Mpox-Related Ophthalmic Disease: A Retrospective Observational Study in a Single Center in Mexico.

Mpox-related ophthalmic disease has been reported as infrequent. We retrospectively describe the ocular manifestations present in 11 of 100 patients with confirmed mpox; 9 were people with HIV. We suggest that an ophthalmological evaluation should be performed in all patients with ocular symptoms or moderate and severe mpox disease.

Executive functions in deaf and hearing children: The mediating role of language skills in inhibitory control.

This study aimed to analyze one of the critical components of inhibitory control-the ability to suppress interference-in deaf and hearing children and to investigate the mediating role of language skills in this central component of executive functions. To this end, a cross-sectional study was carried out with 40 deaf children with and without cochlear implants (CIs) and 21 hearing children age 7-10 years. The ability to suppress interference was assessed with the children's version of the Attentional Network Test (child-ANT), and language skills were assessed with a computerized version of the Carolina Picture Vocabulary Test (CPVT), a receptive vocabulary test. As a measure of control of nonverbal cognitive abilities, we used the Test of Nonverbal Intelligence (TONI-2). The results showed that deaf children had lower nonverbal IQ than hearing children. In addition, deaf children, compared with hearing children and regardless of whether they used CIs, showed a lower range of receptive vocabulary and a poorer ability to suppress the interference of distractors in the child-ANT. Linear regression mediation analyses revealed that this more significant interference effect was mediated by receptive vocabulary level and not by hearing deprivation. These results are consistent with the hypothesis that language is one of the critical factors in the development of executive functions.

Genetic homogeneity coupled with morphometric variability suggests high phenotypic plasticity in the sea louse Caligus rogercresseyi (Boxshall and Bravo, 2000), infecting farmed salmon (Salmo salar) along a wide latitudinal range in southern Chile.

The sea louse Caligus rogercresseyi is the most important pathogen causing "caligidosis" in the Chilean salmon industry. In this study, using cox1 gene, we evaluate the genetic variation of C. rogercresseyi from farmed Salmo salar along a latitudinal range (40°-52°S) in south Chile to determine whether morphological differences are explained by genetic or environmental factors. Female parasites were randomly collected from S. salar at five farms. Body variation was examined using multivariate analyses and genetic heterogeneity was explored with AMOVA. C. rogercresseyi exhibited significant morphometric variability among sites and parasites collected from >54°S were the longest ones. Parasites did not show genetic structure among farms. Thus, C. rogercresseyi infesting salmons is panmictic along an extensive latitudinal range in south Chile. The same genetic pattern can be explained by the frequent movement of parasitized S. salar among farms in that region. Phenotypic plasticity in parasites could be explained by natural or aquaculture-mediated environment variability. C. rogercreseyi from 54°S could favor the local spread of this disease, suggesting an immediate health risk for the recent salmon industry in that region. Further research is required to confirm genetic homogeneity of this parasite along its geographical distribution using more powerful markers (e.g. SNPs).

Dissimilatory reduction of Fe(III) by a novel Serratia marcescens strain with special insight into the influence of prodigiosin.

A novel pigmented bacterium, initially identified as 11E, was isolated from a site historically known to have various iron-related ores. Phylogenetic analysis of this bacterial strain showed that it belongs to Serratia marcescens. This pigmented S. marcescens 11E cultured individually with glucose, acetate, and glycerol as electron donors along with the soluble electron acceptor iron (Fe) (III) citrate offered a large reduction extent (45.3 %, 31.4 %, and 13.5 %, respectively). On the other hand, when iron oxide (Fe2O3) is used as electron acceptor, the pigmented strain produced a null reduction extent. Surprisingly, the absence of prodigiosin on the bacterial surface (non-pigmented strain) resulted in a large reduction extent of the non-soluble iron form (20-49%). All these extents were comparable and, in some cases, superior to those presented in the literature. Additionally, in the present study, it was found that anthraquinone sulfonate (AQS) stimulated Fe(III) reduction of soluble and non-soluble Fe species only with pigmented S. marcescens. In contrast, in the culture media with the non-pigmented strain, the presence of AQS did not stimulate the Fe(III) reduction. These results suggest that the pigmented phenotype of S. marcescens 11E may perform non-soluble Fe(III) reduction by electron shuttling. In contrast, for the non-pigmented phenotype of this bacterium, non-soluble Fe(III) reduction seems to proceed by direct contact. Our study demonstrates that this bacterium may be used in bioreduction process of heavy metals or as a biocatalyst in bioelectrochemical devices.

