One Digit Interruption: The Altered Force Patterns during Functionally Cylindrical Grasping Tasks in Patients with Trigger Digits

Most trigger digit (TD) patients complain that they have problems using their hand in daily or occupational tasks due to single or multiple digits being affected. Unfortunately, clinicians do not know much about how this disease affects the subtle force coordination among digits during manipulation. Thus, this study examined the differences in force patterns during cylindrical grasp between TD and healthy subjects. Forty-two TD patients with single digit involvement were included and sorted into four groups based on the involved digits, including thumb, index, middle and ring fingers. Twelve healthy subjects volunteered as healthy controls. Two testing tasks, holding and drinking, were performed by natural grasping with minimal forces. The relations between the force of the thumb and each finger were examined by Pearson correlation coefficients. The force amount and contribution of each digit were compared between healthy controls and each TD group by the independent t test. The results showed all TD groups demonstrated altered correlation patterns of the thumb relative to each finger. Larger forces and higher contributions of the index finger were found during holding by patients with index finger involved, and also during drinking by patients with affected thumb and with affected middle finger. Although no triggering symptom occurred during grasping, the patients showed altered force patterns which may be related to the role of the affected digit in natural grasping function. In conclusion, even if only one digit was affected, the subtle force coordination of all the digits was altered during simple tasks among the TD patients. This study provides the information for the future studies to further comprehend the possible injuries secondary to the altered finger coordination and also to adopt suitable treatment strategies.     

Regulation of anoikis by Cdc42 and Rac1

Ras family small GTPases play a critical role in malignant transformation, and Rho subfamily members contribute significantly to this process. Anchorage-independent growth and the ability to avoid detachment-induced apoptosis (anoikis) are hallmarks of transformed epithelial cells. In this study, we have demonstrated that constitutive activation of Cdc42 inhibits anoikis in Madin-Darby canine kidney (MDCK) epithelial cells. We showed that activated Cdc42 stimulates the ERK, JNK, and p38 MAPK pathways in suspension condition; however, inhibition of these signaling does not affect Cdc42-stimulated cell survival. However, we demonstrated that inhibition of phosphatidylinositol 3-kinase (PI3K) pathway abolishes the protective effect of Cdc42 on anoikis. Taking advantage of a double regulatory expression system, we also showed that Cdc42-stimulated cell survival in suspension condition is, at least in part, mediated by Rac1. We also provide evidence for a positive feedback loop involving Rac1 and PI3K. In addition, we show that the survival functions of both constitutively active Cdc42 and Rac1 GTPases are abrogated by Latrunculin B, an actin filament-depolymerizing agent, implying an important role for the actin cytoskeleton in mediating survival signaling activated by Cdc42 and Rac1. Together, our results indicate a role for Cdc42 in anchorage-independent survival of epithelial cells. We also propose that this survival function depends on a positive feedback loop involving Rac1 and PI3K.     

Prognostic value of X-chromosome inactivation in symptomatic female carriers of dystrophinopathy

Background

Neonatal screening for Pompe disease has been introduced in Taiwan and a few U.S. states, while other jurisdictions including some European countries are piloting or considering this screening. First-tier screening flags both classic infantile and late-onset Pompe disease, which challenges current screening criteria. Previously, advocacy groups have sometimes supported expanded neonatal screening more than professional experts, while neutral citizens' views were unknown. This study aimed to measure support for neonatal screening for Pompe disease in the general public and to compare it to support among (parents of) patients with this condition. The study was done in the Netherlands, where newborns are not currently screened for Pompe disease. Newborn screening is not mandatory in the Netherlands but current uptake is almost universal.

Methods

A consumer panel (neutral group) and (parents of) patients with Pompe disease (Pompe group) were sent information and a questionnaire. Responses were analyzed of 555 neutral and 58 Pompe-experienced informants who had demonstrated sufficient understanding.

Results

87% of the neutral group and 88% of the Pompe group supported the introduction of screening (95% CI of difference -10 to 7%). The groups were similar in their moral reasoning about screening and acceptance of false positives, but the Pompe-experienced group expected greater benefit from neonatal detection of late-onset disease. Multivariate regression analysis controlling for demographics confirmed that approval of the introduction of screening was independent of having (a child with) Pompe disease. Furthermore, respondents with university education, regardless of whether they have (a child with) Pompe disease, were more likely to be reluctant about the introduction of screening than those with less education, OR for approval 0.29 (95% CI 0.18 to 0.49, p < 0.001).

Conclusions

This survey suggests a rather high level of support for newborn screening for Pompe disease, not only among those who have personal experience of the disease but also among the general public in the Netherlands. Optional screening on the basis of informed parental consent is probably unrealistic, underlining the need for new guidelines to help policymakers in their consideration of newborn screening for broad phenotype conditions.     

