Microbiota dictate T cell clonal selection to augment graft-versus-host disease after stem cell transplantation.

Allogeneic T cell expansion is the primary determinant of graft-versus-host disease (GVHD), and current dogma dictates that this is driven by histocompatibility antigen disparities between donor and recipient. This paradigm represents a closed genetic system within which donor T cells interact with peptide-major histocompatibility complexes (MHCs), though clonal interrogation remains challenging due to the sparseness of the T cell repertoire. We developed a Bayesian model using donor and recipient T cell receptor (TCR) frequencies in murine stem cell transplant systems to define limited common expansion of T cell clones across genetically identical donor-recipient pairs. A subset of donor CD4+ T cell clonotypes differentially expanded in identical recipients and were microbiota dependent. Microbiota-specific T cells augmented GVHD lethality and could target microbial antigens presented by gastrointestinal epithelium during an alloreactive response. The microbiota serves as a source of cognate antigens that contribute to clonotypic T cell expansion and the induction of GVHD independent of donor-recipient genetics.

Intestinal microbiota controls graft-versus-host disease independent of donor-host genetic disparity.

Acute graft-versus-host disease (aGVHD) remains a major limitation of allogeneic stem cell transplantation (SCT), and severe intestinal manifestation is the major cause of early mortality. Intestinal microbiota control MHC class II (MHC-II) expression by ileal intestinal epithelial cells (IECs) that promote GVHD. Here, we demonstrated that genetically identical mice of differing vendor origins had markedly different intestinal microbiota and ileal MHC-II expression, resulting in discordant GVHD severity. We utilized cohousing and antibiotic treatment to characterize the bacterial taxa positively and negatively associated with MHC-II expression. A large proportion of bacterial MHC-II inducers were vancomycin sensitive, and peri-transplant oral vancomycin administration attenuated CD4+ T cell-mediated GVHD. We identified a similar relationship between pre-transplant microbes, HLA class II expression, and both GVHD and mortality in a large clinical SCT cohort. These data highlight therapeutically tractable mechanisms by which pre-transplant microbial taxa contribute to GVHD independently of genetic disparity.

On your mark, get SET(D2), go! H3K36me3 primes DNA mismatch repair.

Trimethylation of histone H3 on Lys36 (H3K36me3) by SETD2 is linked to actively transcribed regions. Li et al. identify a novel role for H3K36me3 that facilitates DNA mismatch repair (MMR) in cells by targeting the MMR machinery to chromatin during the cell cycle, thereby explaining certain cases of MMR-defective cancers.

LBR and lamin A/C sequentially tether peripheral heterochromatin and inversely regulate differentiation.

Eukaryotic cells have a layer of heterochromatin at the nuclear periphery. To investigate mechanisms regulating chromatin distribution, we analyzed heterochromatin organization in different tissues and species, including mice with mutations in the lamin B receptor (Lbr) and lamin A (Lmna) genes that encode nuclear envelope (NE) proteins. We identified LBR- and lamin-A/C-dependent mechanisms tethering heterochromatin to the NE. The two tethers are sequentially used during cellular differentiation and development: first the LBR- and then the lamin-A/C-dependent tether. The absence of both LBR and lamin A/C leads to loss of peripheral heterochromatin and an inverted architecture with heterochromatin localizing to the nuclear interior. Myoblast transcriptome analyses indicated that selective disruption of the LBR- or lamin-A-dependent heterochromatin tethers have opposite effects on muscle gene expression, either increasing or decreasing, respectively. These results show how changes in NE composition contribute to regulating heterochromatin positioning, gene expression, and cellular differentiation during development.

Regulatory T cells suppress myeloma-specific immunity during autologous stem cell mobilization and transplantation.

