Disease-associated mutations in WDR34 lead to diverse impacts on the assembly and function of dynein-2.

The primary cilium is a sensory organelle, receiving signals from the external environment and relaying them into the cell. Mutations in proteins required for transport in the primary cilium result in ciliopathies, a group of genetic disorders that commonly lead to the malformation of organs such as the kidney, liver and eyes and skeletal dysplasias. The motor proteins dynein-2 and kinesin-2 mediate retrograde and anterograde transport, respectively, in the cilium. WDR34 (also known as DYNC2I2), a dynein-2 intermediate chain, is required for the maintenance of cilia function. Here, we investigated WDR34 mutations identified in Jeune syndrome, short-rib polydactyly syndrome and asphyxiating thoracic dysplasia patients. There is a poor correlation between genotype and phenotype in these cases, making diagnosis and treatment highly complex. We set out to define the biological impacts on cilia formation and function of WDR34 mutations by stably expressing the mutant proteins in WDR34-knockout cells. WDR34 mutations led to different spectrums of phenotypes. Quantitative proteomics demonstrated changes in dynein-2 assembly, whereas initiation and extension of the axoneme, localization of intraflagellar transport complex-B proteins, transition zone integrity and Hedgehog signalling were also affected.

Human cells lacking CDC14A and CDC14B show differences in ciliogenesis but not in mitotic progression.

The budding yeast phosphatase Cdc14 has a central role in mitotic exit and cytokinesis. Puzzlingly, a uniform picture for the three human CDC14 paralogues CDC14A, CDC14B and CDC14C in cell cycle control has not emerged to date. Redundant functions between the three CDC14 phosphatases could explain this unclear picture. To address the possibility of redundancy, we tested expression of CDC14 and analysed cell cycle progression of cells with single and double deletions in CDC14 genes. Our data suggest that CDC14C is not expressed in human RPE1 cells, excluding a function in this cell line. Single- and double-knockouts (KO) of CDC14A and CDC14B in RPE1 cells indicate that both phosphatases are not important for the timing of mitotic phases, cytokinesis and cell proliferation. However, cycling CDC14A KO and CDC14B KO cells show altered ciliogenesis compared to wild-type cells. The cilia of cycling CDC14A KO cells are longer, whereas CDC14B KO cilia are more frequent and disassemble faster. In conclusion, this study demonstrates that the cell cycle functions of CDC14 proteins are not conserved between yeast and human cells.

The human phosphatase CDC14A modulates primary cilium length by regulating centrosomal actin nucleation.

CDC14A codes for a conserved proline-directed phosphatase, and mutations in the gene are associated with autosomal-recessive severe to profound deafness, due to defective kinocilia. A role of CDC14A in cilia formation has also been described in other organisms. However, how human CDC14A impacts on cilia formation remains unclear. Here, we show that human RPE1 hCDC14APD cells, encoding a phosphatase dead version of hCDC14A, have longer cilia than wild-type cells, while hCDC14A overexpression reduces cilia formation. Phospho-proteome analysis of ciliated RPE1 cells identified actin-associated and microtubule binding proteins regulating cilia length as hCDC14A substrates, including the actin-binding protein drebrin. Indeed, we find that hCDC14A counteracts the CDK5-dependent phosphorylation of drebrin at S142 during ciliogenesis. Further, we show that drebrin and hCDC14A regulate the recruitment of the actin organizer Arp2 to centrosomes. In addition, during ciliogenesis hCDC14A also regulates endocytosis and targeting of myosin Va vesicles to the basal body in a drebrin-independent manner, indicating that it impacts primary cilia formation in a multilayered manner.

Human phosphatase CDC14A regulates actin organization through dephosphorylation of epithelial protein lost in neoplasm.

CDC14 is an essential dual-specificity phosphatase that counteracts CDK1 activity during anaphase to promote mitotic exit in Saccharomyces cerevisiae Surprisingly, human CDC14A is not essential for cell cycle progression. Instead, it regulates cell migration and cell adhesion. Little is known about the substrates of hCDC14A and the counteracting kinases. Here, we combine phospho-proteome profiling and proximity-dependent biotin identification to identify hCDC14A substrates. Among these targets were actin regulators, including the tumor suppressor eplin. hCDC14A counteracts EGF-induced rearrangements of actin cytoskeleton by dephosphorylating eplin at two known extracellular signal-regulated kinase sites, serine 362 and 604. hCDC14APD and eplin knockout cell lines exhibited down-regulation of E-cadherin and a reduction in α/ß-catenin at cell-cell adhesions. Reduction in the levels of hCDC14A and eplin mRNA is frequently associated with colorectal carcinoma and is correlated with poor prognosis. We therefore propose that eplin dephosphorylation by hCDC14A reduces actin dynamics to restrict tumor malignancy.

