Nuclear stiffening and chromatin softening with progerin expression leads to an attenuated nuclear response to force.

Progerin is a mutant form of the nucleoskeletal protein lamin A, and its expression results in the rare premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS). Patients with HGPS demonstrate several characteristic signs of aging including cardiovascular and skeletal dysfunction. Cells from HGPS patients show several nuclear abnormalities including aberrant morphology, nuclear stiffening and loss of epigenetic modifications including heterochromatin territories. However, it is unclear why these changes disproportionately impact mechanically-responsive tissues. Using micropipette aspiration, we show that nuclei in progerin-expressing cells are stiffer than control cells. Conversely, our particle tracking reveals the nuclear interior becomes more compliant in cells from HGPS patients or with progerin expression, as consistent with decreased chromatin condensation as shown previously. Additionally, we find the nuclear interior is less responsive to external mechanical force from shear or compression likely resulting from damped force propagation due to nucleoskeletal stiffening. Collectively our findings suggest that force is similarly transduced into the nuclear interior in normal cells. In HGPS cells a combination of a stiffened nucleoskeleton and softened nuclear interior leads to mechanical irregularities and dysfunction of mechanoresponsive tissues in HGPS patients.

Probing the biophysical properties of primary breast tumor-derived fibroblasts.

As cancer progresses, cells must adapt to a new and stiffer environment, which can ultimately alter how normal cells within the tumor behave. In turn, these cells are known to further aid tumor progression. Therefore, there is potentially a unique avenue to better understand metastatic potential through single-cell biophysical assays performed on patient-derived cells. Here, we perform biophysical characterization of primary human fibroblastic cells obtained from mammary carcinoma and normal contralateral tissue. Through a series of tissue dissociation, differential centrifugation and trypsinization steps, we isolate an adherent fibroblastic population viable for biomechanical testing. 2D TFM and 3D migration measurements in a collagen matrix show that fibroblasts obtained from patient tumors generate more traction forces and display improved migration potential than their counterparts from normal tissue. Moreover, through the use of an embedded spheroid model, we confirmed the extracellular matrix (ECM) remodeling behavior of primary cells isolated from carcinoma. Overall, correlating biophysical characterization of normal- and carcinoma-derived samples from individual patient along with patient outcome may become a powerful approach to further our comprehension of metastasis and ultimately design drug targets on a patient-specific basis.

Repair of peripheral nerve with vein wrapping.

OBJECTIVE: The post-traumatic neuro-anastomosis must be protected from the surrounding environment. This barrier must be biologically inert, biodegradable, not compressing but protecting the nerve. Formation of painful neuroma is one of the major issues with neuroanastomosis; currently there is no consensus on post-repair neuroma prevention. Aim of this study is to evaluate the efficacy of neuroanastomosis performed with venous sheath to reduce painful neuromas formation, improve the electrical conductivity of the repaired nerve, and reduce the discrepancies of the sectioned nerve stumps. PATIENTS AND METHODS: From a trauma population of 320 patients treated in a single centre between January 2008 and December 2011, twenty-six patients were identified as having an injury to at least one of the peripheral nerves of the arm and enrolled in the study. Patients were divided into two groups. In the group A (16 patients) the end-to-end nerve suture was wrapped in a vein sheath and compared with the group B (10 patients) in which a simple end-to-end neurorrhaphy was performed. The venous segment used to cover the nerve micro-suture was harvested from the superficial veins of the forearm. The parameters analyzed were: functional recovery of motor nerves, sensitivity and pain. RESULTS. Average follow-up was 14 months (range: 12-24 months). The group A showed a more rapid motor and sensory recovery and a reduction of the painful symptoms compared to the control group (B). CONCLUSIONS: The Authors demonstrated that, in their experience, the venous sheath provides a valid solution to avoid the dispersion of the nerve fibres, to prevent adherent scars and painful neuromas formation. Moreover it can compensate the different size of two nerve stumps, allowing, thereby, a more rapid functional and sensitive recovery without expensive devices.

