Electron tomography reveals Rab6 is essential to the trafficking of trans-Golgi clathrin and COPI-coated vesicles and the maintenance of Golgi cisternal number.

We have shown previously that Rab6, a small, trans-Golgi-localized GTPase, acts upstream of the conserved oligomeric Golgi complex (COG) and ZW10/RINT1 retrograde tether complexes to maintain Golgi homeostasis. In this article, we present evidence from the unbiased and high-resolution approach of electron microscopy and electron tomography that Rab6 is essential to the trans-Golgi trafficking of two morphological classes of coated vesicles; the larger corresponds to clathrin-coated vesicles and the smaller to coat protein I (COPI)-coated vesicles. On the basis of the site of coated vesicle accumulation, cisternal dilation and the normal kinetics of cargo transport from the endoplasmic reticulum (ER) to Golgi followed by delayed Golgi to cell surface transport, we suggest that Golgi function in cargo transport is preferentially inhibited at the trans-Golgi/trans-Golgi network (TGN). The >50% increase in Golgi cisternae number in Rab6-depleted HeLa cells that we observed may well be coupled to the trans-Golgi accumulation of COPI-coated vesicles; depletion of the individual Rab6 effector, myosin IIA, produced an accumulation of uncoated vesicles with if anything a decrease in cisternal number. These results are the first evidence for a Rab6-dependent protein machine affecting Golgi-proximal, coated vesicle accumulation and probably transport at the trans-Golgi and the first example of concomitant cisternal proliferation and increased Golgi stack organization under inhibited transport conditions.

Delineating genotypes and phenotypes of individual cells from long-read single cell transcriptomes.

Single-cell nanopore sequencing of full-length mRNAs (scNanoRNAseq) is transforming singlecell multi-omics studies. However, challenges include computational complexity and dependence on short-read curation. To address this, we developed a comprehensive toolkit, scNanoGPS to calculate same-cell genotypes-phenotypes without short-read guidance. We applied scNanoGPS onto 23,587 long-read transcriptomes from 4 tumors and 2 cell lines. Standalone, scNanoGPS accurately deconvoluted error-prone long-reads into single-cells and single-molecules. Further, scNanoGPS simultaneously accessed both phenotypes (expressions/isoforms) and genotypes (mutations) of individual cells. Our analyses revealed that tumor and stroma/immune cells often expressed significantly distinct combinations of isoforms (DCIs). In a kidney tumor, we identified 924 genes with DCIs involved in cell-type-specific functions such as PDE10A in tumor cells and CCL3 in lymphocytes. Moreover, transcriptome-wide mutation analyses identified many cell-type-specific mutations including VEGFA mutations in tumor cells and HLA-A mutations in immune cells, highlighting critical roles of different populations in tumors. Together, scNanoGPS facilitates applications of single-cell long-read sequencing.

High throughput single cell long-read sequencing analyses of same-cell genotypes and phenotypes in human tumors.

Single-cell nanopore sequencing of full-length mRNAs transforms single-cell multi-omics studies. However, challenges include high sequencing errors and dependence on short-reads and/or barcode whitelists. To address these, we develop scNanoGPS to calculate same-cell genotypes (mutations) and phenotypes (gene/isoform expressions) without short-read nor whitelist guidance. We apply scNanoGPS onto 23,587 long-read transcriptomes from 4 tumors and 2 cell-lines. Standalone, scNanoGPS deconvolutes error-prone long-reads into single-cells and single-molecules, and simultaneously accesses both phenotypes and genotypes of individual cells. Our analyses reveal that tumor and stroma/immune cells express distinct combination of isoforms (DCIs). In a kidney tumor, we identify 924 DCI genes involved in cell-type-specific functions such as PDE10A in tumor cells and CCL3 in lymphocytes. Transcriptome-wide mutation analyses identify many cell-type-specific mutations including VEGFA mutations in tumor cells and HLA-A mutations in immune cells, highlighting the critical roles of different mutant populations in tumors. Together, scNanoGPS facilitates applications of single-cell long-read sequencing technologies.

Resident memory T cell development is associated with AP-1 transcription factor upregulation across anatomical niches.

Tissue-resident memory T (T RM ) cells play a central role in immune responses to pathogens across all barrier tissues after infection. However, the underlying mechanisms that drive T RM differentiation and priming for their recall effector function remains unclear. In this study, we leveraged both newly generated and publicly available single-cell RNA-sequencing (scRNAseq) data generated across 10 developmental time points to define features of CD8 T RM across both skin and small-intestine intraepithelial lymphocytes (siIEL). We employed linear modeling to capture temporally-associated gene programs that increase their expression levels in T cell subsets transitioning from an effector to a memory T cell state. In addition to capturing tissue-specific gene programs, we defined a consensus T RM signature of 60 genes across skin and siIEL that can effectively distinguish T RM from circulating T cell populations, providing a more specific T RM signature than what was previously generated by comparing bulk T RM to naïve or non-tissue resident memory populations. This updated T RM signature included the AP-1 transcription factor family members Fos, Fosb and Fosl2 . Moreover, ATACseq analysis detected an enrichment of AP-1-specific motifs at open chromatin sites in mature T RM . CyCIF tissue imaging detected nuclear co-localization of AP-1 members Fosb and Junb in resting CD8 T RM >100 days post-infection. Taken together, these results reveal a critical role of AP-1 transcription factor members in T RM biology and suggests a novel mechanism for rapid reactivation of resting T RM in tissue upon antigen encounter.

