Identifying the midline thalamus in humans in vivo.

The midline thalamus is critical for flexible cognition, memory, and stress regulation in humans and its dysfunction is associated with several neurological and psychiatric disorders, including Alzheimer's disease, schizophrenia, and depression. Despite the pervasive role of the midline thalamus in cognition and disease, there is a limited understanding of its function in humans, likely due to the absence of a rigorous noninvasive neuroimaging methodology to identify its location. Here, we introduce a new method for identifying the midline thalamus in vivo using probabilistic tractography and k-means clustering with diffusion weighted imaging data. This approach clusters thalamic voxels based on data-driven cortical and subcortical connectivity profiles and then segments the midline thalamus according to anatomical connectivity tracer studies in rodents and macaques. Results from two different diffusion weighted imaging sets, including adult data (22-35 years) from the Human Connectome Project (n = 127) and adolescent data (9-14 years) collected at Florida International University (n = 34) showed that this approach reliably classifies midline thalamic clusters. As expected, these clusters were most evident along the dorsal/ventral extent of the third ventricle and were primarily connected to the agranular medial prefrontal cortex (e.g., anterior cingulate cortex), nucleus accumbens, and medial temporal lobe regions. The midline thalamus was then bisected based on a human brain atlas into a dorsal midline thalamic cluster (paraventricular and paratenial nuclei) and a ventral midline thalamic cluster (rhomboid and reuniens nuclei). This anatomical connectivity-based identification of the midline thalamus offers the opportunity for necessary investigation of this region in vivo in the human brain and how it relates to cognitive functions in humans, and to psychiatric and neurological disorders.

Influence of free fatty acid content and degree of fat saturation in laying hen diets on egg quality, yolk fatty acid profile, and cholesterol content.

The aim of the present study was to evaluate the effect of dietary free fatty acid (FFA) content and the degree of saturation on egg quality, yolk fatty acid (FA) profile, and yolk cholesterol content. For a 15-wk period, a total of 144 laying hens (19-wk-old) were randomly assigned to 8 treatments arranged in a 2 × 4 factorial design, with 2 sources of crude oil (soybean oil and palm oil) and 4 levels of FFA (10, 20, 30, and 45%). The dietary treatments were achieved by progressively substituting the original oils with equivalent amounts of their corresponding acid oils (soybean acid oil and palm fatty acid distillate, respectively). No differences in ADFI or egg mass were found. However, dietary FFA reduced egg production (linear, P < 0.05) and increased the feed conversion ratio (linear, P < 0.05). Higher levels of FFA in soybean diets resulted in higher egg weight with higher albumen and yolk weights (linear, P < 0.01). Palm diets presented higher yolk:albumen ratio than soybean diets (P < 0.001), but the effect of FFA did not follow a linear trend. Hens fed soybean diets laid eggs with higher Haugh units (HU) than palm diets (P < 0.001), although increasing the dietary FFA% reduced the HU values in both (linear, P < 0.001). Palm diets enhanced shell quality with greater resistance to breakage, and higher dry matter and ash content than soybean diets (P < 0.05). No differences in egg chemical composition and yolk cholesterol content were found (P > 0.05). The saturation degree had a significant effect on all the analyzed yolk FA (P < 0.001) except for arachidonic acid (C20:4 n-6), whereas increasing the FFA content did not affect to a great extent. These results show that varying dietary FFA level did not affect egg quality and yolk composition as much as the dietary fat source did, supporting the use of acid oils and fatty acid distillates as fat ingredients for feed.

Colonic metabolites from digested Moringa oleifera leaves induced HT-29 cell death via apoptosis, necrosis, and autophagy.

Colorectal cancer is an important concern in modern society. Risk factors such as the diet indicate the need to find healthy food products displaying additional health benefits. This study aimed to characterise and evaluate the impact of the colonic metabolites from the fermented non-digestible fraction of Moringa oleifera (MO) leaves (FNFM) on cell death mechanisms from HT-29 cells. MO leaves were digested in vitro, and the 12 h-colonic extract was obtained. FNFM mainly contained morin and chlorogenic acids (41.97 and 25.33 µg/g sample). Butyric acid was ranked as the most important metabolite of FNFM. The FNFM exerted antiproliferative effect against HT-29 colorectal cancer cells (half lethal concentration, LC50: 5.9 mL/100 mL). Compared to untreated control, LC50 increased H2O2 production (149.43%); induced apoptosis (119.02%), autophagy (75.60%), and necrosis (87.72%). These results suggested that digested MO colonic metabolites exert antiproliferative effect against HT-29 cells, providing additional health benefits associated with MO consumption.