Phase Entropy Analysis of Electrohysterographic Data at the Third Trimester of Human Pregnancy and Active Parturition.

Phase Entropy (PhEn) was recently introduced for evaluating the nonlinear features of physiological time series. PhEn has been demonstrated to be a robust approach in comparison to other entropy-based methods to achieve this goal. In this context, the present study aimed to analyze the nonlinear features of raw electrohysterogram (EHG) time series collected from women at the third trimester of pregnancy (TT) and later during term active parturition (P) by PhEn. We collected 10-min longitudinal transabdominal recordings of 24 low-risk pregnant women at TT (from 35 to 38 weeks of pregnancy) and P (>39 weeks of pregnancy). We computed the second-order difference plots (SODPs) for the TT and P stages, and we evaluated the PhEn by modifying the k value, a coarse-graining parameter. Our results pointed out that PhEn in TT is characterized by a higher likelihood of manifesting nonlinear dynamics compared to the P condition. However, both conditions maintain percentages of nonlinear series higher than 66%. We conclude that the nonlinear features appear to be retained for both stages of pregnancy despite the uterine and cervical reorganization process that occurs in the transition from the third trimester to parturition.

Influence of 1,3,5-triazine Core and Electron Donor Group in Photophysical Properties of BODIPY Dyes.

The relationship between the number of BODIPY in a compound and the increase on its fluorescence has been established such as an aggregation induced by multiple BODIPY. We aimed to determine the influence of an electron donor substituent in the BODIPY-triazine system. In this sense, as a first step, we collected data such as photophysical characteristics about BODIPY without substituent and meso-triazine-BODIPY system. Then, three more meso-triazine-BODIPY were synthetized by Lyndsey method. In addition, absorption and emission spectra, fluorescence quantum yields and time-resolved fluorescence data were obtained. Furthermore, solvatochromism was determined by solvent descriptors and photophysical parameters. Finally, the results showed that the triazine core stabilized the system and we observed that the number of BODIPY increased fluorescence mainly in polar solvents. While electron donation maintained the conjugation that reduced the influence of the solvent on the photophysical characteristics.

Histopathological lesions, pathogens and parasites as health indicators of an edible clam (Protothaca thaca) inhabiting a bay exposed to anthropogenic activities in Northern Chile.

San Jorge Bay (23° S) is characterised by a permanent coastal upwelling front and a thermal front that influence water circulation into the bay. This bay constantly receives effluents from different mining activities. Several studies have demonstrated different levels of heavy metals in waters and sediments within the bay. The clam Protothaca thaca is a bivalve with sedentary habits, which is distributed along the Peruvian and Chilean coasts and is exploited commercially. During 2010, clams were collected inside the bay: north (La Chimba), centre (Paraíso) and south (Llacolén), as well as from a site outside the bay (Bolsico). Haematological parameters, condition factor index, lesions, parasites and pathogens and heavy metal concentrations in clam tissues were determined. The health indicators of clams inhabiting the bay varied between sites. Clams inside the bay showed higher prevalence and intensity of parasites and lesions than those clams inhabiting the site outside the bay, which could be indicating the presence of some environmental stressor (e.g. heavy metals). This study is the first to evaluate the health status of organisms from San Jorge Bay, and our results support the hypothesis that clams P. thaca can be used to evaluate environmental quality.

Maltrato infantil: aspectos jurídicos en México.

In Mexico, the complexity of the comprehensive care of a girl, boy or adolescent who is the victim of any form of child abuse requires the necessary legal knowledge for comprehensive management. Physicians probably lack sufficient knowledge of the laws to understand and correctly address the legal aspects immersed in the comprehensive care of this population group. Considering that child abuse has been characterized as a world-wide medical-social-legal problem, it is necessary to know the essential legal mechanisms to protect minors who are victims of it and understand the legal status of their families and of the probable aggressor. The purpose of this article is to present the existing legal framework in Mexico and the actions of world organizations in this area. It is necessary for the Political Constitution of the United Mexican States to be the basis on which legal behaviors related to child abuse, crime investigation and administration of justice are founded, as well as for international agreements on the subject, which have been signed by the government of Mexico, to be adhered to.

