Combination of common mtDNA variants results in mitochondrial dysfunction and a connective tissue dysregulation.

Mitochondrial dysfunction can be associated with a range of clinical manifestations. Here, we report a family with a complex phenotype including combinations of connective tissue, neurological, and metabolic symptoms that were passed on to all surviving children. Analysis of the maternally inherited mtDNA revealed a novel genotype encompassing the haplogroup J - defining mitochondrial DNA (mtDNA) ND5 m.13708G>A (A458T) variant arising on the mtDNA haplogroup H7A background, an extremely rare combination. Analysis of transmitochondrial cybrids with the 13708A-H7 mtDNA revealed a lower mitochondrial respiration, increased reactive oxygen species production (mROS), and dysregulation of connective tissue gene expression. The mitochondrial dysfunction was exacerbated by histamine, explaining why all eight surviving children inherited the dysfunctional histidine decarboxylase allele (W327X) from the father. Thus, certain combinations of common mtDNA variants can cause mitochondrial dysfunction, mitochondrial dysfunction can affect extracellular matrix gene expression, and histamine-activated mROS production can augment the severity of mitochondrial dysfunction. Most important, we have identified a previously unreported genetic cause of mitochondrial disorder arising from the incompatibility of common, nonpathogenic mtDNA variants.

Permethylation of Ribonucleosides Provides Enhanced Mass Spectrometry Quantification of Post-Transcriptional RNA Modifications.

Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, and degradation, as well as human disease states, such as cancer. However, the analysis of ribonucleoside modifications is hampered by the hydrophilicity of the ribonucleoside molecules. In this work, we used solid-phase permethylation to first efficiently derivatize the ribonucleosides and quantitatively analyze them by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. We identified and quantified more than 60 RNA modifications simultaneously by ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. The increased hydrophobicity of permethylated ribonucleosides significantly enhanced their retention, separation, and ionization efficiency, leading to improved detection and quantification. We further demonstrate that this novel approach is capable of quantifying cytosine methylation and hydroxymethylation in complex RNA samples obtained from mouse embryonic stem cells with genetic deficiencies in the ten-eleven translocation (TET) enzymes. The results match previously performed analyses and highlight the improved sensitivity, efficacy, and robustness of the new method. Our protocol is quantitative and robust and thus provides an augmented approach for comprehensive analysis of RNA modifications in biological samples.

One minute analysis of 200 histone posttranslational modifications by direct injection mass spectrometry.

DNA and histone proteins define the structure and composition of chromatin. Histone posttranslational modifications (PTMs) are covalent chemical groups capable of modeling chromatin accessibility, mostly due to their ability in recruiting enzymes responsible for DNA readout and remodeling. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein PTMs, including histones. High sensitivity proteomics requires online MS coupling with relatively low throughput and poorly robust nano-liquid chromatography (nanoLC) and, for histone proteins, a 2-d sample preparation that includes histone purification, derivatization, and digestion. We present a new protocol that achieves quantitative data on about 200 histone PTMs from tissue or cell lines in 7 h from start to finish. This protocol includes 4 h of histone extraction, 3 h of derivatization and digestion, and only 1 min of MS analysis via direct injection (DI-MS). We demonstrate that this sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates. We also engineered the sequence of a synthetic "histone-like" peptide to spike into the sample, of which derivatization and digestion benchmarks the quality of the sample preparation. We ensure that DI-MS does not introduce biases in histone peptide ionization as compared to nanoLC-MS/MS by producing and analyzing a library of synthetically modified histone peptides mixed in equal molarity. Finally, we introduce EpiProfileLite for comprehensive analysis of this new data type. Altogether, our workflow is suitable for high-throughput screening of >1000 samples per day using a single mass spectrometer.

Determinants associated with viable genital or rectal Chlamydia trachomatis bacterial load (FemCure).

BACKGROUND: Chlamydia trachomatis (CT) is routinely diagnosed by nucleic acid amplification tests (NAATs), which are unable to distinguish between nucleic acids from viable and non-viable CT organisms. OBJECTIVES: We applied our recently developed sensitive PCR (viability PCR) technique to measure viable bacterial CT load and explore associated determinants in 524 women attending Dutch sexual health centres (STI clinics), and who had genital or rectal CT. METHODS: We included women participating in the FemCure study (Netherlands, 2016-2017). At the enrolment visit (pre-treatment), 524 were NAAT positive (n=411 had genital and rectal CT, n=88 had genital CT only and n=25 had rectal CT only). We assessed viable rectal and viable genital load using V-PCR. We presented mean load (range 0 (non-viable) to 6.5 log10 CT/mL) and explored potential associations with urogenital symptoms (coital lower abdominal pain, coital blood loss, intermenstrual bleeding, altered vaginal discharge, painful or frequent micturition), rectal symptoms (discharge, pain, blood loss), other anatomical site infection and sociodemographics using multivariable regression analyses. RESULTS: In genital (n=499) CT NAAT-positive women, the mean viable load was 3.5 log10 CT/mL (SD 1.6). Genital viable load was independently associated with urogenital symptoms-especially altered vaginal discharge (Beta=0.35, p=0.012) and with concurrent rectal CT (aBeta=1.79; p<0.001). Urogenital symptoms were reported by 50.3% of women; their mean genital viable load was 3.6 log10 CT/mL (vs 3.3 in women without symptoms). Of 436 rectal CT NAAT-positive women, the mean rectal viable load was 2.2 log10 CT/mL (SD 2.0); rectal symptoms were reported by 2.5% (n=11) and not associated with rectal viable load. CONCLUSION: Among women diagnosed with CT in an outpatient clinical setting, viable genital CT load was higher in those reporting urogenital symptoms, but the difference was small. Viable genital load was substantially higher when women also had a concurrent rectal CT. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT02694497.

Regulation of nuclear epigenome by mitochondrial DNA heteroplasmy.

Diseases associated with mitochondrial DNA (mtDNA) mutations are highly variable in phenotype, in large part because of differences in the percentage of normal and mutant mtDNAs (heteroplasmy) present within the cell. For example, increasing heteroplasmy levels of the mtDNA tRNALeu(UUR) nucleotide (nt) 3243A > G mutation result successively in diabetes, neuromuscular degenerative disease, and perinatal lethality. These phenotypes are associated with differences in mitochondrial function and nuclear DNA (nDNA) gene expression, which are recapitulated in cybrid cell lines with different percentages of m.3243G mutant mtDNAs. Using metabolic tracing, histone mass spectrometry, and NADH fluorescence lifetime imaging microscopy in these cells, we now show that increasing levels of this single mtDNA mutation cause profound changes in the nuclear epigenome. At high heteroplasmy, mitochondrially derived acetyl-CoA levels decrease causing decreased histone H4 acetylation, with glutamine-derived acetyl-CoA compensating when glucose-derived acetyl-CoA is limiting. In contrast, α-ketoglutarate levels increase at midlevel heteroplasmy and are inversely correlated with histone H3 methylation. Inhibition of mitochondrial protein synthesis induces acetylation and methylation changes, and restoration of mitochondrial function reverses these effects. mtDNA heteroplasmy also affects mitochondrial NAD+/NADH ratio, which correlates with nuclear histone acetylation, whereas nuclear NAD+/NADH ratio correlates with changes in nDNA and mtDNA transcription. Thus, mutations in the mtDNA cause distinct metabolic and epigenomic changes at different heteroplasmy levels, potentially explaining transcriptional and phenotypic variability of mitochondrial disease.

Evidence for Local Spots of Viscous Electron Flow in Graphene at Moderate Mobility.

Dominating electron-electron scattering enables viscous electron flow exhibiting hydrodynamic current density patterns, such as Poiseuille profiles or vortices. The viscous regime has recently been observed in graphene by nonlocal transport experiments and mapping of the Poiseuille profile. Herein, we probe the current-induced surface potential maps of graphene field-effect transistors with moderate mobility using scanning probe microscopy at room temperature. We discover micrometer-sized large areas appearing close to charge neutrality that show current-induced electric fields opposing the externally applied field. By estimating the local scattering lengths from the gate dependence of local in-plane electric fields, we find that electron-electron scattering dominates in these areas as expected for viscous flow. Moreover, we suppress the inverted fields by artificially decreasing the electron-disorder scattering length via mild ion bombardment. These results imply that viscous electron flow is omnipresent in graphene devices, even at moderate mobility.

Assessment of rectal Chlamydia trachomatis viable load in women by viability-PCR.

OBJECTIVES: In recent years, studies have demonstrated frequent rectal Chlamydia trachomatis (CT) detection in women, irrespective of reported anal sex or rectal symptoms. However, the clinical relevance and public health implication of rectal CT detection in women remain under debate. Therefore, evaluating CT viability may provide more insight into the relevance of standard routine nucleic acid amplification test (NAAT)-positive results. METHODS: In this cross-sectional explorative study, a convenience sample of female patients at our STI clinic aged 18 years or older, diagnosed with vaginal and/or rectal CT, were invited to participate. On return for treatment, rectal CT-diagnosed women were instructed to self-collect rectal swab samples before being treated. Standard COBAS 4800 CT/NG routine NAAT testing was applied for CT diagnosis. Rectal viable CT load was evaluated by using viability-PCR (V-PCR). RESULTS: 53 women with rectal CT were included in this study; 86.8% (46/53) had a quantifiable rectal total CT load. Of women with quantifiable samples, 52.2% (24/46) had viable CT detected from rectal swabs by V-PCR, with a mean rectal viable CT load of 3.31 log10 CT/mL (range 1.16-6.22). No statistically significant difference (p=0.73) was observed in the mean rectal viable CT load of women with an indication for rectal testing (n=9) and without (n=15), 3.20 log10 CT/mL (range 2.06-4.36) and 3.38 log10 CT/mL (range 1.16-6.22), respectively. CT culture yielded positive test results from rectal swabs in 22.6% (12/53) of rectal CT NAAT-diagnosed women. Of women with viable rectal CT by V-PCR (n=24), 50% (12/24) were positive by CT culture. CONCLUSIONS: Overall, the detection of high rectal viable CT loads in this study indicates that rectal CT in some women might represent a currently ongoing infection rather than just the presence of remnant DNA from dead bacteria or only contamination from an active vaginal CT infection.

Spontaneous clearance of Chlamydia trachomatis accounting for bacterial viability in vaginally or rectally infected women (FemCure).

OBJECTIVES: Spontaneous clearance of Chlamydia trachomatis (CT) infections can occur between diagnosis and treatment. We followed CT patients to assess clearance using a conventional definition (no total CT-DNA, assessed by routine quantitative PCR methods) and a definition accounting for viability, assessed by viability PCR testing. METHODS: Three outpatient STI clinics included CT-diagnosed women (The Netherlands, 2016-2017, FemCure study); participants had vaginal CT (vCT) and rectal CT (rCT) (group A: n=155), vCT and were rectally untested (group B: n=351), single vCT (group C: n=25) or single rCT (group D: n=29). Follow-up (median interval 9 days) vaginal and rectal samples underwent quantitative PCR testing (detecting total CT-DNA). When PCR positive, samples underwent V-PCR testing to detect 'viable CT' (CT-DNA from intact CT organisms; V-PCR positive). 'Clearance' was the proportion PCR-negative patients and 'clearance of viable CT' was the proportion of patients testing PCR negative or PCR positive but V-PCR negative. We used multivariable logistic regression analyses to assess diagnosis group (A-D), age, days since initial CT test (diagnosis) and study site (STI clinic) in relation to clearance and clearance of viable CT. RESULTS: Clearance and clearance of viable CT at both anatomic sites were for (A) 0.6% and 3.9%; (B) 5.4% and 9.4%; (C) 32.0% and 52.0% and (D) 27.6% and 41.4%, respectively. In multivariate analyses, women with single infections (groups C and D) had higher likelihood of clearance than women concurrently infected with vCT and rCT (p<0.001).Of rectally untested women (group B), 76.9% had total CT-DNA and 46.7% had viable CT (V-PCR positive) at the rectal site. CONCLUSIONS: Of untreated female vCT patients who had CT also at the rectal site, or who were rectally untested, only a small proportion cleared CT (in fact many had viable CT) at their follow-up visit (median 9 days). Among single site infected women clearance was much higher. TRIAL REGISTRATION NUMBER: NCT02694497.

EpiProfile 2.0: A Computational Platform for Processing Epi-Proteomics Mass Spectrometry Data.

Epigenetics has become a fundamental scientific discipline with various implications for biology and medicine. Epigenetic marks, mostly DNA methylation and histone post-translational modifications (PTMs), play important roles in chromatin structure and function. Accurate quantification of these marks is an ongoing challenge due to the variety of modifications and their wide dynamic range of abundance. Here we present EpiProfile 2.0, an extended version of our 2015 software (v1.0), for accurate quantification of histone peptides based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. EpiProfile 2.0 is now optimized for data-independent acquisition through the use of precursor and fragment extracted ion chromatography to accurately determine the chromatographic profile and to discriminate isobaric forms of peptides. The software uses an intelligent retention time prediction trained on the analyzed samples to enable accurate peak detection. EpiProfile 2.0 supports label-free and isotopic labeling, different organisms, known sequence mutations in diseases, different derivatization strategies, and unusual PTMs (such as acyl-derived modifications). In summary, EpiProfile 2.0 is a universal and accurate platform for the quantification of histone marks via LC-MS/MS. Being the first software of its kind, we anticipate that EpiProfile 2.0 will play a fundamental role in epigenetic studies relevant to biology and translational medicine. EpiProfile is freely available at https://github.com/zfyuan/EpiProfile2.0_Family .

Design of the FemCure study: prospective multicentre study on the transmission of genital and extra-genital Chlamydia trachomatis infections in women receiving routine care.

BACKGROUND: In women, anorectal infections with Chlamydia trachomatis (CT) are about as common as genital CT, yet the anorectal site remains largely untested in routine care. Anorectal CT frequently co-occurs with genital CT and may thus often be treated co-incidentally. Nevertheless, post-treatment detection of CT at both anatomic sites has been demonstrated. It is unknown whether anorectal CT may play a role in post-treatment transmission. This study, called FemCure, in women who receive routine treatment (either azithromycin or doxycycline) aims to understand the post-treatment transmission of anorectal CT infections, i.e., from their male sexual partner(s) and from and to the genital region of the same woman. The secondary objective is to evaluate other reasons for CT detection by nucleic acid amplification techniques (NAAT) such as treatment failure, in order to inform guidelines to optimize CT control. METHODS: A multicentre prospective cohort study (FemCure) is set up in which genital and/or anorectal CT positive women (n = 400) will be recruited at three large Dutch STI clinics located in South Limburg, Amsterdam and Rotterdam. The women self-collect anorectal and vaginal swabs before treatment, and at the end of weeks 1, 2, 4, 6, 8, 10, and 12. Samples are tested for presence of CT-DNA (by NAAT), load (by quantitative polymerase chain reaction -PCR), viability (by culture and viability PCR) and CT type (by multilocus sequence typing). Sexual exposure is assessed by online self-administered questionnaires and by testing samples for Y chromosomal DNA. Using logistic regression models, the impact of two key factors (i.e., sexual exposure and alternate anatomic site of infection) on detection of anorectal and genital CT will be assessed. DISCUSSION: The FemCure study will provide insight in the role of anorectal chlamydia infection in maintaining the CT burden in the context of treatment, and it will provide practical recommendations to reduce avoidable transmission. Implications will improve care strategies that take account of anorectal CT. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02694497 .

From whence it came: Mitochondrial mRNA leaves, a protein returns.

Small peptides have previously been reported to be encoded in mitochondrial rRNA and translated by cytosolic ribosomes. In this issue of Cell Metabolism, Hu et al. use mass spectrometry to identify a cytosolically translated protein, encoded instead in mitochondrial mRNA, that is surprisingly targeted back into the mitochondrial matrix.

A mitochondrial surveillance mechanism activated by SRSF2 mutations in hematologic malignancies.

Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2P95H/+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2P95H-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2P95H/+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.

A mitochondrial surveillance mechanism activated by SRSF2 mutations in hematologic malignancies.

Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2P95H/+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2P95H-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2P95H/+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.

Data-Independent Acquisition for the Detection of Mononucleoside RNA Modifications by Mass Spectrometry.

RNA is dynamically modified in cells by a plethora of chemical moieties to modulate molecular functions and processes. Over 140 modifications have been identified across species and RNA types, with the highest density and diversity of modifications found in tRNA (tRNA). The methods used to identify and quantify these modifications have developed over recent years and continue to advance, primarily in the fields of next-generation sequencing (NGS) and mass spectrometry (MS). Most current NGS methods are limited to antibody-recognized or chemically derivatized modifications and have limitations in identifying multiple modifications simultaneously. Mass spectrometry can overcome both of these issues, accurately identifying a large number of modifications in a single run. Here, we present advances in MS data acquisition for the purpose of RNA modification identification and quantitation. Using this approach, we identified multiple tRNA wobble position modifications in Arabidopsis thaliana that are upregulated in salt-stressed growth conditions and may stabilize translation of salt stress induced proteins. This work presents improvements in methods for studying RNA modifications and introduces a possible regulatory role of wobble position modifications in A. thaliana translation.

Combinatorial Histone H3 Modifications Are Dynamically Altered in Distinct Cell Cycle Phases.

The cell cycle is a highly regulated and evolutionary conserved process that results in the duplication of cell content and the equal distribution of the duplicated chromosomes into a pair of daughter cells. Histones are fundamental structural components of chromatin in eukaryotic cells, and their post-translational modifications (PTMs) benchmark DNA readout and chromosome condensation. Aberrant regulation of the cell cycle associated with dysregulation of histone PTMs is the cause of critical diseases such as cancer. Monitoring changes of histone PTMs could pave the way to understanding the molecular mechanisms associated with epigenetic regulation of cell proliferation. Previously, our lab established a novel middle-down workflow using porous graphitic carbon (PGC) as a stationary phase to analyze histone PTMs, which utilizes the same reversed-phase chromatography for gradient separation as canonical proteomics coupled with online mass spectrometry (MS). Here, we applied this novel workflow for high-throughput analysis of histone modifications of H3.1 and H3.2 during the cell cycle. Collectively, we identified 1133 uniquely modified canonical histone H3 N-terminal tails. Consistent with previous findings, histone H3 phosphorylation increased significantly during the mitosis (M) phase. Histone H3 variant-specific and cell-cycle-dependent expressions of PTMs were observed, underlining the need to not combine H3.1 and H3.2 together as H3. We confirmed previously known H3 PTM crosstalk (e.g., K9me-S10ph) and revealed new information in this area as well. These findings imply that the combinatorial PTMs play a role in cell cycle control, and they may serve as markers for proliferation.

Dynamic changes in RNA-protein interactions and RNA secondary structure in mammalian erythropoiesis.

Two features of eukaryotic RNA molecules that regulate their post-transcriptional fates are RNA secondary structure and RNA-binding protein (RBP) interaction sites. However, a comprehensive global overview of the dynamic nature of these sequence features during erythropoiesis has never been obtained. Here, we use our ribonuclease-mediated structure and RBP-binding site mapping approach to reveal the global landscape of RNA secondary structure and RBP-RNA interaction sites and the dynamics of these features during this important developmental process. We identify dynamic patterns of RNA secondary structure and RBP binding throughout the process and determine a set of corresponding protein-bound sequence motifs along with their dynamic structural and RBP-binding contexts. Finally, using these dynamically bound sequences, we identify a number of RBPs that have known and putative key functions in post-transcriptional regulation during mammalian erythropoiesis. In total, this global analysis reveals new post-transcriptional regulators of mammalian blood cell development.

TET2 chemically modifies tRNAs and regulates tRNA fragment levels.

The ten-eleven translocation 2 (TET2) protein, which oxidizes 5-methylcytosine in DNA, can also bind RNA; however, the targets and function of TET2-RNA interactions in vivo are not fully understood. Using stringent affinity tags introduced at the Tet2 locus, we purified and sequenced TET2-crosslinked RNAs from mouse embryonic stem cells (mESCs) and found a high enrichment for tRNAs. RNA immunoprecipitation with an antibody against 5-hydroxymethylcytosine (hm5C) recovered tRNAs that overlapped with those bound to TET2 in cells. Mass spectrometry (MS) analyses revealed that TET2 is necessary and sufficient for the deposition of the hm5C modification on tRNA. Tet2 knockout in mESCs affected the levels of several small noncoding RNAs originating from TET2-bound tRNAs that were enriched by hm5C immunoprecipitation. Thus, our results suggest a new function of TET2 in promoting the conversion of 5-methylcytosine to hm5C on tRNA and regulating the processing or stability of different classes of tRNA fragments.

N6-methyladenosine and RNA secondary structure affect transcript stability and protein abundance during systemic salt stress in Arabidopsis.

After transcription, a messenger RNA (mRNA) is further post-transcriptionally regulated by several features including RNA secondary structure and covalent RNA modifications (specifically N6-methyladenosine, m6A). Both RNA secondary structure and m6A have been demonstrated to regulate mRNA stability and translation and have been independently linked to plant responses to soil salinity levels. However, the effect of m6A on regulating RNA secondary structure and the combinatorial interplay between these two RNA features during salt stress response has yet to be studied. Here, we globally identify RNA-protein interactions and RNA secondary structure during systemic salt stress. This analysis reveals that RNA secondary structure changes significantly during salt stress, and that it is independent of global changes in RNA-protein interactions. Conversely, we find that m6A is anti-correlated with RNA secondary structure in a condition-dependent manner, with salt-specific m6A correlated with a decrease in mRNA secondary structure during salt stress. Taken together, we suggest that salt-specific m6A deposition and the associated loss of RNA secondary structure results in increases in mRNA stability for transcripts encoding abiotic stress response proteins and ultimately increases in protein levels from these stabilized transcripts. In total, our comprehensive analyses reveal important post-transcriptional regulatory mechanisms involved in plant long-term salt stress response and adaptation.