The spatiotemporal structure of the human microbiome1,2, proteome3 and metabolome4,5 reflects and determines regional intestinal physiology and may have implications for disease6. Yet, little is known about the distribution of microorganisms, their environment and their biochemical activity in the gut because of reliance on stool samples and limited access to only some regions of the gut using endoscopy in fasting or sedated individuals7. To address these deficiencies, we developed an ingestible device that collects samples from multiple regions of the human intestinal tract during normal digestion. Collection of 240 intestinal samples from 15 healthy individuals using the device and subsequent multi-omics analyses identified significant differences between bacteria, phages, host proteins and metabolites in the intestines versus stool. Certain microbial taxa were differentially enriched and prophage induction was more prevalent in the intestines than in stool. The host proteome and bile acid profiles varied along the intestines and were highly distinct from those of stool. Correlations between gradients in bile acid concentrations and microbial abundance predicted species that altered the bile acid pool through deconjugation. Furthermore, microbially conjugated bile acid concentrations exhibited amino acid-dependent trends that were not apparent in stool. Overall, non-invasive, longitudinal profiling of microorganisms, proteins and bile acids along the intestinal tract under physiological conditions can help elucidate the roles of the gut microbiome and metabolome in human physiology and disease.

Regulation of replication and expression of mitochondrial DNA (mt DNA ) is essential for cellular energy conversion via oxidative phosphorylation. The mitochondrial transcription elongation factor ( TEFM ) has been proposed to regulate the switch between transcription termination for replication primer formation and processive, near genome‐length transcription for mt DNA gene expression. Here, we report that Tefm is essential for mouse embryogenesis and that levels of promoter‐distal mitochondrial transcripts are drastically reduced in conditional Tefm ‐knockout hearts. In contrast, the promoter‐proximal transcripts are much increased in Tefm knockout mice, but they mostly terminate before the region where the switch from transcription to replication occurs, and consequently, de novo mt DNA replication is profoundly reduced. Unexpectedly, deep sequencing of RNA from Tefm knockouts revealed accumulation of unprocessed transcripts in addition to defective transcription elongation. Furthermore, a proximity‐labeling (Bio ID ) assay showed that TEFM interacts with multiple RNA processing factors. Our data demonstrate that TEFM acts as a general transcription elongation factor, necessary for both gene transcription and replication primer formation, and loss of TEFM affects RNA processing in mammalian mitochondria.

Pathogenic variants in FBXL 4 cause a severe encephalopathic syndrome associated with mt DNA depletion and deficient oxidative phosphorylation. To gain further insight into the enigmatic pathophysiology caused by FBXL 4 deficiency, we generated homozygous Fbxl4 knockout mice and found that they display a predominant perinatal lethality. Surprisingly, the few surviving animals are apparently normal until the age of 8–12 months when they gradually develop signs of mitochondrial dysfunction and weight loss. One‐year‐old Fbxl4 knockouts show a global reduction in a variety of mitochondrial proteins and mt DNA depletion, whereas lysosomal proteins are upregulated. Fibroblasts from patients with FBXL 4 deficiency and human FBXL 4 knockout cells also have reduced steady‐state levels of mitochondrial proteins that can be attributed to increased mitochondrial turnover. Inhibition of lysosomal function in these cells reverses the mitochondrial phenotype, whereas proteasomal inhibition has no effect. Taken together, the results we present here show that FBXL 4 prevents mitochondrial removal via autophagy and that loss of FBXL 4 leads to decreased mitochondrial content and mitochondrial disease.