En México, la complejidad de la atención integral de una niña, niño o adolescente víctima de cualquier modalidad de maltrato infantil requiere el conocimiento jurídico indispensable para su manejo integral. Los médicos probablemente no tienen un conocimiento suficiente de las leyes para entender y atender correctamente los aspectos legales involucrados en la atención integral a este grupo de la población. Considerando que el maltrato infantil ha sido catalogado como un problema médico-social-legal de alcance mundial, se hace necesario el conocimiento de los mecanismos jurídicos indispensables para proteger a los menores que son víctimas y entender la condición legal de su familia y del probable agresor. El objetivo de este artículo es presentar el marco jurídico existente en México y las acciones en este rubro de las organizaciones mundiales. Se precisa que la Constitución Política de los Estados Unidos Mexicanos sea la base en la que se fundamenten las conductas legales vinculadas con el maltrato infantil, la investigación de delitos y la administración de justicia, así como los convenios internacionales sobre el tema, los cuales han sido suscritos por el gobierno de México.

Intraocular Penetration of a vNAR: In Vivo and In Vitro VEGF165 Neutralization.

Variable new antigen receptor domain (vNAR) antibodies are novel, naturally occurring antibodies that can be isolated from naïve, immune or synthetic shark libraries. These molecules are very interesting to the biotechnology and pharmaceutical industries because of their unique characteristics related to size and tissue penetrability. There have been some approved anti-angiogenic therapies for ophthalmic conditions, not related to vNAR. This includes biologics and chimeric proteins that neutralize vascular endothelial growth factor (VEGF)165, which are injected intravitreal, causing discomfort and increasing the possibility of infection. In this paper, we present a vNAR antibody against human recombinant VEGF165 (rhVEGF165) that was isolated from an immunized Heterodontus francisci shark. A vNAR called V13, neutralizes VEGF165 cytokine starting at 75 µg/mL in an in vitro assay based on co-culture of normal human dermal fibroblasts (NHDFs) and green fluorescence protein (GFP)-labeled human umbilical vein endothelial cells (HUVECs) cells. In the oxygen-induced retinopathy model in C57BL/6:Hsd mice, we demonstrate an endothelial cell count decrease. Further, we demonstrate the intraocular penetration after topical administration of 0.1 µg/mL of vNAR V13 by its detection in aqueous humor in New Zealand rabbits with healthy eyes after 3 h of application. These findings demonstrate the potential of topical application of vNAR V13 as a possible new drug candidate for vascular eye diseases.

Reduction of chromium (VI) from aqueous solution by biomass of Cladosporium cladosporioides.

The capacity of Cladosporium cladosporioides biomass for removal of Cr(VI) in aqueous solutions was evaluated. A 2 × 2 factorial experiment design was used to study the effects of pH and biomass doses. Lower pH values and larger biomass doses increased the capacity of C. cladosporioides biomass for removal of Cr(VI), reaching a reduction capacity of 492.85 mg g-1, a significantly higher value compared to other biomass reported. Cr(VI) removal kinetic rates followed a pseudo-second order model, like other fungal biomass reported previously. The apparent adsorption process was described well by the Freundlich isothermal model. However, determination of total chromium indicated that adsorption of Cr(VI) was followed by a redox reaction that released proportional quantities of Cr(III) into the experimental supernatant, suggesting a parallel adsorption-reduction process. Comparison of Fourier transform infrared spectroscopy spectra of C. cladosporioides biomass before and after the reduction process demonstrated the involvement of positively charged amino groups in the Cr(VI) adsorption-reduction process.

A high proportion of cells carrying trisomy 12 is associated with a worse outcome in patients with chronic lymphocytic leukemia.

