local structures; (4) two mutations may affect the dimer interface (G78D and A235E); (5) one mutation (P90S) occurred in a residue that is part of, or immediately adjacent to, the EcoRI active site and which is conserved in the distantly related EcoRV endonuclease. Finally, one class of mutants restricted phage in vivo and was active in vitro, whereas a second class did not restrict and was inactive in vitro. The two classes of mutants may differ in kinetic properties or cleavage mechanism. In summary, these mutations provide insights into EcoRI structure and function, and complement previous genetic, biochemical, and structural analyses., (Copyright 1997 Academic Press Limited.)

Serum vitamin A was determined before and 30-45 d after the administration of 60.6 mg (212 mumol) vitamin A to 544 Brazilian children residing in slum areas of Recife. The frequency-distribution curves were compared in a subgroup of children whose vitamin A status was assessed initially by the relative-dose-response (RDR) test. The curves of children with negative (adequate status) and positive (inadequate status) RDR tests were different. The difference disappeared after supplementation. The shape of the distribution curve after supplementation was close to normal with a mean, median, and 95% confidence interval of 1.78 +/- 0.49, 1.68, and 1.02-2.90 mumol/L, respectively. The postsupplementation curve derived from this underprivileged child population may serve as a reference for diagnostic, surveillance, and program-evaluation purposes.

Vitamin A status was determined using fasting plasma levels and the relative dose response (RDR) procedure before and 30, 120, and 180 days after administration of an oral massive (200,000 IU) dose of vitamin A. The study was carried out in Recife, Brazil among 93 children of 7 yr or less from low income families who attended two day-care programs. The RDR procedure is conducted by obtaining a fasting blood (A0), feeding 450 retinol equivalents and obtaining a second blood specimen after 5 h (A5). The RDR-(A5-A0)/A5 X 100. A single massive oral dose of vitamin A was given after conducting the base-line RDR test. The RDR procedure was repeated at 30, 120, and 180 days. Weight and height measurements were obtained at each observation and the bloods were analyzed for Hb, total protein, and serum iron, as well as vitamin A. Serum albumin was determined in 120- and 180-day bloods. Serum iron levels were improved 30 days after supplementation with the massive dose of vitamin A. The RDR procedure was found practical to apply in mildly undernourished children under nonclinical condition. In this group of low income children presumed to be habitually ingesting minimally adequate diets, a serum vitamin A level of 20 micrograms/dl or less invariably was associated with an elevated RDR test. Blood levels between 20 to 40 micrograms/dl were not consistently predictive of the RDR response. All elevated RDR tests reverted to normal after supplementation with vitamin A, presumably indirectly indicating a presupplementation inadequate vitamin A status. Hence, the RDR was a more sensitive indicator of inadequate vitamin A status than was only a serum level of vitamin A when blood levels were above 20 micrograms/dl.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of an infective episode of chickenpox on the vitamin A status of preschool-aged children was evaluated by use of the relative dose response (RDR) test. Status was determined before and 30, 120, and 180 d after administration of a single oral high-dosage (200,000 IU) supplement of vitamin A. No differences in mean blood levels of retinol or percentage of children showing a positive RDR were apparent until after the infective episode that occurred approximately 90 d after dosing. At 180 d postsupplementation, 74% of children who had been infected tested positive by the RDR, indicative of an inadequate liver reserve of vitamin A, in contrast to only 10% who had not been infected. Paired RDR observations at 0 and 180 d postsupplementation confirmed that the infective episode caused an accelerated depletion of liver reserves of vitamin A.