Mice Lacking Alkbh1 Display Sex-Ratio Distortion and Unilateral Eye Defects

Escherichia coli AlkB is a 2-oxoglutarate- and iron-dependent dioxygenase that reverses alkylated DNA damage by oxidative demethylation. Mouse AlkB homolog 1 (Alkbh1) is one of eight members of the newly discovered family of mammalian dioxygenases.In the present study we show non-Mendelian inheritance of the Alkbh1 targeted allele in mice. Both Alkbh1(-/-) and heterozygous Alkbh1(+/-) offspring are born at a greatly reduced frequency. Additionally, the sex-ratio is considerably skewed against female offspring, with one female born for every three to four males. Most mechanisms that cause segregation distortion, act in the male gametes and affect male fertility. The skewing of the sexes appears to be of paternal origin, and might be set in the pachythene stage of meiosis during spermatogenesis, in which Alkbh1 is upregulated more than 10-fold. In testes, apoptotic spermatids were revealed in 5-10% of the tubules in Alkbh1(-/-) adults. The deficiency of Alkbh1 also causes misexpression of Bmp2, 4 and 7 at E11.5 during embryonic development. This is consistent with the incompletely penetrant phenotypes observed, particularly recurrent unilateral eye defects and craniofacial malformations.Genetic and phenotypic assessment suggests that Alkbh1 mediates gene regulation in spermatogenesis, and that Alkbh1 is essential for normal sex-ratio distribution and embryonic development in mice

Repressing the hedgehog signalling pathway

The Hedgehog (Hh) signalling pathway is essential for metazoan development and aberrant activation of the pathway is found in tumours. Mutations in Hh pathway components are found in several tumour types, including basal cell carcinoma (BCC) and medulloblastoma (MB). The outline of the Hh pathway is evolutionary conserved, with Patched (Ptch) as the Hh receptor and Smoothened (Smo) as the signal transducer that relays the signal into the cytoplasm to activate the transcription factors Ci/Gli, which regulate gene transcription and cellular responses leading to cell proliferation and/or differentiation. These studies focus on the function of Ptch1 and Suppressor of fused (Sufu), both negative regulators of Hh signalling. In paper I, we showed that genetic ablation of Sufu in mice results in embryonic lethality around embryonic day 9 with failure to close the neural tube. Sufu-/- embryos showed high Gli activity that was not repressable at the level of Smo. Sufu+/- mice develop jaw keratocysts and a skin phenotype with BCC-like lesions, alopecia, increased skin pigmenation, and abberant sebaceous gland morphology. Our data show that in contrast to the situation in Drosophila, Sufu has a central role in mammalian Hh signalling, and its loss-offunction leads to ligand-independent activation of the Hh pathway. In paper II, we have investigated the possibility that Sufu would modulate the phenotype of Ptch1+/- mice or vice-versa. We found that the frequency of MB was not altered in Sufu+/-Ptch+/- mice compared to Ptch1+/- mice. All MB in Ptch1+/- mice and all but one MB in Sufu+/-Ptch1+/- mice had lost expression of the Ptch1 wt allele, indicating that this is the critical genetic change leading to MB formation in these mice. Skin from Sufu+/-, Ptch1+/- and Sufu+/-Ptch1+/- mice developed BCClike epidermal lesions. The number of such lesions was increased in Sufu+/-Ptch1+/- mice compared to Sufu+/- and Ptch1+/- mice. Our data indicate a differential importance of Sufu and Ptch1 as tumour suppressors in MB versus the BCC-like lesions. In paper III, we investigated the unique properties of PTCH1 isoforms generated by alternative first exon usage. All isoforms functioned as Hh receptors. However, the 4 isoforms induced by Hh signalling inhibited pathway activity to a higher extent than those not regulated by Hh signalling. In situ hybridization allowed the detection of the Ptch1 isoforms in specific structures of mouse embryos. This study supports a role of splicing variation and/or promoter choice for Hh signalling regulation. In paper IV, we created a conditional Ptch1 allele in mice and deleted Ptch1 by using the ARR2PBi-Cre transgenic line. Ptch1 was deleted in prostate and seminal vesicles but we found no abberant phenotype in these tissues up to the age of 12 months. In contrast, BCC-like epidermal lesions were initiated by Cre-mediated Ptch1 recombination in solitary cells in interfollicular epidermis and hair follicles. Skin with BCC-like lesions showed upregulation of Gli1, an indicator of Hh pathway activity. The skin proliferations arose both from interfollicular epidermis and hair follicles. Our results indicate that loss of Ptch1 in keratinocytes drives them into a hair follicle fate

Protein-Functionalized Gold Nanoparticles as Refractometric Nanoplasmonic Sensors for the Detection of Proteolytic Activity of Porphyromonas gingivalis