The prognosis of chronic lymphocytic leukemia (CLL) patients displaying trisomy 12 (+12) remains unclear. In this study, we analyzed the influence of the proportion of cells with +12, and other clinical and biologic factors, in time to first therapy (TTFT) and overall survival (OS), in 289 patients diagnosed with CLL carrying +12. Median OS was 129 months. One hundred seventy-four patients (60.2%) presented +12 in <60% of cells. TTFT and OS for this subgroup were longer than for the subgroup with +12 in ≥60% of cells, with a median TTFT of 49 months (CI95%, 39-58) vs 30 months (CI95%, 22-38) (P = 0.001); and a median OS of 159 months (CI95%, 119-182), vs 96 months (CI95%, 58-134) (P = 0.015). Other factors associated with a shorter TTFT were: Binet stage, B symptoms, lymphadenopathy, splenomegaly, high lymphocyte count, 11q-, high ß2 microglobulin, and high LDH. In the multivariate analysis, clinical stage, +12 in ≥60% of cells, high lymphocyte count, B symptoms, and 11q- in addition, resulted of significance in predicting shorter TTFT. Significant variables for OS were: Binet stage, lymphadenopathy, splenomegaly, high LDH, high ß2 microglobulin, 11q-, and CD38. In the multivariate analysis, only Binet stage, 11q-, and high ß2microglobulin significantly predicted shorter OS. CLL with +12 entails a heterogeneous group with intermediate prognosis. However, a high proportion of cells carrying +12 separates a subgroup of patients with poor outcome. Copyright © 2015 John Wiley & Sons, Ltd.

Parasite richness in fish larvae from the nearshore waters of central and northern Chile.

In the present study, we determine the presence of parasites in fish larvae collected from nearshore waters along the northern and central coast of Chile. The parasites were identified to the lowest possible taxonomic level based on morphological and molecular analyses. The fish sample was composed of 5 574 fish larvae. Of these, 3% harboured only larval ectoparasitic copepods whereas no endoparasites were found in the 1 141 fish evaluated for this group of parasites. The parasitic copepods collected were initially classified as 'morphotypes' according to differences in morphological characteristics. They were then analysed using molecular techniques based on the 28S and COI genes. Seven morphotypes of parasitic copepods (mostly at chalimus stages) were recognised: two of the morphotypes belonged to Pennellidae Burmeister, 1835, three to Caligidae Burmeister, 1835 and two were not identified. Only five morphotypes of copepods were analysed using molecular sequences, which confirmed the existence of six species: two pennellids of the genus Trifur Wilson, 1917 and two caligids of the genus Caligus Müller, 1785, plus two additional species that were morphologically different from these taxa. The pennellids were present in several fish species, being generally more prevalent than the caligids, in both the central and northern localities of Chile. Multispecies infections in larval fish were infrequent (< 1%). We conclude that fish larvae were rich in parasites, considering that these hosts exhibited small body sizes and were very young. We suggest that fish larvae could play a role, as intermediate hosts, in the life cycle of the parasitic copepods found.

Interstitial 13q14 deletions detected in the karyotype and translocations with concomitant deletion at 13q14 in chronic lymphocytic leukemia: different genetic mechanisms but equivalent poorer clinical outcome.

Deletion of 13q14 as the sole abnormality is a good prognostic marker in chronic lymphocytic leukemia (CLL). Nonetheless, the prognostic value of reciprocal 13q14 translocations [t(13q)] with related 13q losses has not been fully elucidated. We described clinical and biological characteristics of 25 CLL patients with t(13q), and compared with 62 patients carrying interstitial del(13q) by conventional G-banding cytogenetics (CGC) [i-del(13q)] and 295 patients with del(13q) only detected by fluorescence in situ hybridization (FISH) [F-del(13q)]. Besides from the CLL FISH panel (D13S319, CEP12, ATM, TP53), we studied RB1 deletions in all t(13q) cases and a representative group of i-del(13q) and F-del(13q). We analyzed NOTCH1, SF3B1, and MYD88 mutations in t(13q) cases by Sanger sequencing. In all, 25 distinct t(13q) were described. All these cases showed D13S319 deletion while 32% also lost RB1. The median percentage of 13q-deleted nuclei did not differ from i-del(13q) patients (73% vs. 64%), but both were significantly higher than F-del(13q) (52%, P < 0.001). Moreover, t(13q) patients showed an increased incidence of biallelic del(13q) (52% vs. 11.3% and 14.9%, P < 0.001) and higher rates of concomitant 17p deletion (37.5% vs. 8.6% and 7.2%, P < 0.001). RB1 involvement was significantly higher in the i-del(13q) group (79%, P < 0.001). Two t(13q) patients (11.8%) carried NOTCH1 mutations. Time to first treatment in t(13q) and i-del(13q) was shorter than F-del(13q) (67, 44, and 137 months, P = 0.029), and preserved significance in the multivariate analysis. In conclusion, t(13q) and del(13q) patients detected by CGC constitute a subgroup within the 13q-deleted CLL patients associated with a worse clinical outcome.

