Yin and Yang: Why did evolution implement and preserve the circadian rhythmicity?

Yin and Yang concept emphasizes the reciprocal and interrelated nature; neither is sufficient, both are needed to sustain the overall balance of the living system. Changing the balance, by implementing deficiency or excess of one of them, upsets the equilibrium (homeostasis) of the whole system. PURPOSE: In this opinion article intermittent exposure is presented as the stimulus for development and evolutionary conservation of circadian rhythm, an endogenous, entrainable oscillation of approximately 24 h, to counteract/balance the cells' natural tendency to attenuate their response during long-term exposure to different endogenous substances. RESULTS: The concept of Yin and Yang duality is an allegory on which the avoidance of attenuation of the cells' responses hypothesis is presented as an explanation for the circadian rhythmicity, which is integrated in all human cells, with the exception of stem and cancer cells. CONCLUSIONS: We hypothesize, that circadian rhythmicity has evolved, during evolution, into a mechanism that prevents disruption of the organism's negative-feedback-loop homeostasis.

Salivary levels of TNF-α and IL-6 in patients with oral premalignant and malignant lesions.

The aim of this study was to determine the salivary concentrations of tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6) in patients with premalignant and malignant lesions. The study involved 57 patients who were examined between 2008 and 2010 at the Department of Oral Medicine and Department of Oral and Maxillofacial Surgery of the Medical Faculty, University of Rijeka, Croatia. They were divided into three groups: 19 patients with oral premalignant lesions, 19 with oral squamous cell carcinoma and 19 healthy control volunteers. Whole saliva was collected and investigated for the presence of TNF-α and IL-6 by enzyme immunoassay at the Department of Dentistry and Maxillofacial Surgery, Medical Faculty, University of Graz, Austria. All groups had statistically significant differences in values of TNF-α and IL-6 (P < 0.001). The results suggest that these proinflammatory cytokines are elevated in the saliva of patients with oral squamous cell carcinoma and oral premalignant lesions as compared to controls, which may have diagnostic and/or prognostic significance.

Oral sensorial complaints, salivary flow rate and mucosal lesions in the institutionalized elderly.

The aims of this study were to determine the prevalence of oral sensorial complaints, salivary flow rate and oral mucosal lesions in the institutionalized and non-institutionalized elderly. The study included 280 institutionalized and 61 non- institutionalized elderly people. Dry mouth, burning mouth sensations, taste disturbances, salivary flow rate and oral mucosal lesions were assessed and compared between groups. A greater number of the institutionalized elderly had dry mouth (P = 0.001) and taste disturbance (P = 0.035) compared to non-institutionalized elderly. The institutionalized elderly also had significantly lower salivary flow rate (P < 0.0001). Positive correlation was found between salivary flow rate and perception of dry mouth in the institutionalized elderly (r(s) = 0.26; P < 0.05), as well as in the non-institutionalized elderly (r(s) = 0.35; P < 0.05). Moreover, positive correlation was observed between salivary flow rate and the sensation of burning mouth in the institutionalized elderly (r(s) = 0.13; P < 0.05) and non-institutionalized elderly (r(s) = 0.31; P < 0.05). The number of institutionalized elderly people with oral mucosal diseases was higher compared with non-institutionalized ones (P = 0.01). The most common oral mucosal lesions in both groups were related to wearing dentures. It can be concluded that the institutionalized elderly are significantly affected with oral sensorial complaints, including dry mouth and taste disturbance, as well as decreased salivary flow rate and oral mucosal diseases compared with the non-institutionalized elderly.

Association between oral lichenoid reactions and amalgam restorations.

