The protease-activated receptor 4 Ala120Thr variant alters platelet responsiveness to low-dose thrombin and protease-activated receptor 4 desensitization, and is blocked by non-competitive P2Y12 inhibition.

Essentials The rs773902 SNP results in differences in platelet protease-activated receptor (PAR4) function. The functional consequences of rs773902 were analyzed in human platelets and stroke patients. rs773902 affects thrombin-induced platelet function, PAR4 desensitization, stroke association. Enhanced PAR4 Thr120 effects on platelet function are blocked by ticagrelor. SUMMARY: Background F2RL3 encodes protease-activated receptor (PAR) 4 and harbors an A/G single-nucleotide polymorphism (SNP) (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP) AYPGKF. Objectives To determine the functional effects of rs773902 on stimulation by a physiological agonist, thrombin, and on antiplatelet antagonist activity. Methods Healthy human donors were screened and genotyped for rs773902. Platelet function in response to thrombin was assessed without and with antiplatelet antagonists. The association of rs773902 alleles with stroke was assessed in the Stroke Genetics Network study. Results As compared with rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation in response to subnanomolar concentrations of thrombin, increased granule secretion, and decreased sensitivity to PAR4 desensitization. In the presence of PAR1 blockade, this genotype effect was abolished by higher concentrations of or longer exposure to thrombin. We were unable to detect a genotype effect on thrombin-induced PAR4 cleavage, dimerization, and lipid raft localization; however, rs773902 AA platelets required a three-fold higher level of PAR4-AP for receptor desensitization. Ticagrelor, but not vorapaxar, abolished the PAR4 variant effect on thrombin-induced platelet aggregation. A significant association of modest effect was detected between the rs773902 A allele and stroke. Conclusion The F2RL3 rs773902 SNP alters platelet reactivity to thrombin; the allelic effect requires P2Y12 , and is not affected by gender. Ticagrelor blocks the enhanced reactivity of rs773902 A platelets. PAR4 encoded by the rs773902 A allele is relatively resistant to desensitization and may contribute to stroke risk.

NADPH-diaphorase-positive neurons in the human inferior colliculus: morphology, distribution and clinical implications.

Using the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reaction with nitroblue tetrazolium, we provided a detailed investigation of the distribution, dimensional characteristics and morphology of NADPH-d-positive neurons in the three main subdivisions of the human inferior colliculus (IC): central nucleus, pericentral nucleus, and external nucleus. In accordance with their perikaryal diameter, dendritic and axonal morphology, these neurons were categorized as large (averaging up to 45 µm in diameter), medium (20-30 µm), small (13-16 µm) and very small (7-10 µm). Their morphological differences could contribute to varying functionality and processing capacity. Our results support the hypothesis that large and medium NADPH-d-positive cells represent projection neurons, while the small cells correspond to interneurons. Heretofore, the very small NADPH-d-positive neurons have not been described in any species. Their functions-and if they are, indeed, the smallest neurons in the IC of humans-remain to be clarified. Owing to their location, we posit that they are interneurons that connect the large NADPH-d-positive neurons and thereby serve as an anatomical substrate for information exchange and processing before feeding forward to higher brain centers. Our results also suggest that the broad distribution of nitric oxide (NO) synthesis in the human IC is closely tied to the neuromodulatory action of NO on collicular neurotransmitters such as GABA and glutamate, and to calcium-binding proteins such as parvalbumin. A deeper understanding of the relationship between NADPH-d-positive fibers in all IC connections and their co-localization with other neurotransmitters and calcium-binding proteins will assist in better defining the function of NO in the context of its interplay with the cerebral cortex, the sequelae of the aging process and neurodegenerative disorders.

Neuropeptide Y immunoreactivity in the cat claustrum: A light- and electron-microscopic investigation.