ABSTRACT: Autologous stem cell transplantation (ASCT) is the standard of care consolidation therapy for eligible patients with myeloma but most patients eventually progress, an event associated with features of immune escape. Novel approaches to enhance antimyeloma immunity after ASCT represent a major unmet need. Here, we demonstrate that patient-mobilized stem cell grafts contain high numbers of effector CD8 T cells and immunosuppressive regulatory T cells (Tregs). We showed that bone marrow (BM)-residing T cells are efficiently mobilized during stem cell mobilization (SCM) and hypothesized that mobilized and highly suppressive BM-derived Tregs might limit antimyeloma immunity during SCM. Thus, we performed ASCT in a preclinical myeloma model with or without stringent Treg depletion during SCM. Treg depletion generated SCM grafts containing polyfunctional CD8 T effector memory cells, which dramatically enhanced myeloma control after ASCT. Thus, we explored clinically tractable translational approaches to mimic this scenario. Antibody-based approaches resulted in only partial Treg depletion and were inadequate to recapitulate this effect. In contrast, a synthetic interleukin-2 (IL-2)/IL-15 mimetic that stimulates the IL-2 receptor on CD8 T cells without binding to the high-affinity IL-2Ra used by Tregs efficiently expanded polyfunctional CD8 T cells in mobilized grafts and protected recipients from myeloma progression after ASCT. We confirmed that Treg depletion during stem cell mobilization can mitigate constraints on tumor immunity and result in profound myeloma control after ASCT. Direct and selective cytokine signaling of CD8 T cells can recapitulate this effect and represent a clinically testable strategy to improve responses after ASCT.

DoUBLing up: ubiquitin and ubiquitin-like proteases in genome stability.

Maintaining stability of the genome requires dedicated DNA repair and signalling processes that are essential for the faithful duplication and propagation of chromosomes. These DNA damage response (DDR) mechanisms counteract the potentially mutagenic impact of daily genotoxic stresses from both exogenous and endogenous sources. Inherent to these DNA repair pathways is the activity of protein factors that instigate repair processes in response to DNA lesions. The regulation, coordination, and orchestration of these DDR factors is carried out, in a large part, by post-translational modifications, such as phosphorylation, ubiquitylation, and modification with ubiquitin-like proteins (UBLs). The importance of ubiquitylation and UBLylation with SUMO in DNA repair is well established, with the modified targets and downstream signalling consequences relatively well characterised. However, the role of dedicated erasers for ubiquitin and UBLs, known as deubiquitylases (DUBs) and ubiquitin-like proteases (ULPs) respectively, in genome stability is less well established, particularly for emerging UBLs such as ISG15 and UFM1. In this review, we provide an overview of the known regulatory roles and mechanisms of DUBs and ULPs involved in genome stability pathways. Expanding our understanding of the molecular agents and mechanisms underlying the removal of ubiquitin and UBL modifications will be fundamental for progressing our knowledge of the DDR and likely provide new therapeutic avenues for relevant human diseases, such as cancer.

Microarray screening reveals two non-conventional SUMO-binding modules linked to DNA repair by non-homologous end-joining.

SUMOylation is critical for numerous cellular signalling pathways, including the maintenance of genome integrity via the repair of DNA double-strand breaks (DSBs). If misrepaired, DSBs can lead to cancer, neurodegeneration, immunodeficiency and premature ageing. Using systematic human proteome microarray screening combined with widely applicable carbene footprinting, genetic code expansion and high-resolution structural profiling, we define two non-conventional and topology-selective SUMO2-binding regions on XRCC4, a DNA repair protein important for DSB repair by non-homologous end-joining (NHEJ). Mechanistically, the interaction of SUMO2 and XRCC4 is incompatible with XRCC4 binding to three other proteins important for NHEJ-mediated DSB repair. These findings are consistent with SUMO2 forming a redundant NHEJ layer with the potential to regulate different NHEJ complexes at distinct levels including, but not limited to, XRCC4 interactions with XLF, LIG4 and IFFO1. Regulation of NHEJ is not only relevant for carcinogenesis, but also for the design of precision anti-cancer medicines and the optimisation of CRISPR/Cas9-based gene editing. In addition to providing molecular insights into NHEJ, this work uncovers a conserved SUMO-binding module and provides a rich resource on direct SUMO binders exploitable towards uncovering SUMOylation pathways in a wide array of cellular processes.

Apoptosis-Decellularized Peripheral Nerve Scaffold Allows Regeneration across Nerve Gap.