Human phosphatase CDC14A is recruited to the cell leading edge to regulate cell migration and adhesion.

Cell adhesion and migration are highly dynamic biological processes that play important roles in organ development and cancer metastasis. Their tight regulation by small GTPases and protein phosphorylation make interrogation of these key processes of great importance. We now show that the conserved dual-specificity phosphatase human cell-division cycle 14A (hCDC14A) associates with the actin cytoskeleton of human cells. To understand hCDC14A function at this location, we manipulated native loci to ablate hCDC14A phosphatase activity (hCDC14A(PD)) in untransformed hTERT-RPE1 and colorectal cancer (HCT116) cell lines and expressed the phosphatase in HeLa FRT T-Rex cells. Ectopic expression of hCDC14A induced stress fiber formation, whereas stress fibers were diminished in hCDC14A(PD) cells. hCDC14A(PD) cells displayed faster cell migration and less adhesion than wild-type controls. hCDC14A colocalized with the hCDC14A substrate kidney- and brain-expressed protein (KIBRA) at the cell leading edge and overexpression of KIBRA was able to reverse the phenotypes of hCDC14A(PD) cells. Finally, we show that ablation of hCDC14A activity increased the aggressive nature of cells in an in vitro tumor formation assay. Consistently, hCDC14A is down-regulated in many tumor tissues and reduced hCDC14A expression is correlated with poorer survival of patients with cancer, to suggest that hCDC14A may directly contribute to the metastatic potential of tumors. Thus, we have uncovered an unanticipated role for hCDC14A in cell migration and adhesion that is clearly distinct from the mitotic and cytokinesis functions of Cdc14/Flp1 in budding and fission yeast.

Genome editing through large insertion leads to the skipping of targeted exon.

BACKGROUND: Highly efficient genome editing can be achieved through targeting an endonuclease to specific locus of interest. Engineered zinc-finger nuclease (ZFN) and CRISPR-associated protein-9 nuclease (Cas9) offer such an elegant approach for genome editing in vertebrate cells. In this study, we have utilized ZFN and Cas9-catalyzed double strand break followed by homologous recombination-mediated incorporation of premature stop codon and selection marker to target human cell division cycle 14A (hCDC14A) and cell division cycle 14B (hCDC14B) genes. RESULTS: Targeting of the hCDC14A and hCDC14B loci in telomerase immortalized human retinal pigment epithelium (hTERT-RPE1) and human colon cancer (HCT116) cells were confirmed by Southern blot hybridization. Nevertheless, DNA sequence analysis of reverse transcription polymerase chain reaction (RT-PCR) products confirmed that in all the single/double allele ablations, the targeted exon was spliced out. The phenomenon of exon skipping was independent of the genome editing approaches exploited, Cas9 or ZFN. Because the exons had a nucleotide number that could be divided by 3, the reading frame of the exon deletion was maintained. This indicates an exon-skipping event possibly due to the insertion of large DNA fragment (1.7 to 2.5 Kb) within the targeted exons. As a proof-of-principle, we have used gene disruption followed by non-homologous end joining (NHEJ) approach. Small alterations in the exon (one to fifteen bases) were transcribed to mRNA without exon skipping. Furthermore, loxP site-mediated removal of selection markers left a 45 bp scar within the targeted exon that can be traced in mRNA without exon skipping. CONCLUSION: From this study, we conclude that insertion of a large DNA fragment into an exon by genome editing can lead to its skipping from the final transcript. Hence, more cautious approach needs to be taken while designing target sites in such that the possible skipping of targeted exon causes a frame-shift mediated incorporation of pre-mature stop codon. On the other hand, exon skipping may be a useful strategy for the introduction of protein deletions.

Oral Administration of Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes) Extract Protects Gingival Tissues against Proinflammatory TNF-α and Oxidative Stress in Periodontitis Model Rats.

