Combinatorial single-cell profiling of major chromatin types with MAbID.

Gene expression programs result from the collective activity of numerous regulatory factors. Studying their cooperative mode of action is imperative to understand gene regulation, but simultaneously measuring these factors within one sample has been challenging. Here we introduce Multiplexing Antibodies by barcode Identification (MAbID), a method for combinatorial genomic profiling of histone modifications and chromatin-binding proteins. MAbID employs antibody-DNA conjugates to integrate barcodes at the genomic location of the epitope, enabling combined incubation of multiple antibodies to reveal the distributions of many epigenetic markers simultaneously. We used MAbID to profile major chromatin types and multiplexed measurements without loss of individual data quality. Moreover, we obtained joint measurements of six epitopes in single cells of mouse bone marrow and during mouse in vitro differentiation, capturing associated changes in multifactorial chromatin states. Thus, MAbID holds the potential to gain unique insights into the interplay between gene regulatory mechanisms, especially for low-input samples and in single cells.

Osteoinductive calcium phosphate with submicron topography as bone graft substitute for maxillary sinus floor augmentation: A translational study.

OBJECTIVES: The aim of this study was the preclinical and clinical evaluation of osteoinductive calcium phosphate with submicron surface topography as a bone graft substitute for maxillary sinus floor augmentation (MSFA). MATERIAL AND METHODS: A preclinical sheep model of MSFA was used to compare a calcium phosphate with submicron needle-shaped topography (BCPN , MagnetOs Granules, Kuros Biosciences BV) to a calcium phosphate with submicron grain-shaped topography (BCPG ) and autologous bone graft (ABG) as controls. Secondly, a 10-patient, prospective, randomized, controlled trial was performed to compare BCPN to ABG in MSFA with two-stage implant placement. RESULTS: The pre-clinical study demonstrated that both BCPN and BCPG were highly biocompatible, supported bony ingrowth with direct bone apposition against the material, and exhibited bone formation as early as 3 weeks post-implantation. However, BCPN demonstrated significantly more bone formation than BCPG at the study endpoint of 12 weeks. Only BCPN reached an equivalent amount of bone formation in the available space and a greater proportion of calcified material (bone + graft material) in the maxillary sinus compared to the "gold standard" ABG after 12 weeks. These results were validated in a small prospective clinical study, in which BCPN was found comparable to ABG in implant stability, bone height, new bone formation in trephine core biopsies, and overall clinical outcome. CONCLUSION: This translational work demonstrates that osteoinductive calcium phosphates are promising bone graft substitutes for MSFA, whereas their bone-forming potential depends on the design of their surface features. Netherlands Trial Register, NL6436.

Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR-Cas effector complexes.

Prokaryotes encode various host defense systems that provide protection against mobile genetic elements. Restriction-modification (R-M) and CRISPR-Cas systems mediate host defense by sequence specific targeting of invasive DNA. T-even bacteriophages employ covalent modifications of nucleobases to avoid binding and therefore cleavage of their DNA by restriction endonucleases. Here, we describe that DNA glucosylation of bacteriophage genomes affects interference of some but not all CRISPR-Cas systems. We show that glucosyl modification of 5-hydroxymethylated cytosines in the DNA of bacteriophage T4 interferes with type I-E and type II-A CRISPR-Cas systems by lowering the affinity of the Cascade and Cas9-crRNA complexes for their target DNA. On the contrary, the type V-A nuclease Cas12a (also known as Cpf1) is not impaired in binding and cleavage of glucosylated target DNA, likely due to a more open structural architecture of the protein. Our results suggest that CRISPR-Cas systems have contributed to the selective pressure on phages to develop more generic solutions to escape sequence specific host defense systems.

NOD1 is required for Helicobacter pylori induction of IL-33 responses in gastric epithelial cells.

Helicobacter pylori (H. pylori) causes chronic inflammation which is a key precursor to gastric carcinogenesis. It has been suggested that H. pylori may limit this immunopathology by inducing the production of interleukin 33 (IL-33) in gastric epithelial cells, thus promoting T helper 2 immune responses. The molecular mechanism underlying IL-33 production in response to H. pylori infection, however, remains unknown. In this study, we demonstrate that H. pylori activates signalling via the pathogen recognition molecule Nucleotide-Binding Oligomerisation Domain-Containing Protein 1 (NOD1) and its adaptor protein receptor-interacting serine-threonine Kinase 2, to promote production of both full-length and processed IL-33 in gastric epithelial cells. Furthermore, IL-33 responses were dependent on the actions of the H. pylori Type IV secretion system, required for activation of the NOD1 pathway, as well as on the Type IV secretion system effector protein, CagA. Importantly, Nod1+/+ mice with chronic H. pylori infection exhibited significantly increased gastric IL-33 and splenic IL-13 responses, but decreased IFN-γ responses, when compared with Nod1-/- animals. Collectively, our data identify NOD1 as an important regulator of mucosal IL-33 responses in H. pylori infection. We suggest that NOD1 may play a role in protection against excessive inflammation.

Poor recovery of households from out-of-pocket payment for assisted reproductive technology.

STUDY QUESTION: How do households recover financially from direct out-of-pocket payment for government subsidized ART? SUMMARY ANSWER: After a mean of 3.8 years, there was poor recovery from initiated financial coping strategies with the poorest households being disproportionatley affected. WHAT IS KNOWN ALREADY: Out-of-pocket payment for health services can create financial burdens for households and inequities in access to care. A previous study conducted at a public-academic institution in South Africa documented that patient co-payment for one cycle of ART resulted in catastrophic expenditure for one in five households, and more frequently among the poorest, requiring diverse financial coping strategies to offset costs. STUDY DESIGN, SIZE, DURATION: An observational follow-up study was conducted ~4 years later to assess financial recovery among the 135 couples who had participated in this previous study. Data were collected over 12 months from 73 informants. PARTICIPANTS/MATERIALS, SETTING, METHOD: The study was conducted at a level three referral hospital in the public-academic health sector of South Africa. At this institution ART is subsidized but requires patient co-payments. A purpose-built questionnaire capturing socio-economic information and recovery from financial coping strategies which had been activated was administered to all informants. Financial recovery was defined as the resolution of strategies initiated for the specific purpose of covering the original ART cycle. Results were analysed by strategy and household with the latter including analysis by tertiles based on socio-economic status at the time of the original expenditure. In addition to descriptive statistics, the Pearson Chi squared test was used to determine differences between socioeconomic tertiles and associations between recovery and other variables. MAIN RESULTS AND THE ROLE OF CHANCE: The participation rate in this follow-up study was 54.1% with equal representation from the three socio-economic tertiles. The average duration of follow-up was 46.1 months (±9.78 SD) and respondents' mean age was 42 years (range 31-52). The recovery rate was below 50% for four of five strategies evaluated: 23.1% of households had re-purchased a sold asset; 23.5% had normalized a previous reduction in household spending, 33.8% had regained their savings, and 48.7% were no longer bolstering income through additional work. Two-thirds of households (60.0%) had repaid all loans and debts. The poorest households showed lower rates of recovery when compared to households in the richest tertile. Complete recovery from all strategies initiated was reported by only 10 households (13.7%): 1 of 19 in the lowest tertile, 3 of 30 in the middle and by 6 of 24 households in the richest tertile (P > 0.05). No association was found between the degree of financial recovery and additional cost burdens incurred, including related to babies born; or between the degree of recovery and ongoing pursuit of ART. LIMITATIONS, REASONS FOR CAUTION: The sample size was limited. The participation rate was just over 50%. Results were dependent on participants' narrative and recall. WIDER IMPLICATIONS OF THE FINDINGS: The willingness of patients to pay for ART does not necessarily imply the ability to pay. As a result, the lack of comprehensive third-party funding for ART can create immediate and long-term financial hardship which is more pronounced among poorer households. While more data on the impact of out-of-pocket payment for ART are needed to illustrate the problem in other low resource settings, the results from South Africa provide useful information for similar developing countries. The current absence of more extensive data should therefore not be a barrier to the promotion of financial risk protection for infertile couples, especially the poorest, in need of ART. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by a Masters Student Grant from the Faculty of Health Sciences, University of Cape Town. The authors had no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Hepatitis C-induced hepatocyte apoptosis following liver transplantation is enhanced by immunosuppressive agents.