Cdc42-dependent modulation of tight junctions and membrane protein traffic in polarized Madin-Darby canine kidney cells

Polarized epithelial cells maintain the asymmetric composition of their apical and basolateral membrane domains by at least two different processes. These include the regulated trafficking of macromolecules from the biosynthetic and endocytic pathway to the appropriate membrane domain and the ability of the tight junction to prevent free mixing of membrane domain-specific proteins and lipids. Cdc42, a Rho family GTPase, is known to govern cellular polarity and membrane traffic in several cell types. We examined whether this protein regulated tight junction function in Madin-Darby canine kidney cells and pathways that direct proteins to the apical and basolateral surface of these cells. We used Madin-Darby canine kidney cells that expressed dominant-active or dominant-negative mutants of Cdc42 under the control of a tetracycline-repressible system. Here we report that expression of dominant-active Cdc42V12 or dominant-negative Cdc42N17 altered tight junction function. Expression of Cdc42V12 slowed endocytic and biosynthetic traffic, and expression of Cdc42N17 slowed apical endocytosis and basolateral to apical transcytosis but stimulated biosynthetic traffic. These results indicate that Cdc42 may modulate multiple cellular pathways required for the maintenance of epithelial cell polarity.     

Oxidative stress induces lipid-raft-mediated activation of Src homology 2 domain-containing protein-tyrosine phosphatase 2 in astrocytes

Several protein phosphatases are involved in neuroprotection in response to ischemic brain injury. Here, we report that reactive oxygen species (ROS)-mediated oxidative stress promotes phosphorylation of endogenous SHP-2 through lipid rafts in rat primary astrocytes. SHP-2 was transiently phosphorylated during hypoxia/reoxygenation, an effect abrogated by a ROS scavenger and an NADPH oxidase inhibitor. Additionally, exogenous treatment with hydrogen peroxide (H₂O₂) triggered SHP-2 phosphorylation in a time- and dose-dependent manner and led to its translocation into lipid rafts. H₂O₂-mediated SHP-2 phosphorylation and translocation were inhibited by filipin III and methyl-β-cyclodextrin (MCD), lipid-raft-disrupting agents. In the presence of H₂O₂, SHP-2 formed a complex with STAT-3 and reduced the steady-state STAT-3 phosphorylation level. Interestingly, the effect of H₂O₂ on SHP-2 phosphorylation was cell-type specific. Remarkably, SHP-2 phosphorylation was induced strongly by H₂O₂ in astrocytes, but barely detectable in microglia. Our results collectively indicate that SHP-2 is activated by ROS-mediated oxidative stress in astrocytes and functions as a component of the raft-mediated signaling pathway that acts through dephosphorylation and inactivation of other phosphotyrosine proteins, such as STAT-3.     

Primary structure of thymosin beta 12, a new member of the beta-thymosin family isolated from perch liver.

A new polypeptide termed thymosin beta 12 has been isolated from perch liver and its primary structure elucidated. This polypeptide contains 43 amino acid residues with a molecular weight of 4822 Da. The content of thymosin beta 12 from perch liver has been determined as 43 micrograms/g of tissue. The amino-terminal end of this polypeptide is blocked by an acetyl group as deciphered by fast-atom bombardment mass spectrometric analysis. Sequence analysis reveals that thymosin beta 12 is 79% homologous to thymosin beta 4, an immunomodulator which was originally isolated from calf thymus. Thymosin beta 12 also shows 84% sequence homology to thymosin beta 11, a beta 4 analog which replaces beta 4 in two species of bony fish, oscar and rainbow trout. The evolutionary implication of such results will be discussed. The isolation of a new beta 4-related peptide from perch liver which differs from beta 11 indicates that beta-thymosin peptides are widely distributed in lower vertebrate classes.     

Astrogliosis Is a Possible Player in Preventing Delayed Neuronal Death

Mitigating secondary delayed neuronal injury has been a therapeutic strategy for minimizing neurological symptoms after several types of brain injury. Interestingly, secondary neuronal loss appeared to be closely related to functional loss and/or death of astrocytes. In the brain damage induced by agonists of two glutamate receptors, N-ethyl-D-aspartic acid (NMDA) and kainic acid (KA), NMDA induced neuronal death within 3 h, but did not increase further thereafter. However, in the KA-injected brain, neuronal death was not obviously detectable even at injection sites at 3 h, but extensively increased to encompass the entire hemisphere at 7 days. Brain inflammation, a possible cause of secondary neuronal damage, showed little differences between the two models. Importantly, however, astrocyte behavior was completely different. In the NMDA-injected cortex, the loss of glial fibrillary acidic protein-expressing (GFAP⁺) astrocytes was confined to the injection site until 7 days after the injection, and astrocytes around the damage sites showed extensive gliosis and appeared to isolate the damage sites. In contrast, in the KA-injected brain, GFAP⁺ astrocytes, like neurons, slowly, but progressively, disappeared across the entire hemisphere. Other markers of astrocytes, including S100β, glutamate transporter EAAT2, the potassium channel Kir4.1 and glutamine synthase, showed patterns similar to that of GFAP in both NMDA- and KA-injected cortexes. More importantly, astrocyte disappearance and/or functional loss preceded neuronal death in the KA-injected brain. Taken together, these results suggest that loss of astrocyte support to neurons may be a critical cause of delayed neuronal death in the injured brain.     

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity – a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.