Invited review: Human, cow, and donkey milk comparison: Focus on metabolic effects.

Milk is an important food of the daily diet. Many countries include it in their dietary recommendations due to its content in several important nutrients that exert beneficial effects on human health. Human milk is a newborn's first food and plays an important role in the growth, development, and future health of every individual. Cow milk is the type of milk most consumed in the world. However, its relatively high content of saturated fats raises concerns about potential adverse effects on human health, although epidemiological studies have disproved this association. Indeed, dairy consumption appear to be linked to a lower risk of mortality and major cardiovascular disease events. In the last few years many researchers have begun to focus their attention on both the production and quality of cow milk as well as the analysis of milk from other animal species to evaluate their effect on human health. The need to investigate the composition and metabolic effects of milk from other animal species arises from the adverse reactions of individuals in several groups to certain components of cow milk. It has emerged that donkey milk compared with that of other animal species, is the nearest to human milk and an excellent substitute for it. Milk from various animal species shows substantial differences in nutritional composition and distinct metabolic effects. In this review, we discussed the main compositional features and metabolic effects of 3 types of milk: human, cow, and donkey milk.

The influence of mandibular stretching on athletes subjected to high intensity workout.

The use of oral appliances is considered effective in improving sport performance. Jaw clenching or stretching, improved breathing or positioning of the jaw have been proposed to explain the improvement. In this study, we reported the effects of regularly practiced mandibular stretching at submaximal opening of the jaw in Crossfit athletes. Seventeen test subjects undergoing mandibular stretching (MS) showed significantly reduced (p0.001) performance time in a high-intensity workout measured before and after seven days of 10-minutes stretching practiced twice daily, while no improvement was observed in control subjects who did not perform the stretching. Additionally, blood lactate levels appeared to recover more quickly in MS subjects.Crossfit is a type of training regimen that pushes the limits of bodily recovery processes. Jaw stretching, eliciting the trigeminocardiac reflex (a reflex decrease in heart rate and blood pressure following trigeminal stimulation) and central oxygen-conserving mechanisms could help relieve post-training stress and improve training recovery.

Learning effect and repeatability of stabilometric measurements: "standard" vs. usual foot placement.

The existence of a learning effect by which subjects progressively reduce body sway over the course of repetitive stabilometric measurements is currently debated. Also, the position and orientation of the feet on the platform can have a substantial influence on the outcome measurements. The aim of the present work was to assess the effect of feet positions on mean total velocity (V) of the center of pressure and the area (AR) covered by its displacements during quiet standing. A group of 35 healthy young subjects was examined during two successive sessions consisting of five recordings with their feet placed either in the recommended (standard, SP) or their usual most comfortable (UP) position. Results show a slight decreasing trend that failed to be statistically significant checked with Friedman's ANOVA (SP AR, χ2(4)=6.10, p=0.19 and V, χ2(4)=8.66, p=0.07 and UP AR, χ2(4)=2.32, p=0.68 and V, χ2(4)=1.19, p=0.88). Nonetheless, values of AR and V showed a notable decrement especially evident in the SP exam reaching, respectively, 24% and 11% from baseline, whereas variability measured by the coefficient of variation was the same in the two exams. Given the results, a learning effect should not be ruled out with confidence. Also, usual foot placement would be preferable to avoid this effect. Further research is needed to take into consideration the great variability of stabilometric measurements and the fact that different subjects could adapt more readily to the test conditions than others.

Changes of sensory and pain thresholds in healthy subjects after mandibular extension at maximum mouth opening: implications for temporomandibular disorders therapy.

There is evidence showing that increasing the vertical dimension of occlusion, or interocclusal distance, reduces pain symptoms in patients with temporomandibular disorders (TMD) and bruxism. The mechanism underlying the analgesic effect is still under investigation. Some researchers propose either a gate mechanism or the activation of the trigemino-cardiac reflex. In this study, sensory and pain thresholds changes in the mandibular district have been evaluated in twenty healthy subjects immediately after 5 minutes of mandibular stretching at maximum opening of the jaw. Results showed a 60% increment in sensory threshold and a 70% increment in pain threshold (p<0.001) compared with the baseline values. The magnitude of the analgesic effect seems proportional to the extent of mandibular opening. These and similar results are discussed as for the implication in the treatment of TMD related symptoms.