BACKGROUND: The aim of this study was to perform a clinical assessment of the association between oral lichenoid reactions (OLR) and amalgam restorations and to determine the salivary concentrations of interleukin-6 (IL-6) and IL-8 before and after replacement of the amalgam restorations. METHODS: The study included 20 patients with OLR and 20 healthy volunteers, who were examined between 2001 and 2005 at the Oral Medicine Unit of the Medical Faculty University of Rijeka. All patients were skin patch tested by an experienced physician. Saliva samples were collected, prepared and analysed for IL-6 and IL-8 concentrations using enzyme-linked immunosorbent assay. RESULTS: Sixteen out of 20 patch-tested patients showed a sensitization to inorganic mercury or amalgam. Total replacement of all amalgam fillings was carried out on 20 patients with fillings based on composite resin, gold, porcelain or a combination of these. Sixteen out of 20 patients showed complete healing of OLR; three patients had marked improvement, whereas one patient showed no improvement. Levels of IL-6 detected before replacement were significantly higher than IL-6 levels following the replacement (P = 0.003). The IL-8 levels measured before replacement procedure were significantly higher than the IL-8 levels after replacement of the fillings (P < 0.001). CONCLUSIONS: On the basis of clinical observations, restorative therapy resulted in tissue healing. Following the replacement of amalgam fillings with fillings based on other restorative materials, levels of both IL-6 and IL-8 shifted towards normal, as measured in healthy subjects.

Effect of root canal sealers on mouse peritoneal macrophage functions.

Three root canal filling materials, viz. calcium hydroxide-based cement (Apexit, resin-based cement (AH-plus) and glass-ionomer based material (Ketac Endo) were tested for their influence on several functions of peritoneal macrophages from Balb/c mice. Macrophage functions were evaluated by the adherence, phagocytic, candidacidal and Nitro blue tetrazolium-dye assays. Ketac-Endo enhanced all macrophage functions in the first 2 d (p < or = 0.05), when compared to the positive control, but this effect had changed after 7 and 14 d, causing inhibition of these functions. Other materials suppressed substrate adherence capacity and phagocytosis, while significantly stimulating macrophage microbicidal activity (p < or = 0.05) in a time-dependent manner.

A new method for selective measurement of joint movement in hand tremor in Parkinson's disease patients.

Parkinson's disease (PD) is a progressive degenerative disorder of the central nervous system with tremor being one of its four main clinical features. Currently used methods can directly evaluate tremor amplitude and frequency but not joint movement in the affected limb. Measurement of joint movement facilitates the location of muscle groups that participate in PD tremor and this is important for treatment with local botulinum toxin injections. We developed and tested a method that measured tremor amplitude and frequency in a specific joint of the hand in PD patients. The tremor analysis method was based on force transducers adapted to record rest tremor of the wrist and metacarpophalangeal joints in two degrees of freedom for each joint. Direct measurements of joint movement in the hand can evaluate tremor amplitude and frequency and also locate the muscle groups that are most active in tremor movement, thus enabling their local treatment.

Increasing the repetition frequency of electric pulse delivery reduces unpleasant sensations that occur in electrochemotherapy.

Involuntary muscle contractions and painful sensations during electric pulse delivery are the most unpleasant side effects of electrochemotherapy. The aim of this study was to determine the nature of pain caused by the application of electric pulses and to evaluate patients tolerance to the standard electric pulses of 1 Hz repetition frequency and the new 5 kHz protocol. A train of eight electric pulses of 1 Hz and 5 kHz repetition frequencies was delivered to the forearms of 40 healthy volunteers. After the conclusion of each protocol the subjects had to complete the short-form McGill Pain Questionnaire with separate visual analog scales for pain intensity and unpleasantness. All subjects selected at least one superficial and one deep pain descriptor; 85% selected at least two superficial descriptor and 60% at least two deep description. The application of 5 kHz electric pulses was less unpleasant than the standard 1 Hz pulses; however, the pain intensity did not differ between the protocols. Significantly more subjects chose the new 5 kHz protocol as their choice of treatment (P = 0.017). The frequent use of deep descriptors in our study indicates that muscle contractions contribute to the discomfort felt by the subjects during the delivery of electric pulses. The new 5 kHz protocol considerably shortens the treatment session and is also better tolerated. Therefore, the new 5 kHz electrochemotherapy protocol should eventually replace the 1 Hz pulses as new standard.

Microleakage along Glassix glass fibre posts cemented with three different materials assessed using a fluid transport system.