Physical biology in cancer. 5. The rocky road of metastasis: the role of cytoskeletal mechanics in cell migratory response to 3D matrix topography.

The tumor microenvironment is a milieu of heterogeneous architectural features that affect tumor growth and metastatic invasion. Pore size, density, stiffness, and fiber architecture change dramatically from location to location throughout the tumor matrix. While many studies have addressed the effects of two-dimensional extracellular matrix structure and composition on cell migration, less is known about how cancer cells navigate complex, heterogeneous three-dimensional (3D) microenvironments. Mechanical structures such as actin and keratin, part of the cytoskeletal framework, and lamins, part of the nucleoskeletal framework, play a key role in migration and are altered during cancer progression. Recent evidence suggests that these changes in cytoskeletal and nucleoskeletal structures may enable cancer cells to efficiently respond to features such as pore size and stiffness to invade and migrate. Here we discuss the role of cell mechanics and the cytoskeleton in the ability of cells to navigate and respond to 3D matrix features and heterogeneities.

Force-induced changes in subnuclear movement and rheology.

Extracellular mechanical forces result in changes in gene expression, but it is unclear how cells are able to permanently adapt to new mechanical environments because chemical signaling pathways are short-lived. We visualize force-induced changes in nuclear rheology to examine short- and long-time genome organization and movements. Punctate labels in the nuclear interior of HeLa, human umbilical vein endothelial, and osteosarcoma (Saos-2) cells allow tracking of nuclear movements in cells under varying levels of shear and compressive force. Under adequate shear stress two distinct regimes develop in cells under mechanical stimulation: an initial event of increased intranuclear movement followed by a regime of intranuclear movements that reflect the dose of applied force. At early times there is a nondirectionally oriented response with a small increase in nuclear translocations. After 30 min, there is a significant increase in nuclear movements, which scales with the amount of shear or compressive stress. The similarities in the nuclear response to shear and compressive stress suggest that the nucleus is a mechanosensitive element within the cell. Thus, applied extracellular forces stimulate intranuclear movements, resulting in repositioning of nuclear bodies and the associated chromatin within the nucleus.

Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.

Syndecan expressions in the human amnion and chorionic plate.

The syndecan family consists of four distinct membrane glycoproteins in mammals. Syndecans control cell proliferation, differentiation, adhesion and migration through participation in cell-cell interactions, anchorage of cells to the extracellular environment, and modulation of multiple growth factors. Therefore, syndecans may play a pivotal role in the regulation of cell behaviour depending on the cellular microenvironment. Here, we demonstrate that syndecan-1, syndecan-2 and syndecan-4 are expressed in fetal membrane tissue with different immunolocalizations. Syndecan-1 is expressed in the amniotic epithelium, localizing at basolateral cell surfaces. Syndecan-2 and syndecan-4, in contrast, are mostly localized in intracellular compartments, in the extravillous cytotrophoblastic cells and in some fibroblasts of the chorionic plate as well as in the amniotic epithelial cells. In the latter, syndecan-4 is mainly localized in the apical part of the cells. Our results strongly suggest a key role of syndecan-1, syndecan-2 and syndecan-4 in the determination of structural and functional characteristics of human amnion and chorionic plate. Since the solute exchanges between fetus and mother take place in fetal membranes, our data suggest that syndecans are important players in the placenta for the establishment of the fetal-maternal inter-communication.

Nitric oxide and peroxynitrite platelet levels in women with small-for-gestational-age fetuses.