An observational study of the effects of using an high oral splint on pain control

Increasing occlusal vertical dimension (OVD) by means of oral splints is a practice widely used in the treatment of temporomandibular disorders (TMD), specifically myofascial pain, although the results are still uncertain. Oral splints with a vertical height that significantly exceeds the clinical rest position are considered by some researchers to be a better therapeutic solution in alleviating TMD symptoms than are "low" splints. In our observational study, 21 patients suffering from myofascial pain were examined for the effects of wearing a "high" oral splint while sleeping for 3 months. To ensure proper splint making, a mandibular stretching procedure was used to induce a relaxation of the patients' masticatory muscles and allow the correct alignment of the jaws. Results showed a marked increase of the interocclusal distance or "free space" (hence of OVD; from 0.64±0.53 mm to 1.42±0.76 mm, p<0.0001) measured by a kinesiograph, followed by a substantial reduction of the intensity of pain in oral and extraoral regions after using the splint. These results support the view that increasing OVD beyond the clinical rest position is not detrimental to patients' health. More importantly, high oral splints has been shown to be a promising therapeutic aid for the treatment of TMD and correlated pain syndromes. This clinical trial was registered on clinicaltrials.gov (Identifier: NCT02908568).

Postural Disorders Produced by School Furniture on a Population of a Junior High School.

In recent years people suffering of backache has significantly increased. This led us to focus our attention on the adequacy of school furniture for the correct development of the adult posture. The standing posture of 67 students of middle school has been analyzed at the beginning and at the end of two consecutive scholastic years using a stabilometric platform. Starting from the second year, about half of the students were provided with the furniture designed following European standard (UNI EN 1729:2006), while the other half maintained the traditional one. The main purpose of this research has been to verify by means of posturometric analysis the effects on postural parameters of the use of traditional furniture in comparison to the furniture following the UNI EN 1729:2006. We observed that prolonged sitting at school changes some posturometric variables of schoolchildren. Since no differences has been found between the two groups, the validity of the European standards is questioned. The present study allowed us to single out four anthropometric parameters that should be considered in order to devise a new model of adjustable furniture. By adjusting every year the furniture of each student, it would be possible to avoid (at least at school) the adoption of wrong postural positions that could be responsible for backache and other common musculoskeletal disorders.

Vitamin D receptor expression and acid sphingomyelinase activity in prefrontal region of a learning animal model.

Contextual fear conditioning (CFC) paradigm is routinely used to study fear-based learning in animals and it provides a useful model for understanding fear and anxiety in human. In the present study, such model was used following the previously established CFC protocol, and immunohistochemistry, enzymatic activity and western blotting analysis approaches were used to identify the expression of acid sphingomyelinase (aSMase) and vitamin D receptor (VDR) in prefrontal region brain of rat. Results revealed an increase of aSMase activity in conditioned rats, suggesting an apoptotic condition in such animals. In addition, an increase of density and organization of axonal neurofilaments and of VDR expression has been observed in brain of conditioned rats, supporting an induction of growth and organization of new neurons in prefrontal regions, whose contribution to various aspects of contextual fear learning is still largely unknown.

Contextual fear conditioning modulates the gene expression over time.

Contextual fear conditioning (CFC) is a quick cognitive test based on the association context-aversive stimulus in which a single training leads to a long-term memory. Previously, we showed that 2 days after conditioning the expression of the genes Napa, Pnf2, Casp3, Pdrg1, Ywhaz, Stmn1, Bpgm, were positively modulated in CFC rats respect to naïve rats, explor rats which had freely explored the experimental apparatus and SO rats to which the same number of aversive shocks used in CFC paradigm had been administered in the same CFC apparatus in less time to prevent the association between painful stimuli and apparatus, whereas the genes Actr3, Pea15 and Tiprl were more expressed in SO rats and Cplx1, Trim32 and Ran genes were more expressed in explor rats. At 2 days, Tomm20 gene expression resulted positively modulated in both CFC and explor rats. Herein, we have tested the expression of these genes for a period longer than 2 days, by monitoring the modulation of transcripts within 20 days after conditioning. The expression of the transcripts was assessed by qRT-PCR.We found that three days after CFC only the genes Tiprl and Trim32 were positively modulated in CFC rats whereas the gene Tomm20 was negatively modulated in CFC rats as well as in SO and explor rats. Ten days after CFC, the expression of Trim32 was still positively modulated whereas the genes Tiprl and Tomm20 returned to the constitutive level, and the gene Ran was significantly more expressed in CFC rats than in naïve, SO and explor rats. Interestingly, 20 days after CFC, the genes Stmn1 and Tiprl again became significantly more expressed in CFC rats compared with naïve, SO and explor rats.

Probiotic mixture supplementation in the preventive management of trinitrobenzenesulfonic acid-induced inflammation in a murine model.