The claustrum is a telencephalic nucleus located ventrolateral to the basal ganglia in the mammalian brain. It has an extensive reciprocal connectivity with most if not all of the cerebral cortex, in particular, primary sensory areas. However, despite renewed and growing interest amongst investigators, there remains a paucity of data concerning its peptidergic profile. The aim of the present study was to examine the presence, morphology, distribution and ultrastructure of neuropeptide Y-immunoreactive (NPY-ir) neurons and fibers in the claustrum of the cat. Ten adult healthy cats from both sexes were used. All animals received human and ethical treatment in accordance with the Principles of Laboratory Animal Care. Subjects were irreversibly anesthetized and transcardially perfused with fixative solution containing glutaraldehyde and paraformaldehyde. Brains were promptly removed, postfixed and sectioned. Slices were incubated with polyclonal anti-NPY antibodies according to the standard avidin-biotin-peroxidase complex method adopted by our Department of Anatomy, Histology and Embryology. NPY-ir neurons and fibers were found to be diffusely distributed throughout the claustrum, with no obvious topographic or functional patterning other than larger numbers in its central/broadest part (stereotaxic planes A12-A16). Neurons were generally classified by diameter into three sizes: small (under 17 µm), medium (17-25 µm) and large (over 25 µm). Staining density is varied with some neurons appearing darker than others. At the electron-microscopic level NPY immunoproduct was observed within neurons, dendrites and terminal boutons, each differing relative to their ultrastructural attributes. Two types of NPY-ir synaptic boutons were found. Lastly, it is of interest to note that gender-specific differences were not observed.

Parvalbumin-immunoreactive neurons in the human claustrum.

The morphology and distribution of parvalbumin-immunoreactive neurons (PV-ir) were studied in the human claustrum. PV-ir neurons were observed throughout the claustrum, with the highest numbers noted in the central (broadest) portion as compared with the dorsal and ventral aspects. Reaction product was evident in the neuronal perikarya, dendritic processes, and spines. In the majority of these labeled neurons, the cytoplasm was devoid of lipofuscin pigment. Cell bodies varied widely in both shape and size, ranging from oval and small, to multipolar and large. PV-ir neurons were classified into two groups, primarily based on dendritic morphology: spiny neurons with long and straight dendrites, and aspiny neurons with thin and curving dendritic processes. PV-ir fibers were seen throughout the neuropil, with many immuno-positive puncta noted.

MicroRNAs in platelet production and activation.

Recent work by the Encyclopedia of DNA Elements project showed that non-protein-coding RNAs account for an unexpectedly large proportion of the human genome. Among these non-coding RNAs are microRNAs (miRNAs), which are small RNA molecules that modulate protein expression by degrading mRNA or repressing mRNA translation. MiRNAs have been shown to play important roles in hematopoiesis including embryonic stem cell differentiation, erythropoiesis, granulocytopoiesis/monocytopoiesis, lymphopoiesis, and megakaryocytopoiesis. Additionally, disordered miRNA biogenesis and quantitative or qualitative alterations in miRNAs and their targets are associated with hematological pathologies. Platelets contain machinery to process pre-miRNAs into mature miRNAs, and specific platelet miRNA levels have been found to correlate with platelet reactivity. This review summarizes the current state of knowledge of miRNAs in megakaryocytes and platelets, and the exciting possibilities for future megakaryocyte-platelet transcriptome research.

Rotator cuff tears: what have we learned from animal models?

Rotator cuff tendon tears are among the most common soft tissue injuries that occur at the shoulder. Despite advancements in surgical repair techniques, rotator cuff repairs experience a high rate of failure. The common occurrence of tears and the frequency of re-tears require a further understanding of the mechanisms associated with injuries, healing, and regeneration of the rotator cuff. This paper reviews in vivo studies using the various animal shoulder models of the rat, rabbit, sheep, canine, and primate. These animal models have been used to study intrinsic and extrinsic factors leading to shoulder degeneration, various suture techniques, effects of post-surgical treatment, numerous biologic and synthetic scaffolds, and an assortment of biologic augmentations used to accelerate healing. These effects can be examined in a controlled manner using animal models without other confounding factors that sometimes limit clinical studies. The clinically impactful results will be explained to highlight the specific knowledge gained from using animal models in rotator cuff research.

The claustrum: a historical review of its anatomy, physiology, cytochemistry and functional significance.