Ganoderma lucidum is a medicinal mushroom that has been used since ancient times. We studied whether chronic oral administration of G. lucidum extract withstands increases in levels of proinflammatory TNF-α and lipid peroxide (LPO), an indicator of oxidative stress, in the gingival tissues of periodontitis model rats. G. lucidum extract was initially examined for inhibition of in vitro oxidative stress, produced by Fenton's reagents in whole homogenates of fresh gum tissues from rats. Prior to in vivo and in vitro experiments with rats, G. lucidum extract was quantitatively tested for its total polyphenol and/or flavonoid contents and ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH)-free radicals. Chronic oral administration of G. lucidum extract (300 mg/kg BW) significantly decreased TNF-α and LPO levels in the gingival tissues of periodontitis model rats. G. lucidum extract also inhibited (P < 0.05) in vitro oxidative stress, as indicated by reduced levels of LPO in G. lucidum extract-preincubated gum tissue homogenates of fresh rats. The in vitro results were, thus, consistent with the in vivo inhibition of lipid peroxidation, DPPH free radical-scavenging effects, and the presence of total polyphenols/flavonoids in G. lucidum extract. Our results provide the evidence, at least partially, for the beneficial effects of G. lucidum on periodontitis, an inflammatory condition of gums which is associated with oxidative stress and preceded by infectious gum diseases.

Level and determinants of birth preparedness and complication readiness among pregnant women: A cross sectional study in a rural area in Bangladesh.

BACKGROUND: Increasing the level of birth preparedness and complication readiness (BP/CR) is one of the key interventions to promote optimal utilization of skilled maternal health services. It is therefore essential to determine the women's ability to recognize the danger signs and the level of BP/CR. This information can be used to design more effective health interventions. OBJECTIVES: This study was conducted to determine the knowledge in recognition of maternal complications, and the level and factors associated with BP/CR in rural Matlab, Bangladesh. METHODS: A community-based cross-sectional survey was conducted from June- October 2015 on a randomly selected 2262 women who delivered live or stillbirth during the year 2014. A pretested and structured questionnaire was used for data collection. Descriptive and analytical statistical methods were used. RESULTS: The proportion of study participants with "good knowledge", measured by the ability to recognise three or more danger signs, in pregnancy and delivery were 26% and 23%, respectively. Out of four BP/CR components, about 15% women saved money, 12% women identified facility for delivery, 9.6% women planned to deliver by skilled birth attendant and 5.3% of women arranged transport. About 12% of women were "well prepared", measured by planning of at least two components, for skilled childbirth and emergency obstetric complications. In the multivariable logistic regression analysis, asset index, antenatal care (ANC) visits and knowledge of danger signs during pregnancy and delivery were associated with BP/CR. The adjusted odds ratio (OR) of "well prepared" was 4.09 (95% confidence interval [CI]: 2.45-6.82) among women with an asset index of five (richest), compared with women in the asset index of one (poorest). The odds of "well prepared" was six times (OR 5.98, 95% CI: 3.85-9.28) higher for women with four or more ANC visits, compared to women with none or one ANC visit. In comparison to women with "poor knowledge" on maternal danger signs during pregnancy and delivery, the odds ratio of "well prepared" among women with good knowledge during pregnancy and in delivery were 1.95 (95% CI: 1.44-2.63) and 1.74 (95% CI: 1.28-2.36), respectively. CONCLUSION: The study revealed a low level of maternal knowledge of danger signs and BP/CR among pregnant women. Further, low socioeconomic status, fewer ANC visits and poor knowledge in recognition of dangers signs on maternal health were associated with low BP/CR. More emphasis should be placed on the quality of information offered to the pregnant women during the prenatal contact and women from low socio-economic gradient should be prioritized to optimize the impact of future BP/CR interventions.

A set of domain-specific markers in the Arabidopsis embryo.

KEY MESSAGE: We describe a novel set of domain-specific markers that can be used in genetic studies, and we used two examples to show loss of stem cells in a monopteros background. Multicellular organisms can be defined by their ability to establish distinct cell identities, and it is therefore of critical importance to distinguish cell types. One step that leads to cell identity specification is activation of unique sets of transcripts. This property is often exploited in order to infer cell identity; the availability of good domain-specific marker lines is, however, poor in the Arabidopsis embryo. Here we describe a novel set of domain-specific marker lines that can be used in Arabidopsis (embryo) research. Based on transcriptomic data, we selected 12 genes for expression analysis, and according to the observed expression domain during embryogenesis, we divided them into four categories (1-ground tissue; 2-root stem cell; 3-shoot apical meristem; 4-post-embryonic). We additionally show the use of two markers from the "stem cell" category in a genetic study, where we use the absence of the markers to infer developmental defects in the monopteros mutant background. Finally, in order to judge whether the established marker lines also play a role in normal development, we generated loss-of-function resources. None of the analyzed T-DNA insertion, artificial microRNA, or misexpression lines showed any apparent phenotypic difference from wild type, indicating that these genes are not nonredundantly required for development, but also suggesting that marker activation can be considered an output of the patterning process. This set of domain-specific marker lines is therefore a valuable addition to the currently available markers and will help to move toward a generic set of tissue identity markers.