OBJECTIVE: The placenta produces reactive oxygen species (ROS) including nitric oxide (NO) and peroxynitrite (ONOO(-)) that have pronounced effects on placental function. Excessive ROS production may occur in pathological pregnancies, such as those complicated by small-for-gestational-age (SGA) fetuses. DESIGN: The aim of the present work was to study NO and ONOO(-) levels in platelets of pregnant women with SGA fetuses compared with a control group. SETTING AND POPULATION: The study was performed on 30 pregnant women with SGA fetuses (SGA group) and on 30 healthy pregnant women (appropriate-for-gestational-age [AGA] group) matched for maternal and gestational age. All women included in this study were in the third trimester of pregnancy. METHODS: Platelets were isolated by differential centrifugation. NO metabolites, after enzymatic conversion followed by the Griess reaction, were measured as nitrite by spectrophotometric detection. Peroxynitrite (ONOO(-)) levels were evaluated using the fluorescence probe 2,7-dichlorofluorescein diacetate (DCFDA). MAIN OUTCOME MEASURES: The following determinations were made: platelet nitric oxide and peroxynitrite levels in the SGA group and controls; inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and nitrotyrosine (N-Tyr) expression in the same groups. RESULTS: Our results show that both platelet NO and ONOO(-) levels were significantly higher in the SGA group than in the controls. CONCLUSION: Increased platelets levels of nitric oxide and peroxynitrite might play a role in the pathophysiology of intrauterine growth restriction. Further investigations are in progress to clarify if these molecules are pathogenetic factors, an epiphenomenon or a pathophysiological marker.

Circadian melatonin concentration rhythm is lost in pregnant women with altered blood pressure rhythm.

We assessed the correlation between the rhythm of melatonin concentration and circadian blood pressure patterns in normal and hypertensive pregnancy. Ambulatory 24-h blood pressure and blood samples every 4 h were monitored in 16 primigravidae who had shown an abnormal circadian blood pressure pattern (eight pre-eclamptic and eight normotensive) in pregnancy and 6-12 months after pregnancy. The circadian rhythm was analyzed by chronobiological measures. Eight normotensive women with maintained blood pressure rhythm served as controls. During pregnancy, melatonin concentration was significantly higher in pre-eclamptic than in normotensive women (pre-eclampsia, 29.4 +/- 1.9 pg/ml, normotensin, altered rhythm, 15.6 +/- 2.1; controls, 22.7 +/- 1.8; p < 0.001). This difference faded after pregnancy, owing to the fall observed in pre-eclampsia (11.8 +/- 3.2 pg/ml, 9.8 +/- 2.1, and 11.1 +/- 2.0, respectively; NS). The rhythm of melatonin concentration was lost in all pregnant women with loss of blood pressure rhythm. After pregnancy, normotensive women showed a reappearance of both melatonin and blood pressure rhythm, whereas pre-eclamptic women showed a reappearance of blood pressure but not melatonin rhythm. The loss of blood pressure rhythm in pregnancy is consistent with the loss of melatonin concentration rhythm. In pre-eclamptic women, the normalization of blood pressure rhythm, while melatonin rhythm remained altered, suggests a temporal or causal priority of circadian concentration of melatonin in the determination of blood pressure trend.

Estrogen downregulates the number of caveolae and the level of caveolin in uterine smooth muscle.

Estrogen and progesterone, while regulating uterine functions, also regulate the number of caveolae and the level of caveolin. Large numbers of caveolae, as well as elevated expression of caveolin-1 and caveolin-2 isoforms in the myometrium of ovariectomised (OVX) rats were detected. 17beta-estradiol (E2) has a downregulating effect: the treatment of OVX rats with E2 (5 microg/animal) reduced the formation of caveolae by approx. 90%. Western blots clearly demonstrated the reduction of membrane caveolin-1 and -2 content. Progesterone treatment (2.5 mg/animal) alone did not cause any substantial change, but prevented the effect of estrogen. Control experiments showed that the quantity of Na+/K+-ATPase, a plasma membrane protein excluded from caveolae, was not downregulated by E2. The administration of the pure estrogen receptor (ERalpha) antagonist ICI 182,780 (1 mg/animal) not only compensated for the inhibitory effect of E2, but further increased the level of caveolin-1 in the myometrium of OVX rats and facilitated the formation of caveolae by approximately 70%. In contrast, the partial antagonist tamoxifen (1 mg/animal) mimicked the effect of estrogen. The amount of caveolin also changed during pregnancy. During the first half of pregnancy the expression of caveolin was suppressed, but it gradually increased until delivery. Our results indicate that the formation and number of caveolae are influenced by the physiological state of the uterus in a hormone dependent manner.