AIM: To evaluate the microleakage along Glassix fibre posts cemented with three different materials. METHODOLOGY: The root canals of maxillary central incisor teeth were filled and restored with Glassix posts (Harald Nordin sa, Chailly/Montreux, Switzerland) cemented with either a zinc-phosphate Harvard cement (Richter & Hoffmann, Harvard Dental GmbH, Berlin, Germany), Fuji PLUS cement (GC Corporation, Tokyo, Japan) or Variolink II cement (Vivadent, Schaan, Lichtenstein) in three groups of 15 canals each. Twenty unrestored canals served as a control group, 10 filled with gutta-percha and sealer (negative control group), the remaining 10 with gutta-percha only (positive control group). Coronal microleakage was evaluated using a fluid transport system. The movement of an air bubble in a capillary glass tube connected to the apex of the experimental root section was measured over 5-min periods. Measurements were performed four times for each specimen and the mean values recorded. ANOVA and Duncan's test were performed. RESULTS: The positive control group had the highest values of microleakage. Amongst experimental groups, the highest values of microleakage occurred in the group with the posts cemented with Harvard cement, followed by Fuji PLUS and Variolink II cements. Groups with Fuji PLUS, Variolink II and the negative control group had significantly (P < 0.00001) less microleakage compared with the Harvard cement group and the positive control group. CONCLUSION: Canals with Glassix posts cemented with Variolink II and Fuji PLUS cement had the least leakage when assessed using a fluid transport system.

Imbalance between apoptosis and proliferation causes late radiation damage of salivary gland in mouse.

Severe xerostomia is a common late radiation consequence, which occurs after irradiation of head and neck malignancies. The aim of the present study was to analyze apoptosis and proliferation and their relationship during the late post-irradiation phase. C57BL/6 mice were locally irradiated in head and neck region with a single dose of 7.5 or 15 Gy and their submandibular glands were collected at 40 and 90 days after irradiation. To identify apoptotic cells, the TUNEL method was employed and immunohistochemistry with proliferating cell nuclear antigen (PCNA) was used for detecting proliferation. Histological changes at day 40 were mild in contrast to day 90 when glands of irradiated mice showed severe atrophy, vacuolization and mononuclear infiltration. Acinar cells, granular and intercalated duct cells of mice irradiated with 7.5 and 15 Gy expressed higher apoptotic index than cells of non-irradiated, control glands at both examined time points. At 40 days, a higher proliferation index in granular and intercalated duct cells was detected only in group irradiated with 7.5 Gy. At 90 days, proliferation index for all cell types in both irradiated groups was similar to the controls. According to our results, the imbalance between apoptosis and proliferation caused by X-irradiation may be the reason for gland impairment during the late post-irradiation phase.

3D finite element model and cervical lesion formation in normal occlusion and in malocclusion.

The aim of this study was to develop a three-dimensional (3D) finite element model (FEM) of the first maxillary premolar in order to compare the stress profiles in the buccal and palatal cervical regions. The 3D geometry of the tooth was reconstructed, the solid model was transferred into a finite element program where a 3D mesh was created, and the stress distribution analysis was performed. Two typical cases have been considered: the tooth under normal occlusion (case I) and the tooth under malocclusion (case II). In case I, larger compressive stresses were found in the cervical enamel and dentine. Tensile stresses were found in the fissure system, adjacent area, and at the vestibular surface of the buccal cusp. The peak values for the principal stress ranged from -259 to +2.25 MPa in the cervical areas. In the case II, larger compressive stresses were found in the palato-cervical enamel and dentine. Tensile stresses were found inside the enamel in the fissure system, adjacent area, at the vestibular surface of the buccal cusp, and in the bucco-cervical enamel. The peak values for the principal stress ranged from -501.947 MPa in palatal region to +82.4 MPa in the buccal region This study implies a role of occlusal forces in development of non-carious lesions. In the case of malocclusion, tensile stresses generated on the cervical areas were higher compared with the stresses generated in the case of normal occlusion and it is probably capable of producing non-carious cervical lesion.

Postural stability of Parkinson's disease patients is improved by decreasing rigidity.