Peripheral nerve injury results in loss of motor and sensory function distal to the nerve injury and is often permanent in nerve gaps longer than 5 cm. Autologous nerve grafts (nerve autografts) utilize patients' own nerve tissue from another part of their body to repair the defect and are the gold standard in care. However, there is a limited autologous tissue supply, size mismatch between donor nerve and injured nerve, and morbidity at the site of nerve donation. Decellularized cadaveric nerve tissue alleviates some of these limitations and has demonstrated success clinically. We previously developed an alternative apoptosis-assisted decellularization process for nerve tissue. This new process may result in an ideal scaffold for peripheral nerve regeneration by gently removing cells and antigens while preserving delicate topographical cues. In addition, the apoptosis-assisted process requires less active processing time and is inexpensive. This study examines the utility of apoptosis-decellularized peripheral nerve scaffolds compared to detergent-decellularized peripheral nerve scaffolds and isograft controls in a rat nerve gap model. Results indicate that, at 8 weeks post-injury, apoptosis-decellularized peripheral nerve scaffolds perform similarly to detergent-decellularized and isograft controls in both functional (muscle weight recovery, gait analysis) and histological measures (neurofilament staining, macrophage infiltration). These new apoptosis-decellularized scaffolds hold great promise to provide a less expensive scaffold for nerve injury repair, with the potential to improve nerve regeneration and functional outcomes compared to current detergent-decellularized scaffolds.

[Decision-Making Behaviour in Physician-Rating Portals: Results of a Vignette Study in the New Federal States of Germany]. / Das Entscheidungsverhalten in Arztbewertungsportalen. Ergebnisse einer Vignettenstudie in den neuen Bundesländern.

BACKGROUND: The extent to which relevant information is taken into consideration in the decision process for or against the choice of certain physicians when searching for physicians via physician-rating portals is largely unknown. In particular, the question of whether longer travel distances to a doctor's office are accepted in favour of a good rating is investigated in this study. METHODS: In a vignette study, 192 respondents from new German states were shown ten hits each from a fictitious search for a primary care physician. A total of 1881 choice probabilities were evaluated with cluster-corrected regressions. RESULTS: A good rating and a short distance to the doctor's office were the most important determinants of a positive probability of choice. Longer distances were not accepted in favour of a good rating. Arabic-sounding names strongly reduced the probability of choice, although the respondents only assigned subordinate importance to the doctors' names as a basis for decision-making. CONCLUSION: The area served by physicians in private practice does not increase if they receive good grades on rating portals. Service quality is important, but not the most important factor in the rating score.

Metabolomic Insights of Biosurfactant Activity from Bacillus niabensis against Planktonic Cells and Biofilm of Pseudomonas stutzeri Involved in Marine Biofouling.

In marine environments, biofilm can cause negative impacts, including the biofouling process. In the search for new non-toxic formulations that inhibit biofilm, biosurfactants (BS) produced by the genus Bacillus have demonstrated considerable potential. To elucidate the changes that BS from B. niabensis promote in growth inhibition and biofilm formation, this research performed a nuclear magnetic resonance (NMR) metabolomic profile analysis to compare the metabolic differences between planktonic cells and biofilms of Pseudomonas stutzeri, a pioneer fouling bacteria. The multivariate analysis showed a clear separation between groups with a higher concentration of metabolites in the biofilm than in planktonic cells of P. stutzeri. When planktonic and biofilm stages were treated with BS, some differences were found among them. In planktonic cells, the addition of BS had a minor effect on growth inhibition, but at a metabolic level, NADP+, trehalose, acetone, glucose, and betaine were up-regulated in response to osmotic stress. When the biofilm was treated with the BS, a clear inhibition was observed and metabolites such as glucose, acetic acid, histidine, lactic acid, phenylalanine, uracil, and NADP+ were also up-regulated, while trehalose and histamine were down-regulated in response to the antibacterial effect of the BS.

The suitability of micronuclei as markers of relative biological effect.