Inflammatory bowel diseases (IBD) are characterized by inflammatory conditions of the intestine. Probiotic bacteria (PB) can have beneficial effects in several gastrointestinal disorders. The objectives of this study were: (i) to provide an acute experimental IBD model induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) in CD-1 mice, and (ii) to assess the preventive effects of Citogenex (Lactobacillus casei and Bifidobacterum lactis) supplementation on intestinal tissues and microbiota. Mice were inoculated intrarectally with saline, ethanol or different TNBS solutions. 1%TNBS induced clinical signs of colitis (P less than 0.01) and histological damage (P less than 0.01). Based on these results, mice were pre-treated with Citogenex or saline for 1, 2 or 3 weeks before 1%TNBS treatment. Probiotic pre-treatment determined a reduction of clinical signs (P less than 0.05), histological alterations of colitis (P less than 0.05) and increased beneficial bacteria (P less than 0.05). This study confirms that TNBS-induced colitis in CD-1 mice is useful for studying the mechanisms involved in IBD pathogenesis, and pre-treatment with Citogenex prevents the intestinal damage induced by TNBS.

Lactobacillus casei and bifidobacterium lactis supplementation reduces tissue damage of intestinal mucosa and liver after 2,4,6-trinitrobenzenesulfonic acid treatment in mice.

Probiotics (PB) are living microorganisms that act as a commensal population in normal intestines and confer numerous beneficial effects on the host. The introduction of probiotics in the treatment of inflammatory bowel disease (IBD) prolongs remission. The aim of this study was to investigate the intestinal and hepatic effects of PB supplementation in an experimental IBD model in mice induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). In the first step of the experimental procedure, CD-1 male mice, 5 to 6 weeks old, were randomly divided into 3 groups and inoculated intrarectally with, respectively, saline, alcohol, or TNBS to assess the experimental IBD model. In the second step, mice treated, or not, with TNBS inoculation, were treated with PB (Lactobacillus Casei, Bifidobacterum Lactis) for 1, 2 or 3 weeks, on a daily basis. Large bowel (colon and rectum) and liver were processed for histological alterations, according to a scoring system. Large bowel was also assessed for apoptosis by TUNEL assay. TNBS induced, as expected, severe damage and inflammation in the large bowel, including nuclear alterations and apoptosis, and, to a lesser extent, to the liver. Administration of PB determined significant reduction of both histological alterations and apoptosis. PB administration in advance protects from inflammation. In conclusion, supplementation with Lactobacillus casei, Bifidobacterum lactis PB is able to ameliorate the colitis by reversing the histological changes caused by TNBS in mice. Experimentation in human subjects in needed to prove their efficacy in reducing histological alterations that may be present in subjects with IBD.

Ribosomal RNA characterization in the leech Hirudo medicinalis.

In the present study the ribosomal RNA of the leech Hirudo medicinalis has been characterized at the aim of identifying possible analogies with other invertebrates. Upon electrophoresis on denaturating gels, ribosomal RNA fraction of H. medicinalis exhibited a remarkable thermal instability by dissociating into two hydrogen-bonded components when heated at 60 degrees C, at variance with the behaviour of the rat rRNA, which does not show this process. This result suggests a functional role in leech ribosome organisation that requires deeper structural studies.

Using real-time PCR to identify pregnancy-associated glycoprotein 2 (PAG-2) in water buffalo (Bubalus bubalis) blood in early pregnancy.

This study investigates for the first time mRNA pregnancy-associated glycoprotein 2 (PAG-2) expression in blood cells during early pregnancy in water buffalo. The PAGs constitute a large family of glycoproteins expressed in the outer epithelial layer of the placenta in eutherian species. All PAGs are not concomitantly expressed throughout pregnancy; some of them are expressed in the earlier phases, whereas others appear later and are expressed over a shorter period. Twenty-one lactating buffaloes were analyzed-17 females were synchronized with PRID and artificially inseminated (AI), whereas four females were synchronized but not inseminated (control group). Blood was collected at Days 0, 18, 28, 40, and 75 from AI (AI = Day 0). Expression of PAG-2 mRNA in blood samples was measured with real-time polymerase chain reaction. Pregnancy diagnosis was performed on Day 28 (D28) and Day 40 (D40) after AI by ultrasonography (US) and by PAG-1 RIA method. The females diagnosed pregnant at D28 and confirmed pregnant at D40 were defined as D28(+)D40(+) group; the females diagnosed pregnant at D28 but not confirmed pregnant at D40 were defined as D28(+)D40(-) group; and the females that were diagnosed as nonpregnant on either days were defined as D28(-)D40(-) group. PAG-2 mRNA at Day 0 was not observed in any groups. The D28(+)D40(+) group showed the highest expression, starting on Day 18 and increasing progressively up to Day 75. PAG-2 mRNA was also expressed on Day 18 in both D28(+)D40(-) and D28(-)D40(-) groups, but their levels were lower than those of D28(+)D40(+) group and almost constant over time. PAG-2 mRNA was never detected in the control group. The significant difference in the expression of PAG-2 mRNA between the D28(+)D40(+) group and the D28(-)D40(-) group, starting from Day 18, suggests that these animals might have conceived, but have experienced early embryonic loss; therefore, the PAG-2 mRNA was still present in blood circulation although at lower levels, as found in the D28(+)D40(-) group. In conclusion, this study shows that PAG-2 mRNA can be detected in peripheral maternal blood cells earlier than circulating PAG-1 molecules and could be useful for studies on early pregnancy and embryonic mortality.