Postural instability has a big impact on the quality of life of patients with Parkinson's disease (PD) as it often leads to an insecure stance and fall. We investigated if postural stability in these patients improves by decreasing rigidity with a dopaminergic agonist. In our study, we tested eight PD patients with no concomitant diseases. Their age was 61 +/- 2 years (mean +/- SE) and their Hoehn-Yahr score was 3 +/- 0.1. The patients were evaluated according to the Unified Parkinson's Disease Rating Scale for motor function (mUPDRS) and with stabilometric measurements of forward-backward and side-to-side body oscillations during free stance with eyes open. Both evaluations were performed in an "off "state and in an apomorphine-induced "on" state. As expected, the mUPDRS score was significantly decreased in the "on" state with posture being improved in six patients, gait in eight patients and postural stability in seven of eight patients. In addition, apomorphine caused a significant reduction of the relative amplitude of lower frequencies and an increase of the relative amplitude of higher frequencies of forward-backward body oscillations. The results of stabilometry and mUPDRS evaluations are in agreement with the effect of apomorphine on rigidity, indicating that postural stability of PD patients is improved by decreasing rigidity.

A novel staining method for quantification and 3D visualisation of capillaries and muscle fibres.

The aim of this study was to introduce a combined fluorescent staining that clearly demonstrates capillaries and distinguishes them from the basal lamina of muscle fibres in skeletal muscle tissue. The triple staining with CD31, Griffonia (Bandeira) simplicifolia lectin (GSL I) and laminin efficiently distinguishes vascular endothelium from the basal lamina of skeletal muscle fibres in physiological and pathological conditions. The presented triple staining method has several advantages, which facilitate quantitative analysis of the capillary network, and its relation to individual muscle fibres.

Influence of calcium hydroxide root-canal sealer on microbial growth in vitro.

The calcium hydroxide-based filling material Apexit, which is often used in endodontic practice, was evaluated for its antibacterial and antifungal effects against microorganisms isolated from oral cavity (Serratia marcescens, Pseudomonas putida, Staphylococcus aureus, Candida albicans). Two different quantitative techniques were employed--the direct-contact test was used to examine the efficacy of freshly mixed material while the broth-survival test was employed to check the antimicrobial properties of 5-d-old material. Apexit inhibited Gram-negative bacteria more effectively than Gram-positive ones but had none or a very weak inhibitory effect on C. albicans.

Selective stimulation of motor fibres in the sciatic nerve of a rat.

The aim of this study was to test the possibility of selective activation of slow and fast fibres within the rat tibialis anterior muscle, via selective stimulation of corresponding motor fibres within the sciatic nerve. For this purpose the implantable cuffs containing two and three platinum ring-electrodes were constructed and surgically fitted on the left and right sciatic nerve in five Wistar rats. In the left nerve, conventional, rectangular stimuli were delivered bipolarly via double-ring cuffs. In the right nerve quasitrapezoidal stimuli were delivered tripolarly via treblering cuffs. After 60 s of simultaneous and continuous stimulation of both nerves the left and right anterior tibialis muscle were isolated and immediately frozen in liquid nitrogen. Each muscle was completely cut in step serial sections. Glycogen depletion was demonstrated by Periodic acid-Schiff (PAS) reaction. In one randomly selected rat an immunohistochemical profile of muscle fibres (type I, IIA, IIB, and IIX) was determined with antibodies against myosin heavy chain isoforms. In other rats typing of glycogen depleted fibres was performed according to muscle fibre diameters. Preliminary results show that glycogen depleted fibres in the selected rat were type IIB fibres in both types of stimulation, except for a few type I fibres, stimulated by quasitrapezoidal impulses. In other rats glycogen depleted fibres were always the fibres of the largest diameter, most probably also type IIB fibres in both types of stimulation. Glycogen depletion of type I fibres was never observed in muscles stimulated by rectangular stimuli.

A Windows computer application for long term data acquisition, storage and presentation of biological signals.

We developed a windows application dedicated to recording, displaying and storing large amounts of biological signals on a standard PC. The application works in Matlab and uses an A/D-converter interface that enables a user to run the same software for signal acquisition, display and processing with different A/D-converter boards. The software program consists of three integrated modules, for setting the protocol for signal acquisition; for commands that can be accessed during data acquisition and for software routines that enable off-line viewing, pre-processing and processing of recorded data. It adapts to different computer hardware configurations by enabling the user to define file size, data resolution and file storage on local and network hard disks during signal acquisition.

Presentation of dermatological images on the Internet.