Caveolin isoforms in resident and elicited rat peritoneal macrophages.

Caveolin--an integral membrane protein--is the principal component of caveolae membranes in vivo. Multiple forms of caveolin have been identified: caveolin-1alpha, caveolin-1beta, caveolin-2 and caveolin-3. They differ in their specific properties and tissue distribution. When we studied the lysate of resident and elicited macrophages isolated from rat peritoneal cavity by Western blot analysis, we identified two different proteins (approximately 29 kDa and approximately 20 kDa) which were labelled with anti-caveolin antibodies. The approximately 20-kDa protein was labelled specifically only by anti-VIP21/caveolin-1, while the approximately 29-kDa protein was labelled by anti-VIP21/caveolin-1 and anti-caveolin-2. The presence of the approximately 29-kDa protein was characteristic of resident macrophages, and only a small amount of the approximately 20-kDa protein was detected in these cells. Elicitation resulted in a significant increase in the amount of the approximately 20-kDa protein labelled by anti-VIP21/caveolin-1 only. According to its molecular mass and antibody-specificity, this protein might be identical with the caveolin-1beta isoform. Our morphological (confocal and electron microscopical) studies have shown that in resident cells caveolin was present in the cytoplasm, in smaller vesicles and multivesicular bodies around the Golgi area. Only a very small amount of caveolae was found on the surface of these cells. In elicited macrophages, caveolae (labelled with the anti-VIP21/caveolin-1 antibody) appeared in large numbers on the cell surface, but caveolin detected by anti-caveolin-2 was also found in small vesicles and multivesicular bodies in the cytoplasm. According to these results, the absence of caveolae in resident cells can be explained by the absence of caveolin-1. The expression of the approximately 29-kDa (caveolin-related) protein in resident macrophages seems to be insufficient for caveolae formation. Elicitation significantly increased the expression of caveolin-1, and the increased amount of caveolin-1 resulted in caveolae formation on the cell surface.

[Dual fluorescence in situ hybridization analysis of synovial sarcoma]. / Synovalis sarcoma kettös jelölésü fluorescens in situ hibridizációs vizsgálata.

Synovial sarcoma have (about in 95% of the cases) the specific and characteristic reciprocal chromosomal translocation t(X; 18) (p11.2; q 11.2). Application of dual-colour fluorescence in situ hybridization (FISH) on interphase nuclei to identify the specific translocation has a diagnostic importance for daily pathological practice. For visualisation of the translocated chromosomal fragments of synovial sarcoma cells on imprint smears, chromosome X painting probes and chromosome 18 centromeric probes were used. Our present study indicates that the precise preoperative diagnosis of synovial sarcoma using dual-colour FISH is possible on smears and this possibility (to identify specific chromosomal translocations in soft tissue tumours) is a landmark in the preoperative diagnosis of soft tissue sarcomas.

Activation of deoxycytidine kinase during inhibition of DNA synthesis by 2-chloro-2'-deoxyadenosine (Cladribine) in human lymphocytes.

Deoxycytidine kinase (dCK, EC.2.7.1.74), a key enzyme in intracellular metabolism of many antileukemic drugs, was shown to be activated during treatment of lymphocytes by 2-chloro-2'-deoxyadenosine (Cl-dAdo, cladribine), a potent inhibitor of DNA synthesis. While 5-[3H]-thymidine (TdR) incorporation into DNA was decreased by 80-90%, dCK activity was doubled as a consequence of incubating the cells with 1 microM 2-chloro-2'-deoxyadenosine. Thymidine kinase (dTK, EC.2.7.1.21) activity was slightly decreased under the same conditions, similarly to 5-[3H]-thymidine incorporation. dCK activation could not be prevented by cycloheximide, and neither the amount of dCK protein nor its mRNA level was increased after 2-chloro-2'-deoxyadenosine treatment. These results suggest a post-translational activation of dCK protein during inhibition of DNA synthesis.