A comprehensive study of extended tetrathiafulvalene cruciform molecules for molecular electronics: synthesis and electrical transport measurements.

Cruciform-like molecules with two orthogonally placed π-conjugated systems have in recent years attracted significant interest for their potential use as molecular wires in molecular electronics. Here we present synthetic protocols for a large selection of cruciform molecules based on oligo(phenyleneethynylene) (OPE) and tetrathiafulvalene (TTF) scaffolds, end-capped with acetyl-protected thiolates as electrode anchoring groups. The molecules were subjected to a comprehensive study of their conducting properties as well as their photophysical and electrochemical properties in solution. The complex nature of the molecules and their possible binding in different configurations in junctions called for different techniques of conductance measurements: (1) conducting-probe atomic force microscopy (CP-AFM) measurements on self-assembled monolayers (SAMs), (2) mechanically controlled break-junction (MCBJ) measurements, and (3) scanning tunneling microscopy break-junction (STM-BJ) measurements. The CP-AFM measurements showed structure-property relationships from SAMs of series of OPE3 and OPE5 cruciform molecules; the conductance of the SAM increased with the number of dithiafulvene (DTF) units (0, 1, 2) along the wire, and it increased when substituting two arylethynyl end groups of the OPE3 backbone with two DTF units. The MCBJ and STM-BJ studies on single molecules both showed that DTFs decreased the junction formation probability, but, in contrast, no significant influence on the single-molecule conductance was observed. We suggest that the origins of the difference between SAM and single-molecule measurements lie in the nature of the molecule-electrode interface as well as in effects arising from molecular packing in the SAMs. This comprehensive study shows that for complex molecules care should be taken when directly comparing single-molecule measurements and measurements of SAMs and solid-state devices thereof.

Does a cyclopropane ring enhance the electronic communication in dumbbell-type C60 dimers?

Two C60 dumbbell molecules have been synthesized containing either cyclopropane or pyrrolidine rings connecting two fullerenes to a central fluorene core. A combination of spectroscopic techniques reveals that the cyclopropane dumbbell possesses better electronic communication between the fullerenes and the fluorene. This observation is underpinned by DFT transport calculations, which show that the cyclopropane dumbbell gives a higher calculated single-molecule conductance, a result of an energetically lower-lying LUMO level that extends deeper into the backbone. This strengthens the idea that cyclopropane behaves as a quasi-double bond.

Drug-induced sleep endoscopy: a two drug comparison and simultaneous polysomnography.

The purpose of the present study was to compare pharyngeal and polysomnographical findings during drug-induced sleep endoscopy (DISE) performed with either propofol or midazolam as a single sedative agent. It is prospective, non-randomized, double-blinded case series study. Sixteen patients with sleep disordered breathing were sedated first with propofol and after full wake up with midazolam. Simultaneous polysomnography (PSG) was performed. We compared the zones of obstruction and vibration found with both drugs using the VOTE classification. Simultaneous PSG findings are also compared. There were 15 men and one woman; the mean age was 42.7 years old, mean body mass index 26.9 kg/m(2). Average DISE duration was 20 min with Propofol and 14.3 min with Midazolam. The induced sleep stage obtained was N2 with both drugs. Outpatient physical exam did not correlate with drug-induced sleep findings. There was a good correlation between DISE results with both drugs in all the areas of collapse except the velum (p < 0.005). Using a continuous perfusion, there is a good agreement in the findings observed in DISE performed with propofol and midazolam and PSG.

Research of visual attention networks in deaf individuals: a systematic review.

The impact of deafness on visual attention has been widely discussed in previous research. It has been noted that deficiencies and strengths of previous research can be attributed to temporal or spatial aspects of attention, as well as variations in development and clinical characteristics. Visual attention is categorized into three networks: orienting (exogenous and endogenous), alerting (phasic and tonic), and executive control. This study aims to contribute new neuroscientific evidence supporting this hypothesis. This paper presents a systematic review of the international literature from the past 15 years focused on visual attention in the deaf population. The final review included 24 articles. The function of the orienting network is found to be enhanced in deaf adults and children, primarily observed in native signers without cochlear implants, while endogenous orienting is observed only in the context of gaze cues in children, with no differences found in adults. Results regarding alerting and executive function vary depending on clinical characteristics and paradigms used. Implications for future research on visual attention in the deaf population are discussed.