Exploring the radiosensitizing potential of magnetotherapy: a pilot study in breast cancer cells.

Aim: To explore the influence of electromagnetic fields (EMFs) on the cell cycle progression of MDA-MB-231 and MCF-7 breast cancer cell lines and to evaluate the radiosensitizing effect of magnetotherapy during therapeutic co-exposure to EMFs and radiotherapy. Material and methods: Cells were exposed to EMFs (25, 50 and 100 Hz; 8 and 10 mT). In the co-treatment, cells were first exposed to EMFs (50 Hz/10 mT) for 30 min and then to ionizing radiation (IR) (2 Gy) 4 h later. Cell cycle progression and free radical production were evaluated by flow cytometry, while radiosensitivity was explored by colony formation assay. Results: Generalized G1-phase arrest was found in both cell lines several hours after EMF exposure. Interestingly, a marked G1-phase delay was observed at 4 h after exposure to 50 Hz/10 mT EMFs. No cell cycle perturbation was observed after repeated exposure to EMFs. IR-derived ROS production was enhanced in EMF-exposed MCF-7 cells at 24 h post-exposure. EMF-exposed cells were more radiosensitive in comparison to sham-exposed cells. Conclusions: These results highlight the potential benefits of concomitant treatment with magnetotherapy before radiotherapy sessions to enhance the effectiveness of breast cancer therapy. Further studies are warranted to identify the subset(s) of patients who would benefit from this multimodal treatment.

Physicochemical and nutraceutical properties of moringa (Moringa oleifera) leaves and their effects in an in vivo AOM/DSS-induced colorectal carcinogenesis model.

Moringa (Moringa oleifera) is a plant that has generated great interest in recent years because of its attributed medicinal properties. The aim of this study was to characterize the bioactive compounds of moringa leaves (MO) and evaluate their effect on a colorectal carcinogenesis model. Twenty-four male CD-1 mice were divided into 4 groups: Group 1 fed with basal diet (negative control/NC); Group 2 received AOM/DSS (positive control); Groups 3 and 4 were fed with basal diet supplemented with moringa leaves (2.5% w/w and 5% w/w, respectively) for 12weeks. Moringa leaves exhibited a high content of dietary fiber (~18.75%) and insoluble dietary fiber (2.29%). There were identified 9 phenolic compounds whereas the chlorogenic and ρ-coumaric acid showed the higher contents (44.23-63.34µg/g and 180.45-707.42µg/g, respectively). Moringa leaves decreased the activity of harmful fecal enzymes (ß-glucosidase, ß-glucuronidase, tryptophanase and urease up to 40%, 43%, 103% and 266%, respectively) as well tumors incidence in male CD1-mice (~50% with 5% w/v of moringa dose). These findings suggest that the bioactive compounds of moringa such as total dietary fiber and phenolic compounds may have chemopreventive capacity. This is the first study of the suppressive effect of moringa leaves in an in vivo model of AOM/DSS-induced colorectal carcinogenesis.

Extensive surgical history prior to cytoreductive surgery and hyperthermic intraperitoneal chemotherapy is associated with poor survival outcomes in patients with peritoneal mucinous carcinomatosis of appendiceal origin.