Periodontitis is an inflammatory oral disease that affects a large part of the adult population, causing significant costs and suffering. The key pathogen, Porphyromonas gingivalis, secretes gingipains, which are highly destructive proteases and the most important virulence factors in the pathogenesis of the disease. Currently, periodontitis is diagnosed mainly by mechanical manual probing and radiography, often when the disease has already progressed significantly. The possibilities of detecting gingipain activity in gingival fluid could enable early-stage diagnosis and facilitate treatment. Here, we describe a sensitive nanoparticle-based nanoplasmonic biosensor for the detection of the proteolytic activity of gingipains. Gold nanoparticles (AuNPs) were self-assembled as a submonolayer in multiwell plates and further modified with casein or IgG. The proteolytic degradation of the protein coating was tracked by monitoring the shift in the localized surface plasmon resonance (LSPR) peak position. The sensor performance was investigated using model systems with trypsin and purified gingipains (subtypes Kgp and RgpB) and further validated using supernatants from cultures of P. gingivalis. Proteolytic degradation by proteases in buffer results in a concentration- and time-dependent blueshift of the LSPR band of about 1-2 nm when using casein as a substrate. In bacterial supernatants, the degradation of the protein coating resulted in unspecific binding of proteins present in the complex sample matrix to the nanoparticles, which instead triggered a redshift of about 2 nm of the LSPR band. A significant LSPR shift was seen only in samples with gingipain activity. The sensor showed a limit of detection &amp;lt; 0.1 mu g/mL (4.3 nM), which is well below gingipain concentrations detected in severe chronic periodontitis cases (similar to 50 mu g/mL). This work shows the possibility of developing cost-effective nanoparticle-based biosensors for rapid detection of protease activity for chair-side periodontal diagnostics.Funding Agencies|Swedish Research Council (VR)Swedish Research Council [2016-04874, 2017-04475]; Swedish Foundation for Strategic Research (SFF)Swedish Foundation for Strategic Research [FFL15-0026]; Knut and Alice Wallenberg FoundationKnut &amp; Alice Wallenberg Foundation [KAW 2016.0231]</p

Protein-Functionalized Gold Nanoparticles as Refractometric Nanoplasmonic Sensors for the Detection of Proteolytic Activity of Porphyromonas gingivalis

Periodontitis is an inflammatory oral disease that affects a large part of the adult population, causing significant costs and suffering. The key pathogen, Porphyromonas gingivalis, secretes gingipains, which are highly destructive proteases and the most important virulence factors in the pathogenesis of the disease. Currently, periodontitis is diagnosed mainly by mechanical manual probing and radiography, often when the disease has already progressed significantly. The possibilities of detecting gingipain activity in gingival fluid could enable early-stage diagnosis and facilitate treatment. Here, we describe a sensitive nanoparticle-based nanoplasmonic biosensor for the detection of the proteolytic activity of gingipains. Gold nanoparticles (AuNPs) were self-assembled as a submonolayer in multiwell plates and further modified with casein or IgG. The proteolytic degradation of the protein coating was tracked by monitoring the shift in the localized surface plasmon resonance (LSPR) peak position. The sensor performance was investigated using model systems with trypsin and purified gingipains (subtypes Kgp and RgpB) and further validated using supernatants from cultures of P. gingivalis. Proteolytic degradation by proteases in buffer results in a concentration- and time-dependent blueshift of the LSPR band of about 1-2 nm when using casein as a substrate. In bacterial supernatants, the degradation of the protein coating resulted in unspecific binding of proteins present in the complex sample matrix to the nanoparticles, which instead triggered a redshift of about 2 nm of the LSPR band. A significant LSPR shift was seen only in samples with gingipain activity. The sensor showed a limit of detection &amp;lt; 0.1 mu g/mL (4.3 nM), which is well below gingipain concentrations detected in severe chronic periodontitis cases (similar to 50 mu g/mL). This work shows the possibility of developing cost-effective nanoparticle-based biosensors for rapid detection of protease activity for chair-side periodontal diagnostics.Funding Agencies|Swedish Research Council (VR)Swedish Research Council [2016-04874, 2017-04475]; Swedish Foundation for Strategic Research (SFF)Swedish Foundation for Strategic Research [FFL15-0026]; Knut and Alice Wallenberg FoundationKnut &amp; Alice Wallenberg Foundation [KAW 2016.0231]</p

Exploring the working life of people with multiple sclerosis during the COVID-19 pandemic in Sweden