Acetyl-L-carnitine affects nonassociative learning processes in the leech Hirudo medicinalis.

Acetyl-L-carnitine is a natural molecule widely distributed in vertebrate and invertebrate nervous system. It is known to have significant effects on neuronal activity playing a role as neuroprotective and anti-nociceptive agent, as well as neuromodulatory factor. About its capability of affecting learning processes the available data are controversial. In the present study, we utilized the simplified model system of the leech Hirudo medicinalis to analyze the effects of acetyl-L-carnitine, assessing whether and how it might affect elementary forms of nonassociative learning processes. In leeches with the head ganglion disconnected from the first segmental ganglion, repetitive application of weak electrical shocks onto the caudal portion of the body wall induces habituation of swim induction whereas brush strokes on the dorsal skin produces sensitization or dishabituation when the nociceptive stimulus is delivered on previously habituated animals. Herein, the effects of different concentrations of acetyl-L-carnitine (2 mM - 0.05 mM) have been tested at different times on both sensitization and dishabituation. The results show that a single treatment of acetyl-L-carnitine blocked the onset of sensitization in a dose- and time-dependent manner. In fact, the most effective concentration able to block this process was 2 mM, which induced its major effects 11 days after the treatment, whereas 0.05 mM was unable to affect the sensitization process at all considered time points. On the contrary, acetyl-L-carnitine did not completely abolish dishabituation at the tested concentrations and at every time point. Finally, acetyl-L-carnitine also impaired the habituation of swim induction, but only 11 days after treatment.

Static and dynamic baropodometry to evaluate patients treated by total knee replacement with a mobile meniscus.

It was the purpose of this study to use static and dynamic baropodometry pre- and postsurgery in patients submitted to knee replacement with a mobile bearing. Baropodometric analysis was accompanied by clinical and subjective evaluation. Data obtained from the baropodometer revealed considerable oscillations occurring between pre- and postsurgery. Static examination showed a significant increase in the percentage of loading on the side treated that was not observed in the dynamic evaluation. An analysis of the Knee Society score showed a considerable increase in values between pre- and postsurgery similar to how the interpretation of correlations presurgery revealed statistically significant values between function and length of step, between pre- and postsurgery data for scores for function and length of step and Knee Score. The short-term results obtained are encouraging but only a long-term follow-up will be able to confirm the use of evaluating total knee replacement.

Teicoplanin pharmacokinetics in patients with chronic renal failure.

The pharmacokinetic profile of teicoplanin, a new glycopeptide antibiotic active against Gram-positive aerobic and anaerobic bacteria, was studied in 5 healthy male volunteers and 29 adult patients with various degrees of renal impairment, given a single 3 mg/kg intravenous dose. Teicoplanin was assayed in plasma and urine specimens by a microbiological method. Pharmacokinetic parameters for teicoplanin were estimated both by a 3-compartment open pharmacokinetic model and by non-compartmental analysis. Elimination half-life increased with the decrease in creatinine clearance and mean values ranged from 41 hours in volunteers to 163 hours in anuric patients. Renal failure did not affect either the volume of distribution of the central compartment (mean approximately 0.09 L/kg) or the steady-state volume of distribution (mean approximately 0.9 L/kg). Both total and renal clearance decreased with severity of disease, particularly the latter, while non-renal clearance was unaffected by renal failure. Average values were from 19 to 6 ml/min for total clearance and from 12 to 0.4 ml/min for renal clearance. There was a linear correlation between the total clearance of teicoplanin and creatinine clearance, as well as between renal clearance and creatinine clearance. The total urinary excretion of active teicoplanin averaged 65% of the administered dose in normal subjects, but was significantly reduced in the presence of renal insufficiency. Guidelines for administration of teicoplanin in patients with renal failure are given.