OBJECTIVE: The aim of this in-vitro study was to compare the tolerance of surgical instruments in surgical guides produced by 3-D printing, without metal sleeves to a surgical guide with conventional metal sleeves from two different manufacturers. MATERIALS AND METHODS: Lateral movements of drill tips caused by tolerance between the sleeve and drill key and between the drill key and the drill were recorded after application of a standardized force to the surgical instruments. Four groups were tested: Control 1 (C1): metal sleeve from commercially available surgical system 1; Test 1 (T1): 3-D-printed sleeve for surgical system 1; Control 2 (C2): metal sleeve from commercially available surgical system 2. Test 2 (T2): 3-D-printed sleeve for surgical system 2. RESULTS: The mean total lateral movement was 0.75 mm (0.5-1.04 mm) in the C1 group and 0.91 mm (0.54-1.34 mm) in the C2 group. The mean amount of movement from tolerance between sleeve and drill-guiding key was 0.31 mm (range 0.22-0.41 mm) in C1 and 0.42 mm (range 0.29-0.56 mm) in C2. This lateral movement was in mean reduced by 0.24 mm (32%) in T1 and by 0.39 mm (43%) in T2 group. This reduction was statistically significant in both groups (P < 0.001). CONCLUSION: The tolerance of surgical instruments and the lateral movements of the drills were significantly reduced by the use of 3-D printing with reduced sleeve diameter. This reduction could improve the overall accuracy in computer-assisted template-guided implant dentistry. The lateral movement of the drill can be further reduced by using a shorter drill and a higher drill key. This can be considered during implant planning and CAD/CAM of surgical guides. AbstractObjective: The aim of this in-vitro study was to compare the tolerance of surgical instruments in surgical guides produced by 3D printing, without metal sleeves to a surgical guide with conventional metal sleeves from two different manufacturers. Materials and methods:Lateral movements of drill tips caused by tolerance between the sleeve and drill key and between the drill key and the drill were recorded after application of a standardized force to the surgical instruments. Four groups were tested: Control 1 (C1):Metal sleeve from commercially available surgical system 1; Test 1 (T1): 3D-printed sleeve for surgical system 1; Control 2 (C2): Metal sleeve from commercially available surgical system 2. Test 2 (T2): 3D-printed sleeve for surgical system 2. Results:The mean total lateral movement was 0.75mm (0.5 to 1.04mm) in the C1 group and 0.91mm (0.54 to 1.34mm) in the C2 group. The mean amount of movement from tolerance between sleeve and drill-guiding key was 0.31mm (range 0.22 to 0.41mm) in C1 and 0.42mm (range 0.29 to 0.56mm) in C2. This lateral movement was in mean reduced by 0.24mm (32%) in T1 and by 0.39mm (43%) in T2 group. This reduction was statistically significant in both groups (p<0.001). Conclusion:The tolerance of surgical instruments and the lateral movements of the drills were significantly reduced by the use of 3D prin...

Summary Neurodegenerative disorders are an increasingly common and irreversible burden on society, often affecting the aging population, but their etiology and disease mechanisms are poorly understood. Studying monogenic neurodegenerative diseases with known genetic cause provides an opportunity to understand cellular mechanisms also affected in more complex disorders. We recently reported that loss-of-function mutations in the autophagy adaptor protein SQSTM1/p62 lead to a slowly progressive neurodegenerative disease presenting in childhood. To further elucidate the neuronal involvement, we studied the cellular consequences of loss of p62 in a neuroepithelial stem cell (NESC) model and differentiated neurons derived from reprogrammed p62 patient cells or by CRISPR/Cas9-directed gene editing in NESCs. Transcriptomic and proteomic analyses suggest that p62 is essential for neuronal differentiation by controlling the metabolic shift from aerobic glycolysis to oxidative phosphorylation required for neuronal maturation. This shift is blocked by the failure to sufficiently downregulate lactate dehydrogenase expression due to the loss of p62, possibly through impaired Hif-1α downregulation and increased sensitivity to oxidative stress. The findings imply an important role for p62 in neuronal energy metabolism and particularly in the regulation of the shift between glycolysis and oxidative phosphorylation required for normal neurodifferentiation.

The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo , we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology.

Single-cell proteomics aims to characterize biological function and heterogeneity at the level of proteins in an unbiased manner. It is currently limited in proteomic depth, throughput and robustness, a challenge that we address here by a streamlined multiplexed workflow using data-independent acquisition (mDIA). We demonstrate automated and complete dimethyl labeling of bulk or single-cell samples, without losing proteomic depth. In single runs of mammalian cells, a three-plex analysis of tryptic peptides quantified 7,700 proteins per channel. The Lys-N enzyme enables five-plex quantification at MS1 and MS2 level. Because the multiplex channels are quantitatively isolated from each other, mDIA accommodates a reference channel that does not interfere with the target channels. Our algorithm RefQuant takes advantage of this feature and confidently quantifies close to 4,000 proteins in single cells with excellent reproducibility, while our workflow currently allows routine analysis of 80 single cells per day. The concept of stable proteome vs. stochastic transcriptome still holds at this deeper proteome coverage.

Induction of the one-carbon cycle is an early hallmark of mitochondrial dysfunction and cancer metabolism. Vital intermediary steps are localized to mitochondria, but it remains unclear how one-carbon availability connects to mitochondrial function. Here, we show that the one-carbon metabolite and methyl group donor S-adenosylmethionine (SAM) is pivotal for energy metabolism. A gradual decline in mitochondrial SAM (mitoSAM) causes hierarchical defects in fly and mouse, comprising loss of mitoSAM-dependent metabolites and impaired assembly of the oxidative phosphorylation system. Complex I stability and iron-sulfur cluster biosynthesis are directly controlled by mitoSAM levels, while other protein targets are predominantly methylated outside of the organelle before import. The mitoSAM pool follows its cytosolic production, establishing mitochondria as responsive receivers of one-carbon units. Thus, we demonstrate that cellular methylation potential is required for energy metabolism, with direct relevance for pathophysiology, aging, and cancer.