Anaplastic transformation in thyroid cancer revealed by single-cell transcriptomics.

The deadliest anaplastic thyroid cancer (ATC) often transforms from indolent differentiated thyroid cancer (DTC); however, the complex intratumor transformation process is poorly understood. We investigated an anaplastic transformation model by dissecting both cell lineage and cell fate transitions using single-cell transcriptomic and genetic alteration data from patients with different subtypes of thyroid cancer. The resulting spectrum of ATC transformation included stress-responsive DTC cells, inflammatory ATC cells (iATCs), and mitotic-defective ATC cells and extended all the way to mesenchymal ATC cells (mATCs). Furthermore, our analysis identified 2 important milestones: (a) a diploid stage, in which iATC cells were diploids with inflammatory phenotypes and (b) an aneuploid stage, in which mATCs gained aneuploid genomes and mesenchymal phenotypes, producing excessive amounts of collagen and collagen-interacting receptors. In parallel, cancer-associated fibroblasts showed strong interactions among mesenchymal cell types, macrophages shifted from M1 to M2 states, and T cells reprogrammed from cytotoxic to exhausted states, highlighting new therapeutic opportunities for the treatment of ATC.

IL1α Antagonizes IL1ß and Promotes Adaptive Immune Rejection of Malignant Tumors.

We assessed the contribution of IL1 signaling molecules to malignant tumor growth using IL1ß-/-, IL1α-/-, and IL1R1-/- mice. Tumors grew progressively in IL1R-/- and IL1α-/- mice but were often absent in IL1ß-/- mice. This was observed whether tumors were implanted intradermally or injected intravenously and was true across multiple distinct tumor lineages. Antibodies to IL1ß prevented tumor growth in wild-type (WT) mice but not in IL1R1-/- or IL1α-/- mice. Antibodies to IL1α promoted tumor growth in IL1ß-/- mice and reversed the tumor-suppressive effect of anti-IL1ß in WT mice. Depletion of CD8+ T cells and blockade of lymphocyte mobilization abrogated the IL1ß-/- tumor suppressive effect, as did crossing IL1ß-/- mice to SCID or Rag1-/- mice. Finally, blockade of IL1ß synergized with blockade of PD-1 to inhibit tumor growth in WT mice. These results suggest that IL1ß promotes tumor growth, whereas IL1α inhibits tumor growth by enhancing T-cell-mediated antitumor immunity.

Differential effects of DEAE negative mode chromatography and gel-filtration chromatography on the charge status of Helicobacter pylori neutrophil-activating protein.

Helicobacter pylori neutrophil-activating protein (HP-NAP) is involved in H. pylori-associated gastric inflammation. HP-NAP is also a vaccine candidate, a possible drug target, and a potential diagnostic marker for H. pylori-associated diseases. Previously, we purified recombinant HP-NAP by one-step diethylaminoethyl (DEAE) negative mode chromatography by collecting the unbound fraction at pH 8.0 at 4°C. It remains unclear why HP-NAP does not bind to DEAE resins at the pH above its isoelectric point during the purification. To investigate how pH affects the surface net charge of HP-NAP and its binding to DEAE resins during the purification, recombinant HP-NAP expressed in Escherichia coli was subjected to DEAE negative mode chromatography at pH ranging from 7.0 to 9.0 at 25°C and the surface charge of purified HP-NAP was determined by capillary electrophoresis. A minimal amount of HP-NAP was detected in the elution fraction of DEAE Sepharose resin at pH 8.5, whereas recombinant HP-NAP was detected in the elution fraction of DEAE Sephadex resin only at pH 7.0 and 8.0. The purified recombinant HP-NAP obtained from the unbound fractions was not able to bind to DEAE resins at pH 7.0 to 9.0. In addition, the surface charge of the purified HP-NAP was neutral at pH 7.0 to 8.0 and was either neutral or slightly negative at pH 8.5 and 9.0. However, recombinant HP-NAP purified from gel-filtration chromatography was able to bind to DEAE Sepharose resin at pH 7.0 to 9.0 and DEAE Sephadex resin at pH 7.0. At pH 8.5 and 9.0, only the negatively charged species of HP-NAP were found. Thus, recombinant HP-NAP with different charge status can be differentially purified by DEAE negative mode chromatography and gel-filtration chromatography. Furthermore, the charge distribution on the surface of HP-NAP, the presence of impure proteins, and the overall net charge of the resins all affect the binding of HP-NAP to DEAE resins during the negative purification.

A randomized controