Myoferlin depletion elevates focal adhesion kinase and paxillin phosphorylation and enhances cell-matrix adhesion in breast cancer cells.

Breast cancer is the second leading cause of malignant death among women. A crucial feature of metastatic cancers is their propensity to lose adhesion to the underlying basement membrane as they transition to a motile phenotype and invade surrounding tissue. Attachment to the extracellular matrix is mediated by a complex of adhesion proteins, including integrins, signaling molecules, actin and actin-binding proteins, and scaffolding proteins. Focal adhesion kinase (FAK) is pivotal for the organization of focal contacts and maturation into focal adhesions, and disruption of this process is a hallmark of early cancer invasive potential. Our recent work has revealed that myoferlin (MYOF) mediates breast tumor cell motility and invasive phenotype. In this study we demonstrate that noninvasive breast cancer cell lines exhibit increased cell-substrate adhesion and that silencing of MYOF using RNAi in the highly invasive human breast cancer cell line MDA-MB-231 also enhances cell-substrate adhesion. In addition, we detected elevated tyrosine phosphorylation of FAK (FAK(Y397)) and paxillin (PAX(Y118)), markers of focal adhesion protein activation. Morphometric analysis of PAX expression revealed that RNAi-mediated depletion of MYOF resulted in larger, more elongated focal adhesions, in contrast to cells transduced with a control virus (MDA-231(LVC) cells), which exhibited smaller focal contacts. Finally, MYOF silencing in MDA-MB-231 cells exhibited a more elaborate ventral cytoskeletal structure near focal adhesions, typified by pronounced actin stress fibers. These data support the hypothesis that MYOF regulates cell adhesions and cell-substrate adhesion strength and may account for the high degree of motility in invasive breast cancer cells.

Placental proteomics: a shortcut to biological insight.

Proteomics analysis of biological samples has the potential to identify novel protein expression patterns and/or changes in protein expression patterns in different developmental or disease states. An important component of successful proteomics research, at least in its present form, is to reduce the complexity of the sample if it is derived from cells or tissues. One method to simplify complex tissues is to focus on a specific, highly purified sub-proteome. Using this approach we have developed methods to prepare highly enriched fractions of the apical plasma membrane of the syncytiotrophoblast. Through proteomics analysis of this fraction we have identified over five hundred proteins several of which were previously not known to reside in the syncytiotrophoblast. Herein, we focus on two of these, dysferlin and myoferlin. These proteins, largely known from studies of skeletal muscle, may not have been found in the human placenta were it not for discovery-based proteomics analysis. This new knowledge, acquired through a discovery-driven approach, can now be applied for the generation of hypothesis-based experimentation. Thus discovery-based and hypothesis-based research are complimentary approaches that when coupled together can hasten scientific discoveries.

Proteomics of the human placenta: promises and realities.

Proteomics is an area of study that sets as its ultimate goal the global analysis of all of the proteins expressed in a biological system of interest. However, technical limitations currently hamper proteome-wide analyses of complex systems. In a more practical sense, a desired outcome of proteomics research is the translation of large protein data sets into formats that provide meaningful information regarding clinical conditions (e.g., biomarkers to serve as diagnostic and/or prognostic indicators of disease). Herein, we discuss placental proteomics by describing existing studies, pointing out their strengths and weaknesses. In so doing, we strive to inform investigators interested in this area of research about the current gap between hyperbolic promises and realities. Additionally, we discuss the utility of proteomics in discovery-based research, particularly as regards the capacity to unearth novel insights into placental biology. Importantly, when considering under studied systems such as the human placenta and diseases associated with abnormalities in placental function, proteomics can serve as a robust 'shortcut' to obtaining information unlikely to be garnered using traditional approaches.

Association of PAT proteins with lipid storage droplets in term fetal membranes.

As depots for neutral lipids, lipid storage droplets (LDs) accumulate with advancing gestation within the fetal membranes. Little is currently known about the proteins associated with the LDs of these cells. The PAT family [perilipin, adipose differentiation-related protein (ADRP), and tail-interacting protein of 47 kilodaltons (TIP47)] represents a unique group of proteins thought to contribute to LD formation and function. We examined the association of each of the PAT proteins with LDs of term fetal membranes. We found that large LDs of amnion epithelial cells were reactive for neutral lipid stains and simultaneously encoated with ADRP and TIP47, but not perilipin. Within the remaining cell types, LDs were frequently co-labeled with antibodies recognizing ADRP and TIP47; however, in cells harboring only small LDs, the majority of TIP47 labeling was cytoplasmic. Structures labeled with perilipin antibodies were present only in chorion laeve trophoblasts. Gene and protein expression analyses suggested this to be a small molecular weight perilipin isoform, such as that seen in steroidogenic cells. We conclude that LDs are heterogeneous among differing cell types of the fetal membranes. Subclassification of LDs based on associated proteins suggests that these organelles may serve specialized functions within individual cells.

In situ immunolabeling allows for detailed localization of prostaglandin synthesizing enzymes within amnion epithelium.

Detailed information regarding the subcellular distribution of proteins within amnion epithelial cells is a goal of numerous placental biologists. In this report, we describe a versatile technique for in situ immunolabeling in amnion that is as technically permissible as traditional immunolabeling of cultured cells and, when coupled with confocal laser scanning microscopy, is similarly capable of providing detailed information regarding subcellular protein distribution. Using antibodies directed against sequential enzymes of the prostaglandin E biosynthesis cascade, we compared this novel method with immunofluorescent labeling using amnion cells in primary culture and cryosections of reflected fetal membrane rolls. By several criteria, we observed morphological variation between the cells cultured in vitro and the tissue specimens. Despite general consistencies in immunostaining patterns between the cryosectioned specimens and those labeled in situ, morphological preservation was superior using the latter technique. Relative to the cryosectioned specimens, in situ immunostaining was advantageous in that it permitted improved sampling efficiency, and allowed regional variations in labeling to be observed in a more global context within the tissue. Our results demonstrate that in situ immunolabeling provides a useful adjunct or alternative to immunolabeling using membrane roll preparations.

Does the addition of tris(hydroxymethyl) aminomethane to 0.5% bupivacaine delay epidural onset in parturients?

A rapid onset of epidural anesthesia may predispose a pregnant patient to hypotension during the first stage of labor. Sodium bicarbonate has been reported to hasten epidural onset of chloroprocaine, lidocaine and bupivacaine while tris(hydroxymethyl) aminomethane (THAM) has been shown to delay the onset of epidurally administered chloroprocaine in parturients. The purpose of this study was to determine whether the addition of THAM to bupivacaine affected the onset, cephalad spread and incidence of hypotension following epidural administration in healthy pregnant patients during the first stage of labor. Three groups each consisting of 20 ASA I or II patients were studied. Group I received 0.5% bupivacaine with saline (0.06 ml/10 ml). Group II received bupivacaine buffered with THAM (0.06 ml/10 ml) to a pH of 6.8. Group III received bupivacaine buffered to a pH of 6.8 with sodium bicarbonate (0.05 ml+0.01 ml saline/10 ml). Statistical analysis used one-way analysis of variance and the appropriate post hoc test for multiple comparisons. A [Formula: see text] value less than 0.05 was considered significant. The onset of analgesia, the cephalad spread, and the incidence of hypotension did not differ between the groups. It is concluded that THAM does not affect the pharmacodynamics of 0.5% bupivacaine administered epidurally.

The laryngeal mask airway facilitates intubation at cesarean section. A case report of difficult intubation.

Failed intubation at cesarean section presents the anesthesiologist with a challenge, and may subject both mother and baby to significant morbidity and in some cases, mortality. We report a case of failed intubation at an emergency cesarean section at which intubation was subsequently achieved using a laryngeal mask airway as a guide.

Phrenic nerve injury following scalenectomy in a patient with thoracic outlet obstruction.

We present a case in which a patient with normal pulmonary reserve experienced orthopnea and hypoxia secondary to unilateral diaphragmatic paralysis following right scalenectomy. This operation was performed in an attempt to relieve neurovascular compromise at the thoracic outlet. To our knowledge, this association has not been previously described in the literature.

Butorphanol for the relief of shivering associated with extradural anesthesia in parturients.

STUDY OBJECTIVE: To assess the efficacy of butorphanol for the relief of shivering following the epidural administration of 2% lidocaine. DESIGN: Randomized, double-blind study. SETTING: Labor and delivery department of a university-affiliated hospital inpatient facility. PATIENTS: Sixty-one healthy labor patients. INTERVENTIONS: Patients who had sustained shivering associated with lidocaine epidural anesthesia were given normal saline or butorphanol 1 mg. Patients were observed for 20 minutes following the administration of a study solution. MEASUREMENTS AND MAIN RESULTS: Shivering ceased within a mean time of 12.9 +/- 3.8 minutes in approximately 81% of the patients who received epidural butorphanol (p < 0.01), while 3% of the patients in the placebo group had no shivering following the administration of epidural saline. No sedation or changes in fetal heart rate were associated with epidural butorphanol. CONCLUSIONS: Epidural butorphanol is effective in the treatment of postepidural shivering associated with epidural lidocaine. Epidural agonist opioids have been reported to be efficacious in the management of postepidural shivering. This study demonstrated that a partial agonist opioid also is effective in the treatment of postepidural shivering.

The effect of pH-adjusted 2-chloroprocaine on the onset of epidural analgesia in pregnant patients in the lying and sitting position during the first stage of labor.

The purpose of this prospective, randomized, double-blind study was to compare the epidural onset time of 2% 2-chloroprocaine with pH-adjusted 2-chloroprocaine administered in either the sitting or supine position in pregnant patients during the first stage of labor. Patients in Groups I and III received the control solution in the sitting and supine position, respectively. Patients in Groups II and IV received the buffered solution in the sitting and supine position, respectively. The pH and pCO2 of the control and buffered solutions differed significantly. The pH and pCO2 of the control and buffered solutions were 4.38 +/- 0.01, 18.4 +/- 2.2 mm Hg and 7.70 +/- 0.04, 114.9 +/- 3.0 mmHg, respectively. A statistically significant reduction in the time of onset of analgesia in the pH-adjusted groups was noted. Groups I and II had onset times of 4 +/- 1.2 and 4.3 +/- 1.0, whereas Groups II and IV had onset times of 2.6 +/- 0.9 and 2.7 +/- 0.6 min., respectively. There were no intergroup differences in the cephalad spread of analgesia or duration of analgesia. Position had no effect on the onset of analgesia at the S2-3 dermatomes nor on the bilateral cephalad spread of the epidural study solutions. Our results indicate that a pregnant patient may be dosed in the lateral supine position without adversely affecting the caudad or cephalad spread of plain or pH-adjusted 2% 2-chloroprocaine, which is clinically important because the incidence of aortocaval compression is increased in the supine position when compared with the lateral supine position.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidural morphine pruritus reduction with hydroxyzine in parturients.

A majority of patients experience pruritus, nausea and/or emesis following epidural morphine administration post-cesarean section. Naloxone or diphenhydramine are commonly used to treat these side effects. Prevention or reduction in the incidence of side effects of epidural morphine is a clinical goal. The purpose of the study was to observe the efficacy of prophylactic administration of hydroxyzine on the incidence and severity of pruritus following the epidural administration of morphine in 40 patients who requested epidural morphine for postoperative pain relief. Group I (n = 20) received saline, while Group II (n = 20) received 50 mg of hydroxyzine ten minutes after the administration of 5 mg epidural morphine. Both solutions were administered by deep intramuscular injection in the thigh area. The results of this investigation demonstrated that hydroxyzine was efficacious in attenuating the incidence of severe pruritus.

The management of oral mexiletine and intravenous lidocaine to treat chronic painful symmetrical distal diabetic neuropathy.

Intravenous local anesthestics administered to patients with chronic pain have been shown to provide significant levels of systemic analgesia. Furthermore, oral mexiletine which is similar in structure has been demonstrated to be efficacious in the treatment of diabetic neuropathy. It is recommended that this combined form of treatment be considered with those patients whose diabetic neuropathy is resistant to more conventional forms of treatment.

Sensory evoked facial muscle electromyography for the quantification of acute labor pain.

Assessment of the adequacy of epidural analgesia for acute pain management can be difficult on occasion. This investigation used non-invasive sensory evoked facial muscle electromyography (SEFE) as well as a Verbal Assessment Scores (VAS) to assess severe pain in healthy parturients during the first stage of active labor. Institutional Review Board approval and patient informed consent were obtained from 12 healthy parturients who were in active labor and who had requested epidural analgesia for labor pain. SEFE microvoltage was recorded prior to epidural placement when a patient reported severe pain and again when a patient reported no pain with a subsequent uterine contraction. VAS assessments (0 = no pain and 10 = the worst pain ever experienced) were also recorded at identical time intervals. Statistical analysis was done using the paired two tailed Student's t-test. Each patient served as their own control. A statistically significant decrease in SEFE microvoltage (p < 0.001) was noted when analgesia was established (VAS = 0) in each patient. It was concluded in this pilot study that SEFE can be effective in quantifying acute severe nociception and thus can provide a continuous objective indicator of the effectiveness of analgesic regimens in an acute obstetric pain setting. Its applicability in other acute pain areas remains to be investigated.

End tidal carbon dioxide and respiratory rate measurement during conscious sedation through a nasal cannula.

During the administration of central nervous system depressant drugs for dentistry or surgery, it may not always be possible to continuously measure respiratory physiology. We present a simple and efficacious method of monitoring respiration during dental or surgical procedures in which a conscious sedation technique is used.

Ineffective ventilation during conscious sedation due to chest wall rigidity after intravenous midazolam and fentanyl.

Chest wall rigidity has been reported after the administration of high-dose intravenous fentanyl. This case report supports the observation that low-dose intravenous fentanyl may also cause chest wall rigidity. The treatment of chest wall rigidity with naloxone or neuromuscular blocking agents is controversial. A discussion of the management of fentanyl-induced chest wall rigidity is presented.

Multidisciplinary approach for the management of post-herpetic neuralgia in elderly patients.

Post-herpetic neuralgia is associated with significant distress and morbidity. The management of acute neuritis and/or post-herpetic neuralgia can be particularly difficult. A multidisciplinary approach is required. A team consisting of the primary care physician, pain specialist, neurologist, geriatrician, pain psychologist, psychiatrist, and a physiatrist with an integrated approach will provide the best results. Early interventional therapy with sympathetic nerve blocks may significantly decrease the need for long-term opioid therapy, as well as long-term use of anticonvulsants, antidepressants, or membrane stabilizers. Early referral to a multidisciplinary pain center may furthermore decrease the behavioral trauma and family disruption associated with this painful condition.

Physiologic effects of stellate ganglion block: a result of complete ganglion blockade or the vertical spread of local anesthetic?

Traditionally, stellate ganglion blockade has been used for the diagnosis and treatment of upper extremity sympathetic pain. However, this treatment has not been shown to provide adequate sympathetic blockade of the upper extremities. This study demonstrates that a carefully performed upper thoracic sympathetic block with imaging guidance can result in a successful sympathetic blockade of the upper extremities. This study furthermore demonstrates that the occurrence of a Horners syndrome is not a testimony to a successful sympathetic block of the upper extremities.

Exocyst complex protein expression in the human placenta.

INTRODUCTION: Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. OBJECTIVE: While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. METHODS: A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. RESULTS: The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion. DISCUSSION/CONCLUSION: Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst's regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion.

IFPA Meeting 2013 Workshop Report II: use of 'omics' in understanding placental development, bioinformatics tools for gene expression analysis, planning and coordination of a placenta research network, placental imaging, evolutionary approaches to understanding pre-eclampsia.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At the IFPA meeting 2013 twelve themed workshops were presented, five of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of new technologies for placenta research: 1) use of 'omics' in understanding placental development and pathologies; 2) bioinformatics and use of omics technologies; 3) planning and coordination of a placenta research network; 4) clinical imaging and pathological outcomes; 5) placental evolution.

IFPA Meeting 2012 Workshop Report I: comparative placentation and animal models, advanced techniques in placental histopathology, human pluripotent stem cells as a model for trophoblast differentiation.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2012 there were twelve themed workshops, three of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of models and technical issues involved in placenta research: 1) comparative placentation and animal models; 2) advanced techniques in placental histopathology; 3) human pluripotent stem cells as a model for trophoblast differentiation.