The claustrum (Cl) is a subcortical structure located in the basolateral telencephalon of the mammalian brain. It has been a subject of inquiry since the mid-nineteenth century. The Cl can be identified in a number of species, and appears as a phylogenetically related nucleus in Insectivores, Prosimians and Marsupials. Ontogenetic investigations have been the subject of much debate over the years. There are three hypotheses for claustral development. To date, the "hybrid theory" has garnered the most support. Pathological conditions specifically associated with the Cl, while few in number, are of interest from a functional perspective. Several cases of claustral agenesis have been reported. The implications of these clinical reports are discussed. Claustral neuroanatomy at the light-microscopic and electron-microscopic level is reviewed. The morphology of the claustral neuron consists of several types, which roughly corresponds to the neuron's location within distinct claustral subdivisions. The interconnectivity of the Cl with the cerebral cortex is rather complex and reflective of complex functional interrelationships. Several researchers have investigated the angioarchitecture of the Cl. It appears that vessels permeating the insula also vascularize the Cl. Literature investigating the neurotransmitters and overall chemical neuroanatomy of the Cl is extensive. These studies clearly demonstrate that the Cl is richly innervated with a wide and diverse array of neurotransmitters and neuromodulators. Lesion, stimulation and recording experiments demonstrate that the functional and physiologic capacity of the Cl is quite robust. A recurring theme of claustral function appears to be its involvement in sensorimotor integration. This may be expected of the Cl, given the degree ofheterotopic, heterosensory convergence and its interconnectivity with the key subcortical nuclei and sensory cortical areas. The Cl remains a poorly understood and under investigated nucleus. Therefore, a review of the world literature through 1986 prior to the advent of the "molecular revolution" is presented. This diverse and extensive body of knowledge is reviewed in the areas ofphylogeny, ontogeny, pathology, angioarchitecture, cytochemistry, anatomy and physiology. Theories of possible claustral function are also noted. It is hoped that this work will stimulate research scientists to further investigate the functional interrelationships of the Cl as well as to aim with far greater precision and accuracy towards a deeper understanding of its raison d'etre. The recent efforts in neurosciences by Sir Francis Crick and Christof Koch implicating the Cl in visual consciousness, is an important step in understanding just what its functions could encompass. Efforts in molecular neurosciences will be indispensable for a mechanistic understanding of these functions. Currently research efforts are underway from many perspectives. In considering the past scientific literature on the Cl, it is interesting to regard that this once obscure brain structure, may serve as a model system for the study of one of the most interesting and complex brain functions-consciousness.

Robot-aided neurorehabilitation: from evidence-based to science-based rehabilitation.

There is no "magic bullet" in rehabilitation. In the absence of direct neural transplants, neurological rehabilitation is an arduous process. We have pioneered the clinical application of robotics in stroke rehabilitation and have shown evidence of the positive impact of targeted exercise on stroke recovery. In this article, we will review results obtained in the initial clinical trials with 96 stroke patients at the Burke Rehabilitation Hospital. We will provide evidence that robot-aided training enhances recovery, that this enhanced recovery is sustained in the long term, and that this recovery is not due to a general physiological improvement--in fact, it appears to be limb and muscle group specific. An evidence-based approach must now segue into a more scientific approach to stroke rehabilitation. Given the length of the required protocols and patients' variability and limited census, the practical limitations of the evidence-based approach are self-evident and extend trials for years. Each patient and lesion is unique in stroke rehabilitation, so there is no reason to believe that a "one-size-fits-all" optimal treatment exists. To optimize therapy for individual patients, we need science-based models. In this article, we will summarize the scientific tools and models that we are investigating and present some of the results to date.

What criteria reliably distinguish melanoma from benign melanocytic lesions?

The differential diagnosis of melanocytic lesions is fraught with difficulty and a common source of litigation either if a lesion misreported as 'benign' recurs locally or re-presents with nodal metastases or if an atypical naevus is called 'malignant' leading to a cosmetically unsatisfactory wider resection, unwarranted anxiety about prognosis and adverse life insurance prospects. Several authors have claimed that there are valid morphological criteria which, alone or in combination, enable reliable distinction between benign and malignant melanocytic lesions. Others question these criteria and, doubting the extent to which unequivocal diagnoses can be rendered in all cases, believe that the diagnosis is purely subjective and that most diagnostic errors are non-negligent. To address these issues, expert opinions were commissioned from three sets of authors. Okun, Edelstein & Kasznica emphasize that a significant minority of melanocytic lesions are so borderline morphologically that diagnostic uncertainty is allowable and that such uncertainty can be handled responsibly. Kirkham, in favouring the methodical use of criteria, concedes that they are 'largely opinion-based rather than evidence-based, but do go beyond mere subjective pattern analysis'. In agreement with Okun and his colleagues. Slater emphasises that no single feature is reliable by itself and that all aspects, including clinical details, should be interpreted together; he has no hesitation in reporting the diagnosis as 'uncertain' in doubtful cases. In the absence of a specific marker pathognomonic of melanocytic malignancy, the diagnosis will continue to rely on the judicious application of morphological criteria with a small proportion of elusive cases in which diagnostic uncertainty should not be concealed.

The Rel/NF-kappaB family directly activates expression of the apoptosis inhibitor Bcl-x(L).