In recurrent hepatitis C (HCV) post-liver transplantation (OLT), the combination of immunosuppressants and HCV is postulated to increase hepatocyte apoptosis and liver fibrosis. We evaluated hepatocyte apoptosis within the liver tissue of patients with postOLT HCV recurrence compared to HCV-negative individuals and correlated these findings with the effects of immunosuppressants on HCV-induced cell death and its inhibition in primary mouse hepatocytes (PMoH). Liver biopsies from patients with and without HCV were evaluated by immunohistochemistry for markers of apoptosis M30 CytoDEATH (M30) and cleaved PARP (clPARP). PMoH from C57BL/6 mice were infected with recombinant adenoviruses (rAdHCV) that expressed HCV proteins in hepatocytes. Infected cells were treated with cyclosporine, tacrolimus, sirolimus and/or MMF with or without pan-caspase inhibitor Q-VD-Oph. Cell viability and apoptosis were evaluated using crystal violet assays and Western immunoblots probed for cleaved caspase-3 (clCas3) and clPARP. Both M30 and clPARP were increased in the liver biopsies of patients with postOLT HCV recurrence compared to HCV-negative individuals. Treatment of rAdHCV-infected PMoH with cyclosporine, tacrolimus or sirolimus reduced cell viability and increased clCas3 and clPARP compared to rAdHCV infection alone. Addition of MMF to cyclosporine, tacrolimus or sirolimus further reduced cell viability and increased clCas3 and clPARP. Q-VD-Oph improved cell viability in HCV-infected PMoH treated with immunosuppressants alone and in combination and reduced clCas3 and clPARP by approximately 90%. Immunosuppressive agents, especially in combination, enhanced apoptosis in HCV-infected hepatocytes. The finding that Q-VD-Oph reversed hepatocyte death suggests that treatments utilizing apoptosis inhibition might reduce liver injury in postOLT HCV recurrence.

CA1-specific deletion of NMDA receptors induces abnormal renewal of a learned fear response.

CA1 hippocampal N-methyl-d-aspartate-receptors (NMDARs) are necessary for contextually related learning and memory processes. Extinction, a form of learning, has been shown to require intact hippocampal NMDAR signalling. Renewal of fear expression can occur after fear extinction training, when the extinguished fear stimulus is presented in an environmental context different from the training context and thus, renewal is dependent on contextual memory. In this study, we show that a Grin1 knock-out (loss of the essential NR1 subunit for the NMDAR) restricted to the bilateral CA1 subfield of the dorsal hippocampus does not affect acquisition of learned fear, but does attenuate extinction of a cued fear response even when presented in the extinction-training context. We propose that failure to remember the (safe) extinction context is responsible for the abnormal fear response and suggest it is a dysfunctional renewal. The results highlight the difference in outcome of extinguished fear memory resulting from a partial rather than complete loss of function of the hippocampus and suggest a potential mechanism for abnormally increased fear expression in PTSD.

Sharpin is a key regulator of skeletal homeostasis in a TNF-dependent manner.

OBJECTIVES: SHARPIN is a subunit of LUBAC and regulates activation of NF-κB, a pivotal transcription factor in skeletal homeostasis. Mutated SHARPIN gene (cpdm) mice develop chronic proliferative dermatitis and systemic inflammation. Cpdm mice have an osteopaenic phenotype characterised by decreased cortical and trabecular bone volume, but whether this is a consequence of the hyper-inflammatory phenotype is unknown. The inflammatory phenotype of cpdm mice is prevented by Tnf deficiency so we examined cpdm.Tnf (-/-) mice to examine the role of SHARPIN in skeletal development. METHODS: This research determined the extent to which SHARPIN and TNF interact within the skeleton through analyses of gene expression, µCT and biomechanical properties of bones of control (CTRL), cpdm, Tnf (-/-) (TNF KO) and cpdm.Tnf (-/-) (cpdm/TNF KO) mice. RESULTS: Gene expression of IL-1ß, TNF and caspase-3 increased in cpdm mice but was comparable to control values in cpdm/TNF KO mice. Decreased cortical and trabecular bone in cpdm mice translated to a loss in bone strength (ultimate stress and peak force). Cpdm/TNF KO mice developed bones similar to, or stronger than, control bones. CONCLUSIONS: Our results suggest that SHARPIN plays a significant role in skeletal homeostasis and that this role is strongly regulated through TNF pathways.

Aged females unilaterally hypersensitize, lack descending inhibition, and overexpress alpha1D adrenergic receptors in a murine posttraumatic chronic pain model.

ABSTRACT: Vulnerability to chronic pain is found to depend on age and sex. Most patients with chronic pain are elderly women, especially with posttraumatic pain after bone fracture that prevails beyond the usual recovery period and develops into a complex regional pain syndrome (CRPS). There, a distal bone fracture seems to initiate a pathophysiological process with unknown mechanism. To investigate whether sex, age, and alpha adrenergic receptors also contribute to a CRPS-like phenotype in animals, we performed experiments on tibia-fractured mice. Those mice commonly are resilient to the development of a CRPS-like phenotype. However, we found them to be vulnerable to long-lasting pain after distal bone fracture when they were of old age. These mice expressed mechanical and thermal hypersensitivity, as well as weight-bearing and autonomic impairment following bone trauma, which persisted over 3 months. Site-specific and body side-specific glycinergic and α1D-noradrenergic receptor expression in the spinal cord and the contralateral locus coeruleus were misbalanced. Aged female tibia-fractured mice lost descending noradrenergic inhibition and displayed enhanced spinal activity on peripheral pressure stimuli. Together, changes in the noradrenergic, hence, glycinergic system towards excitation in the pain pathway-ascending and descending-might contribute to the development or maintenance of long-lasting pain. Conclusively, changes in the noradrenergic system particularly occur in aged female mice after trauma and might contribute to the development of long-lasting pain. Our data support the hypothesis that some patients with chronic pain would benefit from lowering the adrenergic/sympathetic tone or antagonizing α1(D).

Catastrophic payment for assisted reproduction techniques with conventional ovarian stimulation in the public health sector of South Africa: frequency and coping strategies.

STUDY QUESTION: How often does out-of-pocket payment (OPP) for assisted reproduction techniques (ART) with conventional ovarian stimulation result in catastrophic expenditure for households? SUMMARY ANSWER: Catastrophic cost was a frequent event affecting 51% of the poorest study participants and one in five couples in total. WHAT IS KNOWN ALREADY: There is increasing concern about catastrophic spending on health by households in low resource settings, but to date no study has evaluated OPP for ART. STUDY DESIGN, SIZE, DURATION: We conducted a prospective observational study comprising 135 couples undergoing ART between March 2009 and June 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was set at an urban, level 3 referral hospital in the public and academic health sector of South Africa. At this institution ART is subsidized but requires co-payment by patients. Couples undergoing ART with conventional ovarian stimulation using GnRH analogs were recruited. A questionnaire capturing information on socioeconomic status, monthly household expenditure, OPP for the index ART cycle and financial coping strategies was administered. Households were categorized into tertiles according to socio-economic status. In addition to descriptive statistics, annualized OPP for ART services as a percentage of annual non-food household expenditure was calculated to estimate catastrophic health care expenditure. The Pearson χ(2) test and a logistic regression were used to identify factors related to incurring catastrophic spending. MAIN RESULTS AND THE ROLE OF CHANCE: In total, one in five couples (22%) incurred catastrophic expenditure (P < 0.01), defined as an OPP of ≥ 40% of annual non-food expenditure. Households used a range of coping strategies including reduced expenditure on items such as clothing and food, use of savings, borrowing money and taking on extra work. Differences were observed between the socio-economic tertiles: in the poorest tertile, 51% of households faced catastrophic costs compared with only 2% of the richest tertile (P < 0.01). Participants in the poorest tertile were more likely to be black (P < 0.01), and less likely to have health insurance (P < 0.01) or female full-time employment (P < 0.01). Longer duration of infertility was an additional risk factor for catastrophic payment (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: No attempt was made to obtain proof of any payment or expenditure, and all information collected relied on participants' verbal account. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to document the frequency of catastrophic expenditure for ART using conventional ovarian stimulation in a low resource setting. Our results show that not all couples unable to afford treatment forfeit infertility care; instead poor couples are willing to suffer catastrophic financial hardship in order to pay. ART counselling therefore needs to include financial risk counselling in the short term. Long-term interventions comprise cost-reducing strategies as well as health systems strategies that reduce or eliminate the need for OPP for ART wherever possible. Robust evidence on mild versus conventional stimulation for ART in low resource settings is also required in the form of local RCTs which address the many clinical and health economic variables and exclude bias. Our data cannot be extrapolated to patients undergoing ART elsewhere or to patients undergoing ART with mild ovarian stimulation. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Medical Research Council of South Africa and the University of Cape Town (University Research Committee and Faculty of Health Sciences Research Committee). The authors had no competing interests. TRIAL REGISTRATION NUMBER: not applicable.

Simultaneous Quantification of Spatial Genome Positioning and Transcriptomics in Single Cells with scDam&T-Seq.

Spatial genome organization is considered to play an important role in mammalian cells, by guiding gene expression programs and supporting lineage specification. Yet it is still an outstanding question in the field what the direct impact of spatial genome organization on gene expression is. To elucidate this relationship further, we have recently developed scDam&T-seq, a method that simultaneously quantifies protein-DNA interactions and transcriptomes in single cells. This method efficiently combines two preexisting methods: DamID for measuring protein-DNA contacts and CEL-Seq2 for quantification of the transcriptome in single cells. scDam&T-seq has been successfully applied to measure DNA contacts with the nuclear lamina, while at the same time revealing the effect of these contacts on gene expression. This method is applicable to many different proteins of interest and can thereby aid in studying the relationship between protein-DNA interactions and gene expression in single cells.

D1/D5 modulation of synaptic NMDA receptor currents.

Converging evidence suggests that salience-associated modulation of behavior is mediated by the release of monoamines and that monoaminergic activation of D(1)/D(5) receptors is required for normal hippocampal-dependent learning and memory. However, it is not understood how D(1)/D(5) modulation of hippocampal circuits can affect salience-associated learning and memory. We have observed in CA1 pyramidal neurons that D(1)/D(5) receptor activation elicits a bidirectional long-term plasticity of NMDA receptor-mediated synaptic currents with the polarity of plasticity determined by NMDA receptor, NR2A/B subunit composition. This plasticity results in a decrease in the NR2A/NR2B ratio of subunit composition. Synaptic responses mediated by NMDA receptors that include NR2B subunits are potentiated by D(1)/D(5) receptor activation, whereas responses mediated by NMDA receptors that include NR2A subunits are depressed. Furthermore, these bidirectional, subunit-specific effects are mediated by distinctive intracellular signaling mechanisms. Because there is a predominance of NMDA receptors composed of NR2A subunits observed in entorhinal-CA1 inputs and a predominance of NMDA receptors composed of NR2B subunits in CA3-CA1 synapses, potentiation of synaptic NMDA currents predominates in the proximal CA3-CA1 synapses, whereas depression of synaptic NMDA currents predominates in the distal entorhinal-CA1 synapses. Finally, all of these effects are reproduced by the release of endogenous monoamines through activation of D(1)/D(5) receptors. Thus, endogenous D(1)/D(5) activation can (1) decrease the NR2A/NR2B ratio of NMDA receptor subunit composition at glutamatergic synapses, a rejuvenation to a composition similar to developmentally immature synapses, and, (2) in CA1, bias NMDA receptor responsiveness toward the more highly processed trisynaptic CA3-CA1 circuit and away from the direct entorhinal-CA1 input.