Micronucleus (MN) formation is routinely used as a biodosimeter for radiation exposures and has historically been used as a measure of DNA damage in cells. Strongly correlating with dose, MN are also suggested to indicate radiation quality, differentiating between particle and photon irradiation. The "gold standard" for measuring MN formation is Fenech's cytokinesis-block micronucleus (CBMN) cytome assay, which uses the cytokinesis blocking agent cytochalasin-B. Here, we present a comprehensive analysis of the literature investigating MN induction trends in vitro, collating 193 publications, with 2476 data points. Data were collected from original studies that used the CBMN assay to quantify MN in response to ionizing radiation in vitro. Overall, the meta-analysis showed that individual studies mostly have a linear increase of MN with dose [85% of MN per cell (MNPC) datasets and 89% of percentage containing MN (PCMN) datasets had an R2 greater than 0.90]. However, there is high variation between studies, resulting in a low R2 when data are combined (0.47 for MNPC datasets and 0.60 for PCMN datasets). Particle type, species, cell type, and cytochalasin-B concentration were suggested to influence MN frequency. However, variation in the data meant that the effects could not be strongly correlated with the experimental parameters investigated. There is less variation between studies when comparing the PCMN rather than the number of MNPC. Deviation from CBMN protocol specified timings did not have a large effect on MN induction. However, further analysis showed less variation between studies following Fenech's protocol closely, which provided more reliable results. By limiting the cell type and species as well as only selecting studies following the Fenech protocol, R2 was increased to 0.64 for both measures. We therefore determine that due to variation between studies, MN are currently a poor predictor of radiation-induced DNA damage and make recommendations for futures studies assessing MN to improve consistency between datasets.

Sesquiterpene Lactones Potentiate Olaparib-Induced DNA Damage in p53 Wildtype Cancer Cells.

Despite notable advances in utilising PARP inhibitor monotherapy, many cancers are not PARP inhibitor-sensitive or develop treatment resistance. In this work, we show that the two structurally-related sesquiterpene lactones, a 2-bromobenzyloxy derivative of dehydrosantonin (BdS) and alantolactone (ATL) sensitise p53 wildtype, homologous recombination-proficient cancer cells to low-dose treatment with the PARP inhibitor, olaparib. Exposure to combination treatments of olaparib with BdS or ATL induces cell-cycle changes, chromosomal instability, as well as considerable increases in nuclear area. Mechanistically, we uncover that mitotic errors likely depend on oxidative stress elicited by the electrophilic lactone warheads and olaparib-mediated PARP-trapping, culminating in replication stress. Combination treatments exhibit moderately synergistic effects on cell survival, probably attenuated by a p53-mediated, protective cell-cycle arrest in the G2 cell-cycle phase. Indeed, using a WEE1 inhibitor, AZD1775, to inhibit the G2/M cell-cycle checkpoint further decreased cell survival. Around half of all cancers diagnosed retain p53 functionality, and this proportion could be expected to increase with improved diagnostic approaches in the clinic. Utilising sublethal oxidative stress to sensitise p53 wildtype, homologous recombination-proficient cancer cells to low-dose PARP-trapping could therefore serve as the basis for future research into the treatment of cancers currently refractory to PARP inhibition.

Photosynthesis and photoprotection characteristics related to ROS production in three Chattonella (Raphidophyceae) species.

The toxic potential of Chattonella is associated with a high production of reactive oxygen species (ROS). Chattonella species can tolerate high irradiance levels but seems not to be efficient in the induction of nonphotochemical chl a fluorescence (NPQ) under light stress conditions. Therefore, we postulated that high ROS production of this microalgal group is related to the lack of effective photoprotection mechanisms. We compared the NPQ induction, xanthophyll cycle interconversion (XC), and the production of the ion superoxide (O2- ) in Chattonella marina var. antiqua, Chattonella sp., and C. marina acclimated to 43 (LL) and 300 µmol photons · m2  · s-1 (HL). We also evaluated the photosynthetic characteristics of the three strains. Photosynthesis saturated at relative high irradiances (above 500 µmol photons · m2  · s-1 ) in LL and HL Chattonella strains. For the first time, we documented the conversion of diadinoxanthin into diatoxanthin in microalgae that have violaxanthin as the major XC carotenoid. The slow NPQ induction indicated that qE (fast component of NPQ) was not present, and this process was related to the interconversion of XC pigments. However, the quenching efficiency (QE) of deepoxidated xanthophylls was low in the three Chattonella strains. The strain with the lowest QE produced the highest amount of a O2- . Therefore, ROS production in Chattonella seems to be related to a low expression of XC-related thermal PSII dissipation.

