SNX12 Role in Endosome Membrane Transport

In this paper, we investigated the role of sorting nexin 12 (SNX12) in the endocytic pathway. SNX12 is a member of the PX domain-containing sorting nexin family and shares high homology with SNX3, which plays a central role in the formation of intralumenal vesicles within multivesicular endosomes. We found that SNX12 is expressed at very low levels compared to SNX3. SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides. We find that overexpression of SNX12 prevents the detachment (or maturation) of multivesicular endosomes from early endosomes. This in turn inhibits the degradative pathway from early to late endosomes/lysosomes, much like SNX3 overexpression, without affecting endocytosis, recycling and retrograde transport. In addition, while previous studies showed that Hrs knockdown prevents EGF receptor sorting into multivesicular endosomes, we find that overexpression of SNX12 restores the sorting process in an Hrs knockdown background. Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes

In vitro activity of cefiderocol and comparators against isolates of Gram-negative pathogens from a range of infection sources: SIDERO-WT-2014–2018 studies in Italy

ABSTRACT: Objectives: : Antimicrobial resistance, particularly carbapenem resistance, in Gram-negative pathogens poses a significant healthcare threat. Carbapenem resistance rates in Italy are among the highest in Europe. We report the in vitro activity of cefiderocol, a novel siderophore cephalosporin, and comparator antibiotics against Gram-negative isolates from Italy as part of the SIDERO-WT studies. Methods: : Isolates were collected between 2014 and 2018. Minimum inhibitory concentrations (MICs) were determined using International Organization for Standardization and EUCAST guidelines. Antimicrobial susceptibilities were interpreted using EUCAST breakpoints; pharmacodynamic/pharmacokinetic breakpoints were used if EUCAST breakpoints were not specified. Results: : The 2472 isolates [1545 (62.5%) Enterobacterales and 927 (37.5%) non-fermenters] represented a range of infection sources, including nosocomial pneumonia (902; 36.5%), complicated urinary tract infection (374; 15.1%), bloodstream infection (596; 24.1%), complicated intra-abdominal infection (257; 10.4%) and other infection sources (343; 13.9%). Cefiderocol was active against the majority of isolates, regardless of infection source (susceptibility, 94.2−97.3%). A high proportion of non-fermenters (97.6%) and Enterobacterales (95.6%) were cefiderocol-susceptible, although susceptibility was lower in Klebsiella pneumoniae (88.1%). Susceptibility to cefiderocol was significantly (P 8 mg/L), comprising 516/927 (55.7%) non-fermenters and 96/1545 (6.2%) Enterobacterales. Cefiderocol (499/516; 96.7%) activity was greater than colistin (440/516; 85.3%), ceftazidime/avibactam (123/516; 23.8%) and ceftolozane/tazobactam (89/516; 17.2%) in meropenem-resistant non-fermenter isolates. Conclusion: : Susceptibility to cefiderocol was significantly greater than meropenem, colistin, ceftazidime/avibactam and ceftolozane/tazobactam overall, regardless of infection source

SNX12 overexpression inhibits EGFR transport and degradation without affecting retrograde and recycling transport routes.

<p>(<b>A-C</b>) After cell surface binding, EGF-biotin coupled to streptavidin^{AlexaFluor488} was endocytosed for 10 min (<b>A</b>) or 50 min (<b>B-C</b>) at 37°C in HeLa cells expressing mRFP1-SNX12. Cells were labeled with anti-EEA1 (<b>A</b>) or Lamp1 (<b>B-C</b>) antibodies and analyzed by triple channel fluorescence. (<b>D</b>) HeLa cells expressing myc-SNX12 or myc-SNX3 or mock-treated were incubated with EGF for the indicated time periods. Cell lysates (100 µg) were analyzed by SDS gel electrophoresis and western blotting with antibodies against EGFR, α-tubulin (a-tub) or myc<b>.</b> (<b>E</b>) After cell surface binding, Shiga toxin B-subunit conjugated to Cy3 was internalized for 10 min or 50 min at 37°C into HeLa cells expressing GFP-SNX12. Cells were labeled with anti-transferrin receptor (TfR) or Rab6 antibodies and analyzed by triple channel fluorescence. (<b>F</b>) After cell surface binding (0 min), transferrin conjugated to AlexaFluor546 (transferrin⁵⁴⁶) was internalized for 30 min or 120 min at 37°C into control cells (upper panels) or cells expressing GFP-SNX12 (lower panels). Cells were then analyzed by fluorescence. (<b>A-C and E-F</b>) Scale bar indicates 10 µm.</p

SNX12 silencing has no effect on VSV infection and EGFR transport and degradation.

