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Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies., Competing Interests: The author’s have read the journal’s policy and have the following competing interests: Jan Chemnitz, Joachim Hauber, Frank Buchholz, Ilona Hauber, Niklas Beschorner and Maike Voges are founding partners of PROVIREX Genome Editing Therapies GmbH, which is commercially developing Brec1 technology. Niklas Beschorner and Maike Voges are full-time employees of PROVIREX Genome Editing Therapies GmbH since 2022, and Jan Chemnitz, Frank Buchholz, Joachim Hauber and Ilona Hauber are part-time employees. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare., (Copyright: © 2024 Beschorner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Active haptic sensation is critical for object identification, but its neural circuit basis is poorly understood. We combined optogenetics, two-photon imaging, and high-speed behavioral tracking in mice solving a whisker-based object orientation discrimination task. We found that orientation discrimination required animals to summate input from multiple whiskers specifically along the whisker arc. Animals discriminated the orientation of the stimulus per se as their performance was invariant to the location of the presented stimulus. Populations of barrel cortex neurons summated across whiskers to encode each orientation. Finally, acute optogenetic inactivation of the barrel cortex and cell-type-specific optogenetic suppression of layer 4 excitatory neurons degraded performance, implying that infragranular layers alone are not sufficient to solve the task. These data suggest that spatial summation over an active haptic array generates representations of an object's orientation, which may facilitate encoding of complex three-dimensional objects during active exploration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Short-term studies have demonstrated that nondirected donors (NDDs) have psychosocial outcomes that are similar to donors who donate directly, but long-term studies have not been done. NDDs at our center were surveyed regarding motivation; support during donation; stress related to donation; regret; financial resources used for donation; preferences about communication with the recipient; and cost reimbursement. Of 100 NDDs who donated at our center in the last 20 years, 95 remain in contact with us, and 77 responded to our survey (mean ± standard deviation [SD] 6.7 ± 4 years postdonation). The most common motivation for donation was the desire to help another (99%). Many NDDs received support from family, friends, and employers. NDDs voiced stress about the possibility of recipient kidney rejection, physical consequences to themselves, and financial burden. Only one donor expressed regret. Almost half wanted some recipient information at donation; 61% preferred routine recipient status updates; 56% believed meeting the recipient should occur at any mutually agreeable time; and 55% endorsed reimbursement for expenses. Stressors for NDDs are analogous to those of directed donors; NDDs prefer having some information about the recipient and prefer to be given a choice regarding the timing for communication with the recipient. NDDs supported donation being financially neutral., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Herpes Simplex Virus type 2 (HSV-2) is a neurotropic human pathogen. Upon de novo infection, the viral infected cell protein 0 (ICP0) is immediately expressed and interacts with various cellular components during the viral replication cycle. ICP0 is a multifunctional regulatory protein that has been shown to be important for both efficient viral replication and virus reactivation from latency. In particular, as previously demonstrated in transfected tissue culture models, ICP0 interacts with the cellular E3 ubiquitin ligase SIAH-1, which targets ICP0 for proteasomal degradation. However, the consequence of this virus-host interaction during the establishment of HSV-2 infection in vivo has not yet been elucidated. Here we confirmed that ICP0 of HSV-2 interacts with SIAH-1 via two conserved PxAxVxP amino acid binding motifs. We also demonstrate in vitro that a SIAH-1 binding-deficient HSV-2 strain, constructed by homologous recombination technology, exhibits an attenuated growth curve and impaired DNA and protein synthesis. This attenuated phenotype was also confirmed in an in vivo ocular infection mouse model. Specifically, viral load of the SIAH-1 binding-deficient HSV-2 mutant was significantly reduced in the trigeminal ganglia and brain stem at day 5 and 7 post infection. Our findings indicate that the interplay between ICP0 and SIAH-1 is important for efficient HSV-2 replication in vivo, thereby affecting viral dissemination kinetics in newly infected organisms, and possibly revealing novel targets for antiviral therapy., Competing Interests: The commercial affiliation of an author of this study [MME] (Biomedizinische Forschungsgesellschaft mbH, Vienna, Austria) does not alter our adherence to all PLOS ONE policies on sharing data and materials.