Lipophilic Toxins in Wild Bivalves from the Southern Gulf of California, Mexico.

Most of the shellfish fisheries of Mexico occur in the Gulf of California. In this region, known for its high primary productivity, blooms of diatoms and dinoflagellates are common, occurring mainly during upwelling events. Dinoflagellates that produce lipophilic toxins are present, where some outbreaks related to okadaic acid and dinophisystoxins have been recorded. From January 2015 to November 2017 samples of three species of wild bivalve mollusks were collected monthly in five sites in the southern region of Bahía de La Paz. Pooled tissue extracts were analyzed using LC-MS/MS to detect lipophilic toxins. Eighteen analogs of seven toxin groups, including cyclic imines were identified, fortunately individual toxins did not exceed regulatory levels and also the total toxin concentration for each bivalve species was lower than the maximum permitted level for human consumption. Interspecific differences in toxin number and concentration were observed in three species of bivalves even when the samples were collected at the same site. Okadaic acid was detected in low concentrations, while yessotoxins and gymnodimines had the highest concentrations in bivalve tissues. Although in low quantities, the presence of cyclic imines and other lipophilic toxins in bivalves from the southern Gulf of California was constant.

SAMHD1 Sheds Moonlight on DNA Double-Strand Break Repair.

SAMHD1 (sterile α motif and histidine (H) aspartate (D) domain-containing protein 1) is known for its antiviral activity of hydrolysing deoxynucleotides required for virus replication. Daddacha et al. identify a hydrolase-independent, moonlighting function of SAMHD1 that facilitates homologous recombination of DNA double-strand breaks (DSBs) by promoting recruitment of C-terminal binding protein interacting protein (CTIP), a DNA-end resection factor, to damaged DNA. These findings could benefit anticancer treatment.

Lymphatic-to-blood vessel transition in adult microvascular networks: A discovery made possible by a top-down approach to biomimetic model development.

OBJECTIVE: Emerging areas of vascular biology focus on lymphatic/blood vessel mispatterning and the regulation of endothelial cell identity. However, a fundamental question remains unanswered: Can lymphatic vessels become blood vessels in adult tissues? Leveraging a novel tissue culture model, the objective of this study was to track lymphatic endothelial cell fate over the time course of adult microvascular network remodeling. METHODS: Cultured adult Wistar rat mesenteric tissues were labeled with BSI-lectin and time-lapse images were captured over five days of serum-stimulated remodeling. Additionally, rat mesenteric tissues on day 0 and day 3 and 5 post-culture were labeled for PECAM + LYVE-1 or PECAM + podoplanin. RESULTS: Cultured networks were characterized by increases in blood capillary sprouting, lymphatic sprouting, and the number of lymphatic/blood vessel connections. Comparison of images from the same network regions identified incorporation of lymphatic vessels into blood vessels. Mosaic lymphatic/blood vessels contained lymphatic marker positive and negative endothelial cells. CONCLUSIONS: Our results reveal the ability for lymphatic vessels to transition into blood vessels in adult microvascular networks and discover a new paradigm for investigating lymphatic/blood endothelial cell dynamics during microvascular remodeling.

Interruption points in the wing gene regulatory network underlying wing polyphenism evolved independently in male and female morphs in Cardiocondyla ants.

Wing polyphenism in ants, which produces a winged female queen caste and a wingless female worker caste, evolved approximately 150 million years ago and has been key to the remarkable success of ants. Approximately 20 million years ago, the myrmicine ant genus Cardiocondyla evolved an additional wing polyphenism among males producing two male morphs: wingless males that fight to enhance mating success and winged males that disperse. Here we show that interruption of rudimentary wing-disc development in larvae of the ant Cardiocondyla obscurior occurs further downstream in the network in wingless males as compared with wingless female workers. This pattern is corroborated in C. kagutsuchi, a species from a different clade within the genus, indicating that late interruption of wing development in males is conserved across Cardiocondyla. Therefore, our results show that the novel male wing polyphenism was not developmentally constrained by the pre-existing female wing polyphenism and evolved through independent alteration of interruption points in the wing gene network. Furthermore, a comparison of adult morphological characters in C. obscurior reveals that developmental trajectories lead to similar morphological trait integration between winged and wingless females, but dramatically different integration between winged and wingless males. This suggests that the alternative sex-specific developmental routes to achieve winglessness in the genus Cardiocondyla may have evolved through different selection regimes acting on wingless males and females.