<p>(<b>A</b>) HeLa cells treated with siRNAs against SNX12 or mock-treated were lysed. Lysates were analyzed by SDS gel electrophoresis and western blotting using with antibodies against myc, SNX3 or actin. (<b>B-C</b>) HeLa cells were treated with SNX12 siRNAs or mock-treated, and then microtubules were depolymerized or not with 10 µM nocodazole for 2 h. VSV (1 MOI) was bound at 4°C at the cell surface and cells were then incubated for 3 h at 37°C to allow VSV infection to proceed. Cells were analyzed by immunofluorescence with antibodies against VSV-G protein. Scale bar indicates 10 µm. Experiments were quantified (<b>B</b>) and representative pictures were shown in (<b>C</b>). Under these conditions ≈50% of the control cells were infected so that changes in infection rate can be best monitored, and values are normalized to the controls. Each condition is the mean of at least three independent experiments; standard errors are indicated. (<b>D</b>) After cell surface binding, EGF-biotin coupled to streptavidin^{AlexaFluor488} was endocytosed for 10 min or 50 min at 37°C in HeLa cells treated siRNAs against SNX12. Cells were labeled with anti-EEA1 or Lamp1 antibodies and analyzed by triple channel fluorescence. (<b>E</b>) HeLa cells were treated with siRNAs against SNX12 or mock treated and then incubated with EGF for the indicated time periods. Cell lysates (100 µg) were analyzed by SDS gel electrophoresis and western blotting with antibodies against EGFR or α-tubulin (a-tub)<b>.</b></p

SNX12 overexpression induces an accumulation of MVB-like structures and rescues the intralumenal vesicles that incorporate the EGF receptor.

<p>(<b>A-B</b>) HeLa cells expressing GFP-SNX12 were fixed and processed for electron microscopy. Cryosections were labeled with antibodies against GFP followed by protein A-gold 10 nm (arrows). Panel A shows a cluster of several multivesicular endosomes, each being labeled with a star, and panel B shows a high magnification view of an individual multivesicular endosome. Scale bar indicates 250 nm. (<b>C</b>) Hrs (<b>D-E</b>) or SNX3 (<b>F-G</b>) was knocked down and mRFP-SNX12 (red) was overexpressed (<b>E-G</b>) or not (<b>D-F</b>). In each condition, HeLa cells were also transfected with GFP-Rab5^Q79L (green) during the last 24 h. After cell surface binding, EGF was internalized for 15 min at 37°C. Cells were processed for immunofluorescence with anti-EGFR antibodies (blue). The relative amount of EGFR in the lumen of endosome was quantified as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038949#pone.0038949-Pons1" target="_blank">[25]</a>. Results are expressed as the percentage of the total amount of EGFR. Each condition is the mean of at least three independent experiments; standard errors are indicated and results were analyzed by paired t test (***, p<0,001). (<b>D-G</b>) Representative pictures of experiments quantified in (<b>C</b>). GFP-Rab5^Q79L enlarged endosomes in each condition showed the EGFR localization after Hrs (<b>D-E</b>) or SNX3 (<b>F-G</b>) was knocked down and mRFP-SNX12 (red) was overexpressed (<b>E-G</b>) or not (<b>D-F</b>).</p

SNX12 is less expressed than SNX3 but is also localized on early endosomes.

<p>(<b>A</b>) Alignment of amino acid sequences of Homo sapiens SNX3 and SNX12. (<b>B</b>) RNA was extracted from different cell lines as indicated and relative amounts of endogenous SNX3 and SNX12 mRNA were quantified by RT-PCR. Values are indicated in the table below the graph since they are very low for SNX12 mRNA. (<b>C</b>) HeLa cells expressing GFP-SNX12 or co-expressing GFP-SNX12 and mRFP1-SNX3 were processed for immunofluorescence using the indicated antibodies. Scale bar indicates 10 µm.</p

Pre-analytical phase in cryoglobulin (CRG) detection: an alternative method for sample transport

No Abstract Avalaibl

Anti-nuclear antibody detection in cryoprecipitates: Distinctive patterns in hepatitis C virus-infected patients

Background: Anti-nuclear antibodies are immunoglobulins directed against nuclear antigens. They are associated with many autoimmune disorders, but are frequently found in patients infected with hepatitis C virus, possibly indicating an underlying common origin. Likewise, mixed cryoglobulinemia often accompanies autoimmune diseases and hepatitis C infection. Aim: To compare anti-nuclear antibodies and immunoglobulin content of cryoprecipitates from hepatitis C virus-positive patients in order to assess their predictive value in the onset of hepatitis C virus-driven extrahepatic disorders. Methods: Serum from 40 hepatitis C virus-positive patients and 50 controls with rheumatoid arthritis was processed for cryoglobulin detection: all subjects presented with Type III mixed cryoglobulinemia. Immunoglobulin content and immunoglobulin subclasses of cryoprecipitates were assessed by immunofixation and tested by ELISA for rheumatoid factor. Cryoprecipitates were also analysed for anti-nuclear antibodies by indirect immuno-fluorescence to identify specific patterns typical of each condition. Results: Anti-nuclear antibody patterns differed significantly; 26 infected subjects (65%) were IgG3 positive: of these, 25 were also anti-nuclear antibody-positive (96.1%). Conclusions: IgG3 are autoreactive clones unrelated to viral recognition and possibly involved in autoimmune disorders. Altogether, these results may represent useful diagnostic device for early detection of hepatitis C virus-induced autoimmune diseases