The distribution coefficients and selectivities required for extraction purposes were predicted with a thermodynamic equation of state for the ternary system formic acid/water/extraction solvent. These predictions were validated with experimental data from the literature and experimental data from the oxidation of biomass to formic acid process measured in this work. Extraction solvents discussed in this work are 1-butanol, 1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, 1-decanol, ethyl n -butyl ether, diisopropyl ether, di- n -butyl ether, benzyl formate, and heptyl formate. The considered temperature ranged from 273 to 363 K under atmospheric pressure. Perturbed-chain statistical associating fluid theory (PC-SAFT) was used for prediction purposes applying an approach as simple as possible and as complex as necessary to achieve trustworthy data for selecting the best extraction solvent. Using PC-SAFT allowed identifying 1-hexanol as the most promising solvent out of the 11 extraction agents. The predicted data were in good agreement with the experimental distribution coefficients and the selectivities, which are very sensitive to experimental uncertainties., Competing Interests: The authors declare no competing financial interest.

Background: HIV is primarily transmitted by sexual intercourse and predominantly infects people in Third World countries. Here an important medical need is self-protection for women, particularly in societies where condoms are not widely accepted. Therefore, availability of antiviral microbicides may significantly reduce sexual HIV transmission in such environments., Methods: Here, we investigated structural characteristics and the antiviral activity of the polypurine tract (PPT)-specific ODN A, a 54-mer oligodeoxynucleotide (ODN) that has been previously shown to trigger the destruction of viral RNA genomes by prematurely activating the retroviral RNase H. The stability of ODN A and mutants thereof was tested at various storage conditions. Furthermore, antiviral effects of ODN A were analyzed in various tissue culture HIV-1 infection models. Finally, circular dichroism spectroscopy was employed to gain insight into the structure of ODN A., Results: We show here that ODN A is a powerful tool to abolish HIV-1 particle infectivity, as required for a candidate compound in vaginal microbicide applications. We demonstrate that ODN A is not only capable to prematurely activate the retroviral RNase H, but also prevents HIV-1 from entering host cells. ODN A also exhibited extraordinary stability lasting several weeks. Notably, ODN A is biologically active under various storage conditions, as well as in the presence of carboxymethylcellulose CMC (K-Y Jelly), a potential carrier for application as a vaginal microbicide. ODN A's remarkable thermostability is apparently due to its specific, guanosine-rich sequence. Interestingly, these residues can form G-quadruplexes and may lead to G-based DNA hyperstructures. Importantly, the pronounced antiviral activity of ODN A is maintained in the presence of human semen or semen-derived enhancer of virus infection (SEVI; i.e. amyloid fibrils), both known to enhance HIV infectivity and reduce the efficacy of some antiviral microbicides., Conclusions: Since ODN A efficiently inactivates HIV-1 and also displays high stability and resistance against semen, it combines unique and promising features for its further development as a vaginal microbicide against HIV.

CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs), act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5' long terminal repeat (LTR), for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination.

Background: Several pathogenic bacteria utilize receptors of the CEACAM family to attach to human cells. Binding to different members of this receptor family can result in uptake of the bacteria. Uptake of Neisseria gonorrhoeae, a gram-negative human pathogen, via CEACAMs found on epithelial cells, such as CEACAM1, CEA or CEACAM6, differs mechanistically from phagocytosis mediated by CEACAM3, a CEACAM family member expressed selectively by human granulocytes., Principal Findings: We find that CEACAM1- as well as CEACAM3-mediated bacterial internalization are accompanied by a rapid increase in phosphatidylinositol-3,4,5 phosphate (PI(3,4,5)P) at the site of bacterial entry. However, pharmacological inhibition of phosphatidylinositol-3' kinase (PI3K) selectively affects CEACAM1-mediated uptake of Neisseria gonorrhoeae. Accordingly, overexpression of the PI(3,4,5)P phosphatase SHIP diminishes and expression of a constitutive active PI3K increases CEACAM1-mediated internalization of gonococci, without influencing uptake by CEACAM3. Furthermore, bacterial uptake by GPI-linked members of the CEACAM family (CEA and CEACAM6) and CEACAM1-mediated internalization of N. meningitidis by endothelial cells require PI3K activity. Sensitivity of CEACAM1-mediated uptake toward PI3K inhibition is independent of receptor localization in cholesterol-rich membrane microdomains and does not require the cytoplasmic or the transmembrane domain of CEACAM1. However, PI3K inhibitor sensitivity requires the Ig(C2)-like domains of CEACAM1, which are also present in CEA and CEACAM6, but which are absent from CEACAM3. Accordingly, overexpression of CEACAM1 Ig(C2) domains blocks CEACAM1-mediated internalization., Conclusions: Our results provide novel mechanistic insight into CEACAM1-mediated endocytosis and suggest that epithelial CEACAMs associate in cis with other membrane receptor(s) via their extracellular domains to trigger bacterial uptake in a PI3K-dependent manner.