BACKGROUND: Patients with PMCA commonly undergo surgery before CRS/HIPEC. We evaluated the role of extensive surgical treatment before CRS/HIPEC in terms of overall survival (OS). METHODS: 105 patients with PMCA who underwent a CRS/HIPEC procedure were identified from a prospective database. Patients were divided into two groups based on Prior Surgery Score (PSS): PSS ≤ 1 limited surgery group (LSG), PSS >1 extensive surgery group (ESG). Survival of lymph node (LN) negative and positive patients was analyzed separately. RESULTS: 40 patients were in LSG and 65 in ESG. Mean time from diagnosis to CRS/HIPEC was 6 and 17 months for LSG and ESG, respectively (p = 0.004). Groups were well balanced in peritoneal cancer index, complete cytoreduction rate, and LN status. One, 3, and 5-year OS among LN negative patients was 95, 83, and 75% for the LSG (n = 22) group and 87, 55, and 32% for the ESG (n = 35), group respectively (p = 0.026). One, 3, and 5-year OS among LN positive patients was 69, 50, and 17% for the LSG (n = 18) group and 80, 21, and 14% for the ESG (n = 30), group respectively (p = 0.613). For all patients 1, 3, and 5-year OS was 84, 65, and 54% for the LSG (n = 40) group and 86, 43, and 26% for the ESG (n = 65) group, respectively (p = 0.029). CONCLUSION: Extensive surgical treatment before CRS/HIPEC is associated with delay of CRS/HIPEC and poorer OS overall, especially among LN negative patients. We recommend early referral of PMCA patients to a peritoneal surface malignancy center.

Systemic chemotherapy (SC) before cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) in patients with peritoneal mucinous carcinomatosis of appendiceal origin (PMCA).

BACKGROUND: The role of SC before CRS/HIPEC for patients with PMCA is unclear. This study explores the effect of SC prior to CRS/HIPEC on overall survival (OS) in patients with PMCA. METHODS: 72 patients with recently diagnosed PMCA who underwent CRS/HIPEC were identified from a prospective database. Thirty patients had SC before CRS/HIPEC (Group 1) and 42 did not (Group 2). Patients who were referred to our center after multiple lines of SC were excluded from this analysis. OS was estimated. RESULTS: Median follow-up was 3.2 years. Groups were similar regarding lymph node positivity, postoperative SC and rate of complete cytoreduction. Twenty-four (80%) patients in Group 1 and 21 (50%) in Group 2 had high grade histology (HG) (p = 0.01). OS from CRS/HIPEC at 1, 2, and 3 years was 93, 68, 51% in Group 1 and 82, 64, 60% in Group 2, respectively (p = 0.74). Among HG patients 3-year survival was 36% in the SC group vs. 35% in the group without SC (p = 0.67). The 3-year OS for patients with low grade (LG) tumors was 100% in the SC group vs. 79% in the group with no prior SC (p = 0.26). Among patients with signet ring cell (SRC) histology, 1, 2 and 3-year survival was 94, 67 and 22% in the SC group vs. 43, 14, 14% in the group with no SC, respectively (p = 0.028). There were only 6 patients with LG PMCA who received prior SC. CONCLUSIONS: Preoperative SC could improve the prognosis of patients with high-grade PMCA with SRC histology.

Target driven preclinical screening for new antimitotic chemotherapy agents.

Currently approved antimitotic therapies used in chemotherapy are microtubule-targeting agents (MTAs). Despite they achieved some level of success, they have limited efficacy as single agents, with issues of slippages and resistance, and cause significant side effects. The advances in the identification of other mitosis-related targets led to the development of new mitotic regulators aimed to perturb mitosis without interfering with microtubule dynamics in non-dividing cells trying to reduce side effects in patients. Some of these compounds like those targeted to entry and mitotic kinases, mitotic kinesins/motor proteins, and multiprotein complexes have been evaluated in vitro and in animal models, and some of them have reached clinical trials. Despite promising preclinical results, in many cases, the efficacy demonstrated by these new antimitotics was not better than current microtubule inhibitors. In this paper we review present and future strategies on the search for new antimitotic compounds based on identification of new protein targets and development of multifunctional inhibitors of mitosis in cancer cells.

Prospective double-blind clinical trial evaluating the effectiveness of Bromelain in the third molar extraction postoperative period.

OBJECTIVES: To evaluate the anti-inflammatory and analgesic effect of Bromelain (pineapple extract) administered orally in the postoperative after extraction of impacted lower molars. STUDY DESIGN: This is a prospective, placebo-controlled, unicentric, double-blind study; the sample size was 34 patients. The pre and postoperative outcomes, evaluated on the third (D3) and eighth day (D8), included inflamtion, pain and oral aperture, as well as the need for analgesics. One group received Bromelain 150mg per day for three days and 100mg on days 4 to 7. The other group received placebo in the same dosage. All outcomes werrecorded quantitatively and analyzed with the Mann-Whitney U test for independent samples. RESULTS: Although there were no statistically significant differences between the treatment groups, a trend towards less inflammation and improved oral aperture was observed in the group that received Bromelain, compared to the group that received placebo. This trend can be attributed completely to random reasons, since there is no statistical difference in the results. CONCLUSIONS: Further studies are necessary to analyze different administration patterns and doses of Bromelain for the use in the postoperative of impacted third molars.