Modulation of gene expression in human osteoblasts by targeting a distal promoter region of human estrogen receptor-alpha gene.

Estrogen receptor (ER) alpha is expressed during osteoblast differentiation; however, both its functional role in bone metabolism and its involvement in osteoporotic pathogenesis caused by estrogen deficiency are not well understood. Loss of ER alpha gene expression could be one of the mechanisms leading to osteoporosis. Therefore, we investigated a possible modulation of ER alpha gene expression in a human osteoblastic cell line and in four primary osteoblast cultures by using a decoy strategy. Double stranded DNA molecules, mimicking a regulatory region of the ER alpha gene promoter (DNA-102) and acting as a 'silencer' in breast cancer cells, were introduced into osteoblasts as 'decoy' cis-elements to bind and functionally inactivate a putative negative transcription factor, and thus to induce ER alpha gene expression. We found that the DNA-102 molecule was able to specifically bind osteoblast nuclear proteins. Before decoy treatment, absence or variable low levels of ER alpha RNAs in the different cultures were detected. When the cells were transfected with the DNA-102 decoy, an increase in expression of ER alpha and osteoblastic markers, such as osteopontin, was observed, indicating a more differentiated osteoblastic phenotype both in the cell line and in primary cultures. These results showed that the DNA-102 sequence competes with endogenous specific negative transcription factors that may be critical for a decrease in or lack of ER alpha gene transcription. Therefore, osteoblastic transfection with the DNA-102 decoy molecule may be considered a tempting model in a putative therapeutic approach for those pathologies, such as osteoporosis, in which the decrease or loss of ER alpha expression plays a critical role in bone function.

Nonassociative learning in the leech Hirudo medicinalis.

In the present study we examined nonassociative learning of the induction of swimming which was evoked by weak electrical stimulation in the leech Hirudo medicinalis. The behavioural response to stimuli applied repeatedly to the body wall at an inter-trial interval (ITI) of 1 min decreased, eventually ceased, and then recovered spontaneously. More rapid reduction of the behavioural response occurred in repeated training sessions. This decrement of response conformed to the operational definition of habituation. Moreover, a noxious stimulus (i.e. brushing on the skin) facilitated the decremented response (dishabituation). In addition, we compared response decrement in naive animals with decrement in dishabituated and in sensitized animals. The analysis of the best fitting functions of the habituation, the habituation of dishabituation and the habituation of sensitization revealed interesting differences in these processes.

Somatostatin enhances neurite outgrowth in PC12 cells.

The rat pheochromocytoma cell line PC12 forms neurites in response to nerve growth factor (NGF), and it was also reported to extend processes in the presence of somatostatin (somatotropin release-inhibiting factor, SRIF), a neuroactive peptide that seems to act as a morphogenetic factor in the developing nervous system. In the present study, we re-evaluated the effects of SRIF on PC12 cell differentiation. Our results indicate that SRIF alone is ineffective in promoting neurite outgrowth. Instead, SRIF or its analogue, octreotide (a SRIF agonist on the receptor subtypes 2, 3 and 5), potentiates neurite extension induced by NGF. These results suggest that SRIF enhances neurite formation in PC12 cells without directly promoting neurite outgrowth. SRIF potentiation of NGF-induced neurite outgrowth persists at least in part in the presence of pertussis toxin (PTX), suggesting the involvement of PTX-insensitive G-proteins. In addition, protein kinase-dependent pathways are likely to mediate SRIF effects on NGF-induced differentiation.

Mechanisms mediating somatostatin-induced reduction of cytosolic free calcium in PC12 cells.

In PC12 cells, somatostatin (SRIF) decreases the Ca2+ influx in response to high K+ through the inhibition of voltage-dependent Ca2+ channels. In the present study, we measured the intracellular Ca2+ concentration ( [Ca2+]i) following the application of SRIF-14 or its analogues which bind to different SRIF subtype (sst) receptors. Their application differentially reduces [Ca2+]i (octreotide > SRIF-14 > CGP23996 > BIM23056) in a dose-dependent manner. Pretreatment with a SRIF antagonist prevents the SRIF-induced inhibition of [Ca2+]i. These results suggest the involvement of specific membrane receptors and are in line with recent findings indicating the presence of receptor mRNAs on PC12 cells. Our results also exclude the possibility that SRIF interacts with opioid receptors and suggest that the SRIF-induced inhibition of [Ca2+]i is mediated by mechanisms involving PTX-sensitive G-proteins and PK-dependent pathways.