BACKGROUND: The COVID-19 pandemic led to vast changes in working life and conditions in which we work. These changes may affect people with multiple sclerosis (PwMS) differently. We aimed to describe the working situation of PwMS during the COVID-19 pandemic and the pandemic's impact on their working lives. METHODS: All individuals aged 20-50 listed in the Swedish Multiple Sclerosis Registry were invited to participate in an online survey in 2021. Closed and open-ended responses linked to individual-level register data were used in this exploratory mixed-methods study. Differences in the proportions reporting specific impacts were assessed with chi-square tests by sex, MS severity, education, and profession. The open-ended answers were analysed through content analysis. RESULTS: Over 8500 PwMS were invited (52% response rate). We included the 3887 respondents who answered questions about the impact of the pandemic on working life. Most (93.7%) reported being in paid work. An impact of the ongoing pandemic to one's daily occupation was reported by 26.2%, with different characteristics observed across the impacts. Four categories of type of answers were identified from the open-ended answers: Direct impact on one's occupation, Disclosing or concealing MS in the workplace, Worry and uncertainty, and Broader impact to life situation. CONCLUSIONS: PwMS navigated the pandemic by interrupting as well as continuing their working lives. Many PwMS reported that the pandemic did not affect their work situation. However, the reported impacts differed among the participants and a sense of uncertainty and worry was often underlying their statements. Lessons from the pandemic may support future work participation

Le Courrier Bordelais apportant toutes les nouvelles de Bordeaux, tant dedans la ville que dehors

Moreau81

Arming of MAIT Cell Cytolytic Antimicrobial Activity Is Induced by IL-7 and Defective in HIV-1 Infection

<div><p>Mucosa-associated invariant T (MAIT) cells represent a large innate-like evolutionarily conserved antimicrobial T-cell subset in humans. MAIT cells recognize microbial riboflavin metabolites from a range of microbes presented by MR1 molecules. MAIT cells are impaired in several chronic diseases including HIV-1 infection, where they show signs of exhaustion and decline numerically. Here, we examined the broader effector functions of MAIT cells in this context and strategies to rescue their functions. Residual MAIT cells from HIV-infected patients displayed aberrant baseline levels of cytolytic proteins, and failed to mobilize cytolytic molecules in response to bacterial antigen. In particular, the induction of granzyme B (GrzB) expression was profoundly defective. The functionally impaired MAIT cell population exhibited abnormal T-bet and Eomes expression patterns that correlated with the deficiency in cytotoxic capacity and cytokine production. Effective antiretroviral therapy (ART) did not fully restore these aberrations. Interestingly, IL-7 was capable of arming resting MAIT cells from healthy donors into cytotoxic GrzB+ effector T cells capable of killing bacteria-infected cells and producing high levels of pro-inflammatory cytokines in an MR1-dependent fashion. Furthermore, IL-7 treatment enhanced the sensitivity of MAIT cells to detect low levels of bacteria. In HIV-infected patients, plasma IL-7 levels were positively correlated with MAIT cell numbers and function, and IL-7 treatment <i>in vitro</i> significantly restored MAIT cell effector functions even in the absence of ART. These results indicate that the cytolytic capacity in MAIT cells is severely defective in HIV-1 infected patients, and that the broad-based functional defect in these cells is associated with deficiency in critical transcription factors. Furthermore, IL-7 induces the arming of effector functions and enhances the sensitivity of MAIT cells, and may be considered in immunotherapeutic approaches to restore MAIT cells.</p></div

Aberrant MAIT cell transcription factor expression in chronic HIV-1 infection correlates with MAIT cell effector dysfunction.

<p>(A) Unstimulated PBMCs from 10 healthy controls and 17 HIV-1 infected, ART-untreated patients were stained for transcription factors, and their MAIT cell T-bet and Eomes expression profile was determined. The effect of long-term ART on MAIT cell T-bet and Eomes expression profile was also determined in paired samples from 14 HIV-infected patients. (B) Comparison of PLZF, RORγt, and Helios levels between the two MAIT cell populations in the same set of HIV-1 infected, ART-untreated patients. (C, D, E, F) Correlations between the levels of T-bet^negEomes^neg MAIT cells in ART-untreated HIV-1 infected patients with MAIT cell numbers <i>ex vivo</i> and effector functions after <i>E</i>. <i>coli</i> stimulation (MOI 10) were calculated using Spearman’s test. (G) T-bet^negEomes^neg MAIT cells were generated following a six day incubation of PBMCs from healthy controls with PFA-fixed <i>E</i>. <i>coli</i> (MOI 10). The expression levels of PLZF, RORγt, and GrzB, as well as measurement of proliferation using a Cell Trace Violet (CTV) dilution method, were determined in both T-bet^dimEomes^hi and T-bet^negEomes^neg MAIT cells (n = 3). Box and whisker plot shows median, IQR and the 10^th to the 90^th percentile. Representative FACS plots are shown. The Mann-Whitney test was used to determine significance between healthy controls and HIV-1 infected, ART-untreated patients, and the Wilcoxon test for paired samples.</p