Efecto del masaje terapéutico en prematuros del Servicio de Neonatología del Hospital San José / Effects of massage in prematures at the neonatology unit of San Jose Hospital

Antecedentes: En Chile, la estadística poblacional reporta 259.069 recién nacidos vivos anualmente, corregidos para el año 2001, de los cuales, 6 por ciento son prematuros. Este grupo presenta la más alta morbimortalidad neonatal, requiere prolongadas hospitalizaciones e involucra un fuerte gasto de recursos para la salud pública del país. Objetivos: Esta investigación evaluó los efectos del masaje como terapia complementaria, al tratamiento habitual de los recién nacidos prematuros del Servicio de Neonatología del Hospital San José. Método: Se realizó un ensayo clínico de casos y controles que incluyó al azar 40 niños menores de 37 semanas de edad gestacional e inferior a 1.700 g al nacer que no presentaban condiciones patológicas al momento del estudio y que fueron agrupados por peso y edad gestacional al inicio de la intervención y pareados posteriormente por edad gestacional y categoría de peso de nacimiento (pequeño, normal o grande para la edad gestacional). Los padres de los niños fueron capacitados en masaje shiatsu y lo aplicaron a sus hijos por 15 minutos, tres veces al día, seis días alternos, por dos semanas. Se registró peso de inicio y diario en ambos grupos y días de hospitalización. El análisis estadístico se realizó mediante test t de Student para datos no pareados. Resultados: Los niños masajeados aumentaron en promedio, 14 gramos diarios más que el grupo control (p=0,0001) y permanecieron 15 días menos hospitalizados (p=0,0169). Conclusión: El masaje shiatsu produce una más rápida ganancia de peso en niños prematuros. Aplicando esta técnica a niños de 1000 gal nacer se ahorraría en promedio $1.216.000 por niño, sólo por concepto de días cama de hospitalización y una potencial ganancia en el desarrollo psicomotor.

Background: In Chile every year 259.069 childs were borned, 6 percent of them are prematures. This group has the highest neonatal mortality and morbidity, spending prolonged hospitalizations and monetary resources for the public health of Chile. Objective: This investigation looks for the effects of massage as a complementan/ therapy to the common treatment of our hospitalized prematures babies at the Neonatal Unit of San José Hospital. Method: We performed a clinic assay of cases and controls that included 40 babies under 37 weeks of gestation and less than 1700 g which had no pathologies. They were classified by weight and gestational age at the beginning of the assay and compared by weight and gestational age at the end of the study. Parents were trained in shiatsu massage and they applied it to their babies 15 minutes, 3 times per day, 6 every other days during 2 weeks. We registered the initial weight and then daily and hospitalization days in both groups. Statistical analysis was performed by test t student for data no paired. Results: The group of babies stimulated by massage, increased their weight 14 g per day in average more than the control group (p=0.0001) and spent 15 days less of hospitalization (p=0.0169). Conclusions: The shiatsu massage produces a fast gain of weight in newborns prematures babies. Applying this therapy to babies under 1000 g, we can save $1,216,000 per baby, just only for hospitalization concept and a potential gain in psychomotor development.

Influence of sodium glycocholate on production of conjugated linoleic acid by cells of Lactobacillus reuteri ATCC 55739.

The possible influence of a bile salt on production of conjugated linoleic acid (CLA) by Lactobacillus reuteri ATCC 55739 was evaluated. Cells of the lactobacilli grown in MRS broth with and without linoleic acid (LA, 0.2%) were harvested and washed. The washed cells were added to buffer containing 0.2% LA and incubated 18 h at 37 degrees C. The cells, which had been grown without LA, transformed LA into CLA (mainly c9t11-C18:2) better than did those cells grown with it. When sodium glycocholate (0.3%) was added to the washed cell suspensions, about the same level of CLA was formed as in its absence regardless of whether or not the cells had been grown in broth supplemented with free LA. Thus, glycocholate that occurs in humans did not influence production of CLA by resting cells of the lactobacilli.