In this paper, we focused on selected problems of integrating and presenting medical images organised in a World Wide Web (WWW) database. To solve these problems we developed a prototype of a bilingual (Slovenian and English) WWW database of medical images for the field of dermatology. This dermatology database includes a graphic interface with four modes of access: (1) browsing, (2) searching, (3) comparison of images, and (4) self-testing. The quantity and quality of requests to this WWW database was estimated with log file analysis. There was a steady increase in the number of users and volume of data transferred from the dermatology WWW database.

Three-dimensional study of the capillary supply of skeletal muscle fibres using confocal microscopy.

Three-dimensional (3D) study of capillary network of individual muscle fibres in rat extensor digitorum longus (EDL) and soleus (SOL) muscles is presented. Stereology and 3D reconstruction techniques were applied to stacks of serial optical sections recorded by a confocal microscope from thick muscle slices. The results suggest that SOL muscle fibres have a larger surface area and volume as well as a larger length of capillaries per fibre length than EDL. On the other hand, these two muscles have a similar ratio of capillary length to fibre surface area. The 3D approach to evaluation of muscle fibre capillarization brings many advantages over traditional measurements made on single muscle sections and could also be applied to the study of angiogenesis in other tissues.

Reversible transitions between synchronization states of the cardiorespiratory system.

Phase synchronization between cardiac and respiratory oscillations is investigated during anesthesia in rats. Synchrograms and time evolution of synchronization indices are used to show that the system passes reversibly through a sequence of different phase-synchronized states as the anesthesia level changes, indicating that it can undergo phase transitionlike phenomena. It appears that the synchronization state may be used to characterize the depth of anesthesia.

The contribution of lumbar sympathetic neurones activity to rat's skin blood flow oscillations.

Skin blood flow on the rat's paws using laser Doppler flowmeter, electrical activity of the heart (ECG) and respiration were measured simultaneously. The signals were recorded for 20 minutes, both before and after denervation, at core temperature 37 degrees C and 38.5 degrees C, that was maintained constant during the recordings. Spinal nerve fibres, at the level L3-L4, were transected. Experiments were performed on 15 adult Wistar rats under general anaesthesia. The oscillations in the measured signals were analysed in the time-frequency domain using wavelet transform. On the frequency region from 0.7 Hz to 5 Hz two characteristic peaks were observed in the skin blood flow spectrum. They correspond to the main peaks in the spectra of the ECG (around 3.3 Hz) and respiration (around 1.3 Hz). Several additional peaks were observed in the low frequency region, from 0.01 to 0.7 Hz, in all measured signals. In this frequency region the relative energy contribution of the blood flow oscillations decreased after denervation only in the denervated left hind paw. This difference was not statistically significant at 37 degrees C (p=0.098, Kruskal-Wallis test) but became statistically significant at 38.5 degrees C (p=0.017). Relative energy contribution of the low frequency region, from 0.01 to 0.7 Hz, decreased 2.5-fold in the blood flow of the denervated paw. Within this region the relative energy contribution decreased significantly in two intervals, from 0.01 to 0.08 Hz and from 0.08 to 0.2 Hz (p=0.023). In the higher frequency region, from 0.7 to 5 Hz, o statistically significant differences were obtained in any paws when compared before and after denervation at the same core temperature. We conclude that the activity of lumbar sympathetic neurones contributes to low frequency skin blood flow oscillations.

Modification of skeletal muscle AChE expression by a novel method of stimulus application to the peripheral nerve.

We have developed a new method for chronic application of electrical stimuli to the rat peripheral nerve in vivo. This method has the following advantages: (1) the amplitude, duration and pattern of stimulation can be adjusted before and during the course of experiment, (2) the set-up allows the animal to move freely during the experiment, and (3) the set-up is constructed from inexpensive, of-the-shelf components that can be reused several times. The new method was used to study the influence of the pattern of muscle activation on acetylcholinesterase (AChE) regulation in extensor digitorum longus (EDL) and tibialis anterior (TA), fast skeletal muscles. Northern blot analysis of the chronically stimulated, fast EDL and TA revealed a rapid decrease of AChE mRNA level to a level typical for a slow, skeletal muscle.