Tissue-Engineered Peripheral Nerve Interfaces.

Research on neural interfaces has historically concentrated on development of systems for the brain; however, there is increasing interest in peripheral nerve interfaces (PNIs) that could provide benefit when peripheral nerve function is compromised, such as for amputees. Efforts focus on designing scalable and high-performance sensory and motor peripheral nervous system interfaces. Current PNIs face several design challenges such as undersampling of signals from the thousands of axons, nerve-fiber selectivity, and device-tissue integration. To improve PNIs, several researchers have turned to tissue engineering. Peripheral nerve tissue engineering has focused on designing regeneration scaffolds that mimic normal nerve extracellular matrix composition, provide advanced microarchitecture to stimulate cell migration, and have mechanical properties like the native nerve. By combining PNIs with tissue engineering, the goal is to promote natural axon regeneration into the devices to facilitate close contact with electrodes; in contrast, traditional PNIs rely on insertion or placement of electrodes into or around existing nerves, or do not utilize materials to actively facilitate axon regeneration. This review presents the state-of-the-art of PNIs and nerve tissue engineering, highlights recent approaches to combine neural-interface technology and tissue engineering, and addresses the remaining challenges with foreign-body response.

Ciguatera in Mexico (1984⁻2013).

Historical records of ciguatera in Mexico date back to 1862. This review, including references and epidemiological reports, documents 464 cases during 25 events from 1984 to 2013: 240 (51.72%) in Baja California Sur, 163 (35.12%) in Quintana Roo, 45 (9.69%) in Yucatan, and 16 (3.44%) cases of Mexican tourists intoxicated in Cuba. Carnivorous fish, such as snapper (Lutjanus) and grouper (Epinephelus and Mycteroperca) in the Pacific Ocean, and great barracuda (Sphyraena barracuda) and snapper (Lutjanus) in the Atlantic (Gulf of Mexico and Caribbean Sea), were involved in all cases. In the Mexican Caribbean, a sub-record of ciguatera cases that occurred before 1984 exists. However, the number of intoxications has increased in recent years, and this food poisoning is poorly studied in the region. Current records suggest that ciguatera fish poisoning in humans is the second most prevalent form of seafood poisoning in Mexico, only exceeded by paralytic shellfish poisoning (505 cases, 21 fatalities in the same 34-year period). In this study, the status of ciguatera in Mexico (epidemiological and treatment), and the fish vectors are reviewed. Dinoflagellate species Gambierdiscus, Ostreopsis, and Prorocentrum are related with the reported outbreaks, marine toxins, ecological risk, and the potential toxicological impact.

Processing-Size Correlations in the Preparation of Magnetic Alginate Microspheres Through Emulsification and Ionic Crosslinking.

Magnetic alginate microspheres are biocompatible due to their alginate matrix, and motion-controllable by applied magnetic fields due to their magnetic character. Therefore, they have the potential of being used as vessels to a broad variety of materials, including drugs and therapeutic agents, facilitating entry to biological systems in a relatively non-invasive manner. Here, magnetic alginate microspheres were prepared through an emulsification and ionic cross-linking process, where a mixture of alginate and iron oxide magnetic nanoparticles was initially dispersed in a continuous phase, followed by gelation of this dispersed phase into microspheres by cross-linking the dispersion with calcium ions. The resulting magnetic alginate microspheres were found to be superparamagnetic and to respond to applied magnetic fields by chain formation. The effect of shear rate, alginate concentration, and magnetic nanoparticle concentration on microsphere size was investigated with the aim to control the size of microspheres with respect to process and formulation parameters. Two of these parameters, shear rate and alginate concentration, were used to correlate experimental results with a theoretical model for the case where the dispersed phase is more viscous than the continuous phase.