Scorpion envenoming in two regions of Colombia: clinical, epidemiological and therapeutic aspects.

To determine clinical and epidemiological features of scorpion stings in two departments of Colombia, a descriptive study was performed in the hospitals of 10 towns from Antioquia (2 256 071 inhabitants) and five from Tolima (630 424 inhabitants). One hundred and twenty-nine cases were admitted during one year, 51 in Antioquia, 78 in Tolima and 41 were children less than 15 years old. Most stings (70.5%) occurred inside the house; 27.9% were on the hands and 26.4% on the feet. The scorpion species involved were Tityus pachyurus (51), Centruroides gracilis (31), T. fuehrmanni (29), T. asthenes (7) and Chactas spp. (1). In 10 cases the scorpion involved was not identified. Systemic envenoming signs (e.g. vomiting, tachypnea) were significantly more frequent in children than in adults (P < 0.05). Four children had hypertension, but none developed pulmonary oedema. One 3-year-old girl, stung by T. asthenes, had acute oedematous pancreatitis. Ninety-eight patients had mild envenoming. Moderate (27 patients) and severe (four patients) envenoming was significantly more frequent in children than in adults (P = 0.003; relative risk = 2.97). A pepsin-digested anti-Centruroides spp. antivenom was administered to 19 of 31 patients presenting systemic envenoming signs. No adverse reactions to antivenom were observed.

Anesthesia technique for outpatient facial laser resurfacing.

BACKGROUND AND OBJECTIVES: Multiple anesthetic approaches exist for full-face laser resurfacing. The purpose of this study was to describe an anesthesia technique based on combination of eutectic mixture local anesthetics (EMLA) and remifentanil sedation, that can be utilized by anesthesiologists in the ambulatory environment. STUDY DESIGN/MATERIALS AND METHODS: Fifty patients elected for facial laser resurfacing. All patients received topical anesthesia in full face with EMLA cream at 60 minutes (min) before laser procedure. On arrival at the operating room, intravenous (IV) sedation was administered with remifentanil (0.20 mcg/kg/minute), midazolam (1.5- 2 mg bolus IV), and propofol infusion (0.5-1 mg/kg/hour). The subsequent infusion rate of remifentanil was varied to maintain an adequate level of sedation and analgesia. Five minutes before the operation conclusion, the sedation infusion was discontinued. Patients were discharged after achieving a minimum criteria for recovery. RESULTS: Almost all the patients were successfully anesthetized by this combination technique, only four patients needed complementary anesthesia with regional nerve blockade. The mean level of sedation scored 2-3 on the Ramsay scale. The mean discharge time was 55 minutes. No complications were observed. CONCLUSIONS: The use of a combination of topical EMLA anesthesia and IV conscious sedation based on remifentanil provided an adequate depth of anesthesia for outpatient facial laser resurfacing without complications.

Attention capture by auditory significant stimuli: semantic analysis follows attention switching.

Event-related potentials (ERPs) were recorded from the scalp to investigate a long-standing controversy in auditory attention research, namely when the 'breakthrough of the unattended' takes place in the human brain. Nine subjects classified visual stimuli appearing 300 ms after task-irrelevant standard tones (80%, i.e. P = 0.8) or novel sounds (20%, i.e. P = 0.2) into odd/even categories. After the recording session, subjects scored the novel sounds as to whether they had any particular meaning (identifiable) or were perceived as a burst of noise (non-identifiable), and performance and ERPs were analysed according to this classification. A control condition, in which the visual stimuli were presented with no sounds, showed that subjects covertly monitored the task-irrelevant sounds during visual task-performance, and a further condition, in which the auditory and visual stimuli appeared regardless of each other, made it possible to trace the processing of the distracters during allocation of attention outside the auditory environment. Results yielded identical N1-enhancement for the two types of novel sounds, indicating similar attention switching triggered to these two types of unexpected sounds. However, there was a stronger orientating of attention towards identifiable novel sounds, as indicated both by behavioural distraction and by larger novelty-P3. Furthermore, this stronger orientating of attention was due to the sounds being contingent on the visual stimuli, as no increase in novelty-P3 to identifiable novel sounds was observed in the control condition, in which the sounds occurred outside the attentional set. Therefore, provided that the N1-enhancement reflects a call for focal attention, and novelty-P3 the effective orientating of attention towards the eliciting sounds, the present results suggest that semantic analysis of significant sounds occurs after a transitory switch of attention towards the eliciting stimuli. Moreover, as the novelty-P3 increase in amplitude was observed only when subjects covertly monitored the sounds, the present data suggest that semantic analysis of irrelevant sounds depends on the top-down cognitive influences of the attentional set.