Persistent infections with human papillomavirus type 16 (HPV16), HPV18, or HPV31 are necessary for the development of cervical cancer, implying that HPVs have evolved immunoevasive mechanisms. Recent global transcriptome analyses indicated that these HPV types downregulate the constitutive expression of interferon (IFN)-stimulated genes (ISGs), but the underlying mechanism is not well understood. Comparative analyses of ISG transcription in keratinocytes with complete HPV16, -18, and -31 genomes revealed that antiviral genes (IFIT1 and MX1), genes involved in IFN signaling (STAT1), proapoptotic genes (TRAIL and XAF1), and pathogen recognition receptors (TLR3, RIG-I, and MDA5) are inhibited to similar extents by HPV16, -18, and -31. The lower expression of pathogen receptors in HPV-positive cells correlated with a greatly impaired induction of IFN-β and also of IFN-λ1, -2, and -3 upon receptor stimulation. IFN-κ is constitutively expressed in normal keratinocytes and is strongly repressed by HPV16, -18, and -31. ISGs downregulated in HPV-positive cells can be reactivated by IFN-κ expression. The viral E6 and E7 oncogenes are sufficient for IFN-κ repression, with E6 being mainly responsible. E6 inhibits IFN-κ transcription independently from binding to PDZ proteins. IFN-κ expression can be activated in only one cell line by E6AP knockdown but can be activated in all tested HPV-positive cells by addition of a DNA methyltransferase inhibitor, suggesting that HPVs modulate DNA methylation. Taken together, these results suggest that carcinogenic HPVs target IFN-κ by different pathways in keratinocytes to inhibit both antiviral ISGs and pathogen recognition receptors, which in turn reduces the expression of inducible IFNs.

Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is an immunoglobulin-related receptor expressed on human granulocytes. CEACAM3 functions as a single chain phagocytotic receptor recognizing gram-negative bacteria such as Neisseria gonorrhoeae, which possess CEACAM-binding adhesins on their surface. The cytoplasmic domain of CEACAM3 contains an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is phosphorylated upon receptor engagement. Here we show that the SH2 domains of the regulatory subunit of phosphatidylinositol 3'-kinase (PI3K) bind to tyrosine residue 230 of CEACAM3 in a phosphorylation-dependent manner. PI3K is rapidly recruited and directly associates with CEACAM3 upon bacterial binding as shown by FRET analysis. Although PI3K activity is not required for efficient uptake of the bacteria by CEACAM3-transfected cells or primary human granulocytes, it is critical for the stimulated production of reactive oxygen species by infected phagocytes and the intracellular degradation of CEACAM-binding bacteria. Together, our results highlight the ability of CEACAM3 to coordinate signaling events that not only mediate bacterial uptake, but also trigger the killing of internalized pathogens.

Background: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), an immunoglobulin (Ig)-related glycoprotein, serves as cellular receptor for a variety of Gram-negative bacterial pathogens associated with the human mucosa. In particular, Neisseria gonorrhoeae, N. meningitidis, Moraxella catarrhalis, and Haemophilus influenzae possess well-characterized CEACAM1-binding adhesins. CEACAM1 is typically involved in cell-cell attachment, epithelial differentiation, neovascularisation and regulation of T-cell proliferation, and is one of the few CEACAM family members with homologues in different mammalian lineages. However, it is unknown whether bacterial adhesins of human pathogens can recognize CEACAM1 orthologues from other mammals., Results: Sequence comparisons of the amino-terminal Ig-variable-like domain of CEACAM1 reveal that the highest sequence divergence between human, murine, canine and bovine orthologues is found in the beta-strands comprising the bacteria-binding CC'FG-face of the Ig-fold. Using GFP-tagged, soluble amino-terminal domains of CEACAM1, we demonstrate that bacterial pathogens selectively associate with human, but not other mammalian CEACAM1 orthologues. Whereas full-length human CEACAM1 can mediate internalization of Neisseria gonorrhoeae in transfected cells, murine CEACAM1 fails to support bacterial internalization, demonstrating that the sequence divergence of CEACAM1 orthologues has functional consequences with regard to bacterial recognition and cellular invasion., Conclusions: Our results establish the selective interaction of several human-restricted bacterial pathogens with human CEACAM1 and suggest that co-evolution of microbial adhesins with their corresponding receptors on mammalian cells contributes to the limited host range of these highly adapted infectious agents.