Simultaneous quantification of protein-DNA interactions and transcriptomes in single cells with scDam&T-seq.

Protein-DNA interactions are essential for establishing cell type-specific chromatin architecture and gene expression. We recently developed scDam&T-seq, a multi-omics method that can simultaneously quantify protein-DNA interactions and the transcriptome in single cells. The method effectively combines two existing methods: DNA adenine methyltransferase identification (DamID) and CEL-Seq2. DamID works through the tethering of a protein of interest (POI) to the Escherichia coli DNA adenine methyltransferase (Dam). Upon expression of this fusion protein, DNA in proximity to the POI is methylated by Dam and can be selectively digested and amplified. CEL-Seq2, in contrast, makes use of poly-dT primers to reverse transcribe mRNA, followed by linear amplification through in vitro transcription. scDam&T-seq is the first technique capable of providing a combined readout of protein-DNA contact and transcription from single-cell samples. Once suitable cell lines have been established, the protocol can be completed in 5 d, with a throughput of hundreds to thousands of cells. The processing of raw sequencing data takes an additional 1-2 d. Our method can be used to understand the transcriptional changes a cell undergoes upon the DNA binding of a POI. It can be performed in any laboratory with access to FACS, robotic and high-throughput-sequencing facilities.

Structure of the MDM2/MDMX RING domain heterodimer reveals dimerization is required for their ubiquitylation in trans.

MDM2, a ubiquitin E3-ligase of the RING family, has a key role in regulating p53 abundance. During normal non-stress conditions p53 is targeted for degradation by MDM2. MDM2 can also target itself and MDMX for degradation. MDMX is closely related to MDM2 but the RING domain of MDMX does not possess intrinsic E3-ligase activity. Instead, MDMX regulates p53 abundance by modulating the levels and activity of MDM2. Dimerization, mediated by the conserved C-terminal RING domains of both MDM2 and MDMX, is critical to this activity. Here we report the crystal structure of the MDM2/MDMX RING domain heterodimer and map residues required for functional interaction with the E2 (UbcH5b). In both MDM2 and MDMX residues C-terminal to the RING domain have a key role in dimer formation. In addition we show that these residues are part of an extended surface that is essential for ubiquitylation in trans. This study provides a molecular basis for understanding how heterodimer formation leads to stabilization of MDM2, yet degradation of p53, and suggests novel targets for therapeutic intervention.

Blocking granule-mediated death by primary human NK cells requires both protection of mitochondria and inhibition of caspase activity.

Human GraB (hGraB) preferentially induces apoptosis via Bcl-2-regulated mitochondrial damage but can also directly cleave caspases and caspase substrates in cell-free systems. How hGraB kills cells when it is delivered by cytotoxic lymphocytes (CL) and the contribution of hGraB to CL-induced death is still not clear. We show that primary human natural killer (hNK) cells, which specifically used hGraB to induce target cell death, were able to induce apoptosis of cells whose mitochondria were protected by Bcl-2. Purified hGraB also induced apoptosis of Bcl-2-overexpressing targets but only when delivered at 5- to 10-fold the concentration required to kill cells expressing endogenous Bcl-2. Caspases were critical in this process as inhibition of caspase activity permitted clonogenic survival of Bcl-2-overexpressing cells treated with hGraB or hNK cells but did not protect cells that only expressed endogenous Bcl-2. Our data therefore show that hGraB triggers caspase activation via mitochondria-dependent and mitochondria-independent mechanisms that are activated in a hierarchical manner, and that the combined effects of Bcl-2 and direct caspase inhibition can block cell death induced by hGraB and primary hNK cells.

DIABLO promotes apoptosis by removing MIHA/XIAP from processed caspase 9.