The transcription factors of the Rel/NF-kappaB family are key regulators of immune and inflammatory responses and contribute to lymphocyte proliferation, survival, and oncogenesis. The absolute correlation between the antiapoptotic and oncogenic activities of the Rel/NF-kappaB oncoprotein v-Rel emphasizes the importance of characterizing the death antagonists under NF-kappaB control. Our recent finding that the prosurvival Bcl-2 homolog Bfl-1 (also called A1) is a direct transcriptional target of NF-kappaB raised the issue of whether NF-kappaB is a specific or global regulator of death antagonists in the Bcl-2 family. Here, we demonstrate that NF-kappaB differentially regulates the expression of particular Bcl-2-related death inhibitors and that it directly activates the expression of Bcl-x(L). While Bcl-x(L) was significantly upregulated by c-Rel and RelA, Bcl-2 was not. Importantly, stimuli that activate endogenous NF-kappaB factors also upregulated bcl-x gene expression and this effect was antagonized by an inhibitor of NF-kappaB activity. The expression of bcl-x suppressed apoptosis in the presence or absence of NF-kappaB activity. Functional analysis of the bcl-x promoter demonstrated that it is directly controlled by c-Rel. These results establish that NF-kappaB directly regulates the expression of distinct prosurvival factors in the Bcl-2 family, such as Bcl-x(L) and Bfl-1/A1. These findings raise the possibility that some of these factors may contribute to oncogenesis associated with aberrant Rel/NF-kappaB activity.

Pili bigeminy induced by low fluence therapy with hair removal alexandrite and ruby lasers.

BACKGROUND: Lasers are being widely used for the hair removal. Several complications including hyperpigmentation, erythma, hypopigmentation, and burns have been reported. OBJECTIVE: To study laser hair removal complications. METHODS: Pubic hairs were treated with alexandrite and ruby lasers. RESULTS: Pili Bigeminy can be induced by low fluence therapy with hair removal Alexandrite and Ruby lasers as a complication. CONCLUSION: To our knowledge, this is the first report of pili bigminy as a complication of laser-assisted hair removal.

The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-kappaB that blocks TNFalpha-induced apoptosis.

Bcl-2-family proteins are key regulators of the apoptotic response. Here, we demonstrate that the pro-survival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-kappaB. We show that bfl-1 gene expression is dependent on NF-kappaB activity and that it can substitute for NF-kappaB to suppress TNFalpha-induced apoptosis. bfl-1 promoter analysis identified an NF-kappaB site responsible for its Rel/NF-kappaB-dependent induction. The expression of bfl-1 in immune tissues supports the protective role of NF-kappaB in the immune system. The activation of Bfl-1 may be the means by which NF-kappaB functions in oncogenesis and promotes cell resistance to anti-cancer therapy.

Overview of clinical trials with MIT-MANUS: a robot-aided neuro-rehabilitation facility.

We are applying robotics and information technology to assist, enhance, and quantify neuro-rehabilitation. Our goal is a new class of interactive, user-affectionate clinical devices designed not only for evaluating patients, but also-for delivering meaningful therapy via engaging "video games". Notably, the novel robot MIT-MANUS has been designed and programmed for clinical neurological applications, and has undergone extensive clinical trials for more than four years at Burke Rehabilitation Hospital - White Plains, NY. This paper will review results of the first clinical trial of 20 patients, which showed that: - Stroke patients treated daily with additional robot-aided therapy during acute rehabilitation had improved outcome in motor activity at hospital discharge, when compared to a control group that received only standard acute rehabilitation treatment. - This improved outcome was sustained after three years. - The neuro-recovery process continued far beyond the commonly accepted 3 months post-stroke interval.

A double-blind placebo-controlled study of 3,4-diaminopyridine in amytrophic lateral sclerosis patients on a rehabilitation unit.

3,4-Diaminopyridine (DAP) enhances acetylcholine release from the nerve terminal and improves conduction in demyelinated axons. In this double-blinded placebo controlled cross over study we examined the effects of DAP combined with inpatient rehabilitation in nine patients with disabling motor weakness due to amyotrophic lateral sclerosis (ALS). A single dose of DAP or placebo was increased daily to the maximum (range: 10-80 mg) tolerated dose; after patients were assessed on the first treatment, the alternate drug was given in the same manner. Functional Independence Measurement (FIM), Ashworth, grip strength, limb strength measurements, nerve conduction studies and speech assessments were initiated 1/2 h after receiving the maximum tolerated dose of DAP or placebo. DAP was tolerated in all patients, but limited by gastrointestinal side effects in four patients. The mean peak serum level was 20.11 (S.D. = 5.11) ng/ml, occurring 1.25 (S.D. = 0.56) h after dose. A statistically significant improvement in FIM and speech assessment scores between admission and discharge occurred. However, no significant differences in clinical or electrophysiologic measures were seen between DAP and placebo treatments. This study suggests that intensive inpatient rehabilitation has a role in the management of patients with ALS, but DAP does not diminish motor impairment.

