An efficient molecular genetic testing strategy for incontinentia pigmenti based on single-tube long fragment read sequencing.

Incontinentia pigmenti (IP) is a rare X-linked dominant neuroectodermal dysplasia that primarily affects females. The only known causative gene is IKBKG, and the most common genetic cause is the recurrent IKBKG△4-10 deletion resulting from recombination between two MER67B repeats. Detection of variants in IKBKG is challenging due to the presence of a highly homologous non-pathogenic pseudogene IKBKGP1. In this study, we successfully identified four pathogenic variants in four IP patients using a strategy based on single-tube long fragment read (stLFR) sequencing with a specialized analysis pipeline. Three frameshift variants (c.519-3_519dupCAGG, c.1167dupC, and c.700dupT) were identified and subsequently validated by Sanger sequencing. Notably, c.519-3_519dupCAGG was found in both IKBKG and IKBKGP1, whereas the other two variants were only detected in the functional gene. The IKBKG△4-10 deletion was identified and confirmed in one patient. These results demonstrate that the proposed strategy can identify potential pathogenic variants and distinguish whether they are derived from IKBKG or its pseudogene. Thus, this strategy can be an efficient genetic testing method for IKBKG. By providing a comprehensive understanding of the whole genome, it may also enable the exploration of other genes potentially associated with IP. Furthermore, the strategy may also provide insights into other diseases with detection challenges due to pseudogenes.

LIM kinase1 regulates mitotic centrosome integrity via its activity on dynein light intermediate chains.

Abnormal centrosome number and function have been implicated in tumour development. LIM kinase1 (LIMK1), a regulator of actin cytoskeleton dynamics, is found to localize at the mitotic centrosome. However, its role at the centrosome is not fully explored. Here, we report that LIMK1 depletion resulted in multi-polar spindles and defocusing of centrosomes, implicating its involvement in the regulation of mitotic centrosome integrity. LIMK1 could influence centrosome integrity by modulating centrosomal protein localization at the spindle pole. Interestingly, dynein light intermediate chains (LICs) are able to rescue the defects observed in LIMK1-depleted cells. We found that LICs are potential novel interacting partners and substrates of LIMK1 and that LIMK1 phosphorylation regulates cytoplasmic dynein function in centrosomal protein transport, which in turn impacts mitotic spindle pole integrity.

The Regulation of Cellular Responses to Mechanical Cues by Rho GTPases.

The Rho GTPases regulate many cellular signaling cascades that modulate cell motility, migration, morphology and cell division. A large body of work has now delineated the biochemical cues and pathways, which stimulate the GTPases and their downstream effectors. However, cells also respond exquisitely to biophysical and mechanical cues such as stiffness and topography of the extracellular matrix that profoundly influence cell migration, proliferation and differentiation. As these cellular responses are mediated by the actin cytoskeleton, an involvement of Rho GTPases in the transduction of such cues is not unexpected. In this review, we discuss an emerging role of Rho GTPase proteins in the regulation of the responses elicited by biophysical and mechanical stimuli.

Vitrification of in vitro matured oocytes: effects on meiotic spindle configuration and mitochondrial function.

BACKGROUND: In the assisted reproductive technique, cryopreserving in vitro-matured oocytes is a new strategy to extend the pool of total oocytes. However, oocyte cryopreservation technique is still unsatisfied. So the assessment of cyro-damage on meiotic spindle and mitochondrial function is necessary to evaluate and refine the current protocols. MATERIAL AND METHODS: The immature oocytes were donated from women undergoing ICSI cycles. Cytoskeleton was assessed by α-tubulin and mitochondria through the fluorescent ΔΨm reporter JC-1. RESULTS: Relative inner membrane potential in MII oocytes from vIVM group sharply decreased, compared with the control (n=30) (1.397 vs. 1.019, P<0.05). 45.2% defective spindles were observed in fIVM group, compared with 48.0% in vIVM group (P>0.05). Oocytes in fIVM (35.5%, 11/31) and vIVM (40.0%, 10/25) displayed abnormal chromosome (P>0.05). CONCLUSION: In vitro maturation (IVM) has an adverse effect on the organization of spindle and chromosome, and no significantly effect on spindle and chromosome was discovered after vitrification-thaw cycle, while there was obvious damage of oocyte mitochondrial function of in vitro-matured oocyte detected after warming, which may be the reason of the low following developmental potential.

Effect of mouse oocyte vitrification on mitochondrial membrane potential and distribution.

The effects of mouse oocyte vitrification on mitochondrial membrane potential and distribution were explored in this study. The collected mouse oocytes were randomly divided into vitrification and control groups. Ethylene glycol (EG) and dimethylsulphoxide (DMSO) were used as cryoprotectants in the vitrification group. The mitochondrial function and distribution in the oocytes were examined by using the fluorescent probes, JC-1 and Mito Tracker green. The results showed that the ratio of red to green fluorescence in mouse oocytes was significantly decreased after thawing in the vitrification group as compared with the control group (1.28 vs. 1.70, P<0.05). The percentage of polarized distribution of the mitochondria in oocytes was conspicuously reduced in the vitrification group when compared with the control group (31% vs. 63%, P<0.05). It was suggested that vitrification significantly affects the mitochondrial function and distribution in oocytes and reduces the potential of oocyte fertilization and embryo development.

Misperceptions among patients with chronic hepatitis B in Singapore.

AIM: To identify the misperceptions among CHB patients, as well as to determine the factors associated with better knowledge. METHODS: A telephone interview was conducted on 192 adult CHB patients, who earlier responded to an advertisement for free screening. The questionnaire included items about socio-demographic factors and a 14-item quiz on knowledge of general aspects, transmission, and management of HBV infection. RESULTS: The mean knowledge score on HBV was 10.4/14. Common misperceptions included availability of treatment for HBV infection and early liver cancer, as well as on transmission. Having completed tertiary education was the only independent factor associated with a high knowledge score, after controlling other demographic factors. CONCLUSION: More educational efforts should be focused on patients' misperceptions and target the less educated HBV carriers.