Mycoplasmal contamination remains a significant impediment to the culture of eukaryotic cells. For certain cultures, attempts to eliminate the infection are feasible alternatives to the normally recommended disposal of the contaminated culture. Here, three antibiotic regimens for mycoplasmal decontamination were compared in a large panel of naturally infected cultures: a 1-wk treatment with the fluoroquinolone mycoplasma removal agent (MRA), a 2-wk treatment with the fluoroquinolone ciprofloxacin, and three rounds of a sequential 1-wk treatment with BM-Cyclin containing tiamulin and minocyclin. These antibiotic treatments had a high efficiency of permanent cure: MRA 69%, ciprofloxacin 75%, BM-Cyclin 87%. Resistance to mycoplasma eradication was observed in some cell cultures: BM-Cyclin 0%, MRA 20%, ciprofloxacin 20%. Nearly all resistant contaminants that could be identified belonged to the species Mycoplasma arginini and M. orale. Detrimental effects of the antibiotics were seen in the form of culture death caused by cytotoxicity (in 5 to 13% of the cultures). Alterations of the cellular phenotypic features or selective clonal outgrowth might represent further untoward side effects of exposure to these antibiotics. Overall, antibiotic decontamination of mycoplasmas is an efficient, inexpensive, reliable, and simple method: 150/200 (75%) chronically and heavily contaminated cultures were cured and 50/200 (25%) cultures could not be cleansed and were either lost or remained infected. It is concluded that eukaryotic cell cultures containing mycoplasmas are amenable to antibiotic treatment and that a cure rate of three-quarters is a reasonable expectation.

Methyl-coenzyme-M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M [2-(methylthio)ethanesulfonate] and 7-mercaptoheptanoylthreonine phosphate in methanogenic archaea. The enzyme contains the nickel porphinoid coenzyme F430 as a prosthetic group. In the active, reduced (red) state, the enzyme displays two characteristic EPR signals, MCR-red1 and MCR-red2, probably derived from Ni(I). In the presence of the substrate methyl-coenzyme M, the rhombic MCR-red2 signal is quantitatively converted to the axial MCR-red1 signal. We report here on the effects of inhibitory substrate analogues on the EPR spectrum of the enzyme. 3-Bromopropanesulfonate (BrPrSO3), which is the most potent inhibitor of MCR known to date (apparent Ki = 0.05 microM), converted the EPR signals MCR-red1 and MCR-red2 to a novel axial Ni(I) signal designated MCR-BrPrSO3. 3-Fluoropropanesulfonate (apparent Ki < 50 microM) and 3-iodopropanesulfonate (apparent Ki < 1 microM) induced a signal identical to that induced by BrPrSO3 without affecting the line shape, despite the fact that the fluorine, bromine and iodine isotopes employed have nuclear spins of I = 1/2, I = 3/2 and I = 5/2, respectively. This finding suggests that MCR-BrPrSO3 is not the result of a close halogen-Ni(I) interaction. 7-Bromoheptanoylthreonine phosphate (BrHpoThrP) (apparent Ki = 5 microM), which is an inhibitory substrate analogue of 7-mercaptoheptanoylthreonine phosphate, converted the signals MCR-red1 and MCR-red2 to a novel axial Ni(I) signal, MCR-BrHpoThrP, similar but not identical to MCR-BrPrSO3. The results indicate that inhibition of MCR by the halogenated substrate analogues investigated above is not via oxidation of Ni(I)F430. The different MCR EPR signals are assigned to different enzyme/substrate and enzyme/inhibitor complexes.

Methyl-coenzyme-M reductase from Methanobacterium thermoautotrophicum (strain Marburg) was purified to a stage where, besides the alpha, beta and gamma subunits, no additional polypeptides were detectable in the preparation. Under appropriate conditions the enzyme was found to catalyze the reduction of methyl-CoM with 7-mercaptoheptanoylthreonine phosphate (H-S-HTP) to CH4 at a specific rate of 2.5 mumol.min-1.mg protein-1. This finding contradicts a recent report that methyl-CoM reductase is only active when some contaminating proteins are present. The two polypeptides encoded by the open reading frames ORF1 and ORF2 of the methyl-CoM reductase transcription unit did not co-purify with the alpha, beta and gamma subunits. They were neither required nor did they stimulate the activity under the assay conditions. 3-Bromopropanesulfonate (apparent Ki = 0.05 microM) and 2-azidoethanesulfonate (apparent Ki = 1 microM) were found to be two new competitive inhibitors of methyl-CoM reductase. Both inhibitors were considerably more effective than the "classical" 2-bromoethanesulfonate (apparent Ki = 4 microM).