MIHA is an inhibitor of apoptosis protein (IAP) that can inhibit cell death by direct interaction with caspases, the effector proteases of apoptosis. DIABLO is a mammalian protein that can bind to IAPs and antagonize their antiapoptotic effect, a function analogous to that of the proapoptotic Drosophila molecules, Grim, Reaper, and HID. Here, we show that after UV radiation, MIHA prevented apoptosis by inhibiting caspase 9 and caspase 3 activation. Unlike Bcl-2, MIHA functioned after release of cytochrome c and DIABLO from the mitochondria and was able to bind to both processed caspase 9 and processed caspase 3 to prevent feedback activation of their zymogen forms. Once released into the cytosol, DIABLO bound to MIHA and disrupted its association with processed caspase 9, thereby allowing caspase 9 to activate caspase 3, resulting in apoptosis.

Lamina Associated Domains and Gene Regulation in Development and Cancer.

The nuclear lamina (NL) is a thin meshwork of filaments that lines the inner nuclear membrane, thereby providing a platform for chromatin binding and supporting genome organization. Genomic regions contacting the NL are lamina associated domains (LADs), which contain thousands of genes that are lowly transcribed, and enriched for repressive histone modifications. LADs are dynamic structures that shift spatial positioning in accordance with cell-type specific gene expression changes during differentiation and development. Furthermore, recent studies have linked the disruption of LADs and alterations in the epigenome with the onset of diseases such as cancer. Here we focus on the role of LADs and the NL in gene regulation during development and cancer.

Identification of mammalian mitochondrial proteins that interact with IAPs via N-terminal IAP binding motifs.

Direct IAP binding protein with low pI/second mitochondrial activator of caspases, HtrA2/Omi and GstPT/eRF3 are mammalian proteins that bind via N-terminal inhibitor of apoptosis protein (IAP) binding motifs (IBMs) to the baculoviral IAP repeat (BIR) domains of IAPs. These interactions can prevent IAPs from inhibiting caspases, or displace active caspases, thereby promoting cell death. We have identified several additional potential IAP antagonists, including glutamate dehydrogenase (GdH), Nipsnap 3 and 4, CLPX, leucine-rich pentatricopeptide repeat motif-containing protein and 3-hydroxyisobutyrate dehydrogenase. All are mitochondrial proteins from which N-terminal import sequences are removed generating N-terminal IBMs. Whereas most of these proteins have alanine at the N-terminal position, as observed for previously described antagonists, GdH has an N-terminal serine residue that is essential for X-linked IAP (XIAP) interaction. These newly described IAP binding proteins interact with XIAP mainly via BIR2, with binding eliminated or significantly reduced by a single point mutation (D214S) within this domain. Through this interaction, many are able to antagonise XIAP inhibition of caspase 3 in vitro.

Postmatch recovery of physical performance and biochemical markers in team ball sports: a systematic review.

BACKGROUND: Insufficient postmatch recovery in elite players may cause an increased risk of injuries, illnesses and non-functional over-reaching. OBJECTIVE: To evaluate postmatch recovery time courses of physical performance and biochemical markers in team ball sport players. STUDY DESIGN: Systematic review. DATA SOURCES: PubMed and Web of Science. ELIGIBILITY CRITERIA FOR SELECTING STUDIES: This systematic review was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The Critical Review Form for Quantitative Studies was used to evaluate quality. Studies were included if they met the following criteria: (1) original research evaluated players' physical recovery postmatch; (2) team/intermittent sports; and (3) at least two postmeasurements were compared with baseline values. RESULTS: Twenty-eight studies were eligible. Mean methodological quality was 11.2±1.11. Most used performance tests and biochemical markers were the countermovement jump test, sprint tests and creatine kinase (CK), cortisol (C) and testosterone (T), respectively. SUMMARY/CONCLUSIONS: The current evidence demonstrates that underlying mechanisms of muscle recovery are still in progress while performance recovery is already reached. CK recovery time courses are up to ≥72 hours. Soccer and rugby players need more time to recover for sprint performance, CK and C in comparison to other team ball sports. There are more high-quality studies needed regarding recovery in various team sports and recovery strategies on an individual level should be evaluated. CLINICAL RELEVANCE: Ongoing insufficient recovery can be prevented by the use of the presented recovery time courses as specific practical recovery guidelines.