Development of full-body rhythmic synchronization in middle childhood.

Rhythmic entrainment is a fundamental aspect of musical behavior, but the skills required to accurately synchronize movement to the beat seem to develop over many years. Motion capture studies of corporeal synchronization have shown immature abilities to lock in to the beat in children before age 5, and reliable synchronization ability in adults without musical training; yet there is a lack of data on full-body synchronization skills between early childhood and adulthood. To document typical rhythmic synchronization during middle childhood, we used a wireless motion capture device to measure period- and phase-locking of full body movement to rhythm and metronome stimuli in 6 to 11 year-old children in comparison with adult data. Results show a gradual improvement with age; however children's performance did not reach adult levels by age 12, suggesting that these skills continue to develop during adolescence. Our results suggest that in the absence of specific music training, full-body rhythmic entrainment skills improve gradually during middle childhood, and provide metrics for examining the continued maturation of these skills during adolescence.

Anti-tumor necrosis factor VNAR single domains reduce lethality and regulate underlying inflammatory response in a murine model of endotoxic shock.

BACKGROUND: In sepsis, tumor necrosis factor (TNF) is the key factor triggering respiratory burst, tissue injury and disseminated coagulation. Anti-TNF strategies based on monoclonal antibodies or F(ab')2 fragments have been used in sepsis with contradictory results. Immunoglobulin new antigen receptors (IgNAR) are a unique subset of antibodies consisting of five constant (CNAR) and one variable domains (VNAR). VNAR domains are the smallest, naturally occurring, antibody-based immune recognition units, having potential use as therapy. Our aim was to explore the impact of an anti-TNF VNAR on survival in an experimental model of endotoxic shock. Also, mRNA expression and serum protein of several inflammatory molecules were measured. RESULTS: Endotoxic shock was induced by lipopolysaccharide (LPS) in male Balb/c mice. Animals were treated with anti-TNF VNAR domains, F(ab')2 antibody fragments, or saline solution 15 minutes before, 2 h and 24 h after lethal dose100 (LD100) LPS administration. TNF blockade with either VNAR domains or F(ab')2 fragments were associated with lower mortality (60% and 75%, respectively) compared to LD100. Challenge with LPS induced significant production of serum TNF and interleukins -10 and -6 at 3 h. After that, significant reduction of IL-6 at 24 h (vs 3 h) was shown only in the VNAR group. Nitrites level also increased in response to LPS. In liver, TNF and IL-10 mRNA expression showed a pro-inflammatory imbalance in response to LPS. Blocking TNF was associated with a shift towards an anti-inflammatory status; however, polarization was more pronounced in animals receiving F(ab')2 fragments than in those with VNAR therapy. With regard to IL-6, gene expression was increased at 3 h in all groups. TNF blockade was associated with rapid and sustained suppression of IL-6 expression, even more evident in the VNAR group. Finally, expression of inducible-nitric oxide synthase (iNOS) increased in response to LPS at 3 h, but this was decreased at 24 h only in the anti-TNF VNAR group. CONCLUSIONS: Anti-TNF VNAR single domains improved survival in a murine model of endotoxic shock. Protection was associated with regulation in the TNF/IL-10 balance, attenuation of IL-6 and iNOS gene expression in the liver as well as decreased serum IL-6 concentration.

The effectiveness of control strategies for dementia-driven wandering, preventing escape attempts: a case report.

One of the most complicated aspects of caring for patients with dementia is dementia-driven wandering due to its adverse ramifications. We report a case of an 80-year-old man who had been previously diagnosed with dementia (with a score of 6 on the Reisberg Global Deterioration Scale - GDS). The patient went to an Adult Day Care Center on a daily basis where he demonstrated wandering behavior with a high rate of escape attempts (the number of times the Center's glass exit door was approached). The objective of this study is to present effective non-pharmacological intervention strategies for dementia-driven wandering; assessed strategies included: environmental (subjective barriers), cognitive/behavioral (cognitive training with differential reinforcement), and combined (subjective barriers + cognitive/behavioral). The results showed that all of these three strategies significantly decreased the number of escape attempts.