Evaluation of extracts from Gevuina avellana hulls as antioxidants.

The antioxidant activity of the extracts from Gevuina avellana hulls was evaluated and compared with that of BHT (butylated hydroxytoluene) and BHA (butylated hydroxyanisole), using the beta-carotene bleaching assay, the accelerated oxidation of crude soybean oil, and the 2,2-diphenyl-beta-picrylhydrazyl (DPPH) radical scavenging method. Solvents of different polarity were used to obtain the extracts. Both the extraction yield and the antioxidant activity were strongly dependent on the solvent. The ethanol and diethyl ether soluble fractions were the most active with the beta-carotene assay. Ethanol and methanol extracts were the most active in hydrogen radical scavenging activity. Water and methanol inhibited more efficiently the oxidation of soybean oil at 70 and 80 degrees C, respectively. As a general trend, increased antioxidant activity was observed for increased extract concentration. Except the acetone extracts, all were stable after 6 months storage at 4 degrees C. The ethanol solubles from G. avellana hulls present antioxidant activity similar to that of synthetic antioxidants and to other reported residual agroindustrial materials.

Non-quassinoid constituents from the twigs and thorns of Castela polyandra.

The structures of two new constituents, a beta-glycoside (1) and a steroid (2), isolated from the twigs and thorns of Castela polyandra, were established by a combination of spectroscopic and single-crystal X-ray analysis.

Quassinoids from the twigs and thorns of Castela polyandra.

The structures of six new C20 quassinoids and one new C19 quassinoid, all isolated from the twigs and thorns of Castela polyandra, were established by a combination of spectroscopic and single-crystal X-ray analysis. Five known quassinoids and one known sterol were also identified.

Appearance of resistance to meropenem during the treatment of a patient with meningitis by Acinetobacter.

A case is reported of a patient who developed Acinetobacter meningitis after an external ventricular drainage system had been fitted for control of intracranial pressure. During the process, nine strains of Acinetobacter isolated from her cerebrospinal fluid were indistinguishable by analysis of total genomic DNA by pulse-field gel electrophoresis. The first eight strains were sensitive to meropenem and imipenem (MICs < 1 g/l). The MIC of the last one, which had been recovered after 32 days during two courses of treatment with meropenem, increased to > 32 g/l for meropenem, while with imipenem the increase was minimal (MIC = 1.5 g/l). The microorganism persisted in the central nervous system despite the administration of different antimicrobials, including intraventricular aminoglycosides and six changes in the external ventricular system. The patient died 68 days after admission to the intensive care unit from bilateral cerebral ischemic lesions, intraventricular hemorrhage and cerebral edema with endocraneal hypertension, the Acinetobacter ventriculitis also contributing to this state.

A comparison of p53 and p16 expression in human tumor cells treated with hyperthermia or ionizing radiation.

To assess the potential relationship between p53 and p16 proteins in the cellular response to stress, we have examined the levels of these proteins in a series of human tumor cell lines after treatment with either ionizing radiation or hyperthermia. We found that cells with abnormal radiation-induced G1 arrest (non-functional p53) had significantly higher constitutive levels of p16 than cells showing a normal G1 arrest (functional p53). Time-course experiments were done to test the effect of gamma-irradiation on intracellular levels of p16. The pattern of changes in p16 response was similar in all cell lines studied, and p16 expression was not related to cellular sensitivity to radiation or to the level of p53 induction after treatment. We also provide evidence that short-term exposure to high temperature causes p53 accumulation. Hyperthermia-induced p53 accumulation was greatest in those cells exhibiting the highest radiation-induced p53 accumulation, suggesting a possible relationship between p53 induction after these 2 different stresses. p16 synthesis was also induced in different cell lines after heat treatment, and this response was independent of p53 functionality. When we compared the level of p16 expression with the extent of G0/G1 arrest induced by heat, a linear correlation was found, raising the possibility that p16 may be involved in the control of cell cycle progression in response to heat treatment.