Aloe vera gel protects liver from oxidative stress-induced damage in experimental rat model.

Aloe vera is a semi-tropical plant of Liliaceae family which has a wide range of applications in traditional medicine. In the present study, we sought to investigate the heptaoprotective potential of Aloe vera gel as a diet supplement. To achieve this goal, we have designed in vitro and in vivo experimental models of chemical-induced liver damage using male Sprague-Dawley rat. In the in vitro model, its effect was evaluated on Fenton's reaction-induced liver lipid peroxidation. Co-incubation with gel significantly reduced the generation of liver lipid peroxide (LPO). Next, to see the similar effect in vivo, gel was orally administered to rats once daily for 21 successive days. Following 1 hour of the last administration of gel, rats were treated with intra-peritoneal injection of CCl4. Dietary gel showed significant hepatoprotection against CCl4-induced damage as evident by restoration of liver LPO, serum transaminases, alkaline phosphatase, and total bilirubin towards near normal. The beneficial effects were pronounced with the doses used (400 and 800 mg/kg body weight). Besides, we did not observe any significant drop in serum albumin, globulin as well as total protein levels of gel-administered rats. Histopathology of the liver tissue further supported the biochemical findings confirming the hepatoprotective potential of dietary gel.

Organophosphorus and carbamate pesticide residues detected in water samples collected from paddy and vegetable fields of the Savar and Dhamrai Upazilas in Bangladesh.

Several types of organophosphorous and carbamate pesticides have been used extensively by the farmers in Bangladesh during the last few decades. Twenty seven water samples collected from both paddy and vegetable fields in the Savar and Dhamrai Upazilas in Bangladesh were analyzed to determine the occurrence and distribution of organo-phosphorus (chlorpyrifos, malathion and diazinon) and carbamate (carbaryl and carbofuran) pesticide residues. A high performance liquid chromatograph instrument equipped with a photodiode array detector was used to determine the concentrations of these pesticide residues. Diazinon and carbofuran were detected in water samples collected from Savar Upazila at 0.9 µg/L and 198.7 µg/L, respectively. Malathion was also detected in a single water sample at 105.2 µg/L from Dhamrai Upazila. Carbaryl was the most common pesticide detected in Dhamrai Upazila at 14.1 and 18.1 µg/L, while another water sample from Dhamrai Upazila was contaminated with carbofuran at 105.2 µg/L. Chlorpyrifos was not detected in any sample. Overall, the pesticide residues detected were well above the maximum acceptable levels of total and individual pesticide contamination, at 0.5 and 0.1 µg/L, respectively, in water samples recommended by the European Economic Community (Directive 98/83/EC). The presence of these pesticide residues may be attributed by their intense use by the farmers living in these areas. Proper handling of these pesticides should be ensured to avoid direct or indirect exposure to these pesticides.

Syzygium cumini seed extract protects the liver against lipid peroxidation with concurrent amelioration of hepatic enzymes and lipid profile of alcoholic rats.

The in vitro oxidative stress induced by ethanol/Fenton's reaction in rat liver homogenates decreased significantly in the presence of Syzygium cumini seed extract, suggesting the protective effect of the seed extract against the oxidative stress in liver. To corroborate the in vitro effects by an in vivo experiment, 24 rats were divided into four groups: control, S. cumini seed-extract-administered (SE), 15% ethanol-fed (Alc) and Alc+SE rats. The oral administration of the extract (400 mg/kg BW.day) for 7 weeks significantly decreased the levels of liver LPO in the Alc+SE rats, suggesting that S. cumini seed not only obstructed the in vitro free radical production and subsequent oxidative stress, but also inhibited their in vivo formation. The oral administration of extract also reduced the enzyme activities of serum gammaglutamyl transferase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase and the levels of serum creatinine and blood urea nitrogen, serum/liver triglycerides and total cholesterol of the alcoholic rats. The levels of fecal cholesterol were increased by the extract. Fatty degenerations in liver and kidney were absent with S. cumini seed extract treatment. The results suggest that S. cumini seed may be a potential therapy for alcoholics and related dysfunctions by restraining oxidative stress.