The pattern of melatonin in amenorrheic women affected by sterility.

The authors analyzed the pattern of melatonin in the menstrual cycles of six amenorrheic women affected by sterility. Only in one ovulatory cycle did melatonin respect its typical secretory behavior, i.e., nadir in the preovulatory period followed by an increase in the luteal phase, even though melatonin values were higher than normal over the entire cycle. The melatonin pattern in the five cycles showing no evidence of follicular recruitment nor regular formation of a main follicle was different from that seen in an ovulatory cycle. The authors support the concept of a correlation between the pineal gland and pituitary gland and hypothalamus, also because previous studies have demonstrated the presence of melatonin in follicular and peritoneal fluid. They conclude that there is a role for melatonin in the regulation of the menstrual cycle even though they have not as yet uncovered the specific mechanism.

The activity of Na+/K(+)-ATPase and abundance of its mRNA are regulated in rat myometrium during pregnancy.

The Na+/K(+)-ATPase activity and expression of mRNAs encoding alpha and beta subunits of the enzyme were examined in rat myometrium at different stages of pregnancy. The enzyme activity appeared to increase during the pregnancy and reached the highest value at the 17th day. Northern blot analysis of total RNA isolated from the same myometrial samples shows the expressions of alpha 1, alpha 3 and beta mRNAs. A2 mRNA was not detectable. By the progression of pregnancy until the 17th day the expression of all the three kinds of mRNA increased. The change in the abundance of mRNA-beta was much more higher (12-fold) than that of mRNA-alpha 1 and -alpha 3 (4 and 2.5-fold, respectively). Furthermore, the expression of mRNA-beta sharply decreased after the 17th day, while the level of alpha subunits mRNAs barely changed. We conclude that transcriptional regulation of the Na+/K(+)-ATPase subunits could be different during this physiological process.

The effect of micromolar Ca2+ on the activities of the different Na+/K(+)-ATPase isozymes in the rat myometrium.

In the present work we show the existence of two Na+/K(+)-ATPase isozymes in rat myometrial microsomes and suggest that they have different Ca2+ sensitivities. The catalytic subunits (alpha 1, alpha 2) of Na+/K(+)-ATPase were labelled by fluorescein-isothiocyanate and separated by SDS gel electrophoresis. The two isozyme Ca2(+)-sensitivities were studied by comparing the kinetics of Ca2+, strophantidin, ouabain and N-ethylmaleimide inhibitions. Our results indicate that the activity of the high ouabain-sensitive part (alpha 2 type) of Na+/K(+)-ATPase enzyme could only be inhibited by micromolar Ca2+. Furthermore, treatment of the microsomal preparation with 1mM N-ethylmaleimide selectively inactivated the high Ca2+ sensitive isoform of myometrial Na+/K(+)-ATPase.

What are the implications of melatonin in the peritoneal fluid?

The Authors evidence the presence of melatonin (MT) in the peritoneal fluid (PF). They researched possible correlations between MT and other protein and steroid hormones in the PF and in the plasma. The only significant correlation found was a "negative" one between plasma MT and plasma E2. The Authors exclude an ovarian source for peritoneal fluid MT. They believe that MT may have a role in menstrual cycle regulation, directly modulating ovarian steroid production. To determine if MT has indeed such a role, they feel it important to study the possible presence of ovarian MT receptors.

Integrated neuropsychopathological approach in patients affected by amenorrhea.

A compared study of neurovegetative modifications (following stress stimula) and of psychopathological aspects in patients affected by amenorrhea was carried out. Such an integrated approach is useful for a better definition of clinical characteristic and for a selection of a suitable therapeutic approach.