The cell and the organism: the role of subdivisional cell replication in the development and maintenance of the multicellular organism.

Evidence of new cell formation from nucleoli (subdivisional cell replication) was observed in vitro and in vivo. Mouse melanoma cells, human fibroblasts, and rat mast cells were observed in tissue culture with phase contrast time-lapse cinematography. Evidence of subdivisional cell replication seen in tissue culture was supported by observations of mast cells, cervical epithelial cells, melanoma cells, keratinocytes, and fungal spores in vivo. Indirect evidence for subdivisional cell replication was the presence of differentiated form and function in nuclei and nucleoli. Synergism between subdivisional replication and mitotic replication (subdivisional expansion) is believed to be a key to morphogenesis, whereby cellular and subdivisional zones act as biologic "molds". It is believed that subdivisional cell replication has a key role in maintenance of differentiated form in multicellular organisms, as well as in morphogenesis.

3,4-diaminopyridine as a treatment for amyotrophic lateral sclerosis.

The slow potassium channel blocker 3,4-diaminopyridine (DAP) enhances acetylcholine release from the nerve terminal and improves conduction in unmyelinated nerve. In this open label pilot study, we examined the effect of DAP combined with inpatient rehabilitation in seven patients with motor weakness due to amyotrophic lateral sclerosis (ALS). A single daily 20 mg oral dose of DAP was gradually increased to the maximum tolerated dose, and serum DAP concentrations were measured. Videotaped motor examination (for subsequent "blinded" review and assignment of a quantitative motor score), Functional Independence Measure (FIM) assessment, nerve conduction studies and neuropsychological evaluations were performed on admission, 1 h after maximum DAP dose, and post-treatment. DAP was tolerated in all patients, though dose was limited by gastrointestinal side effects in five patients. The mean peak serum level was 128 (+/- 50) ng/ml, occurring 1.0 (+/- 0.50) h after dose. A modest but statistically significant (p = 0.045) peak in motor score occurred on DAP. A significant (p = 0.045) improvement from baseline in FIM performance was apparent with DAP. Nerve conduction studies showed small increases in evoked response amplitudes and conduction velocities on DAP, but they did not reach statistical significance. No cognitive or affective changes were apparent. This unblinded pilot study shows that DAP is tolerated in ALS patients, and may be associated with functional and electrophysiologic improvement.

Plexiform neurofibromatosis of the liver and mesentery in a child.

Plexiform neurofibromatosis of the liver was recognized by needle biopsy of the liver in an 11-yr-old boy who had a 2-yr history of diarrhea, intermittent abdominal pain, failure to gain weight and progressive abdominal distention. Imaging studies demonstrated a large retroperitoneal mass; a laparotomy was performed. At surgery, the mesentery was greatly thickened by neurofibromas, and plexiform neurofibroma extended through the hilum of the liver. Light and electron microscopy demonstrated that in addition to the direct involvement by tumor, neural hyperplasia existed throughout the liver. The most distal ramifications of the portal spaces were filled with Schwann cells, bundles of unmyelinated nerves and perineurium-surrounded nerves containing myelinated and unmyelinated fibers. The ultrastructural findings were consistent with stimulation of proliferation of all the portal neural elements and tumoral tissue. The nontumoral response was more than simple hyperplasia because it appeared to result in fibrotic changes in the most involved areas and active breaching of the limiting plate with destruction of hepatocytes and collagen deposition throughout the liver.

When a child dies.

The death of a child has a significant and far-reaching impact on all remaining members of the family. Each individual undergoes the process of bereavement, adjusting to this new and unwanted reality. The stages of this process include disorganization, the initial impact; the struggle of holding on/letting go, the lengthy period of keeping memories and certain aspects of the dead child, while relinquishing the child as a living member of the family; and reorganization, the integration of the loss into an ongoing life structure. Reactions to a loss of this magnitude often include a variety of normal and pathologic symptoms, some of which indicate the need for professional therapeutic intervention. In addition to professional help, there are numerous needs that can also be met by other family members, clergy, and support groups that facilitate the bereavement process. Importantly for the professionals in the service fields, the intense experience of these family members gives rise to problems and dilemmas in the helper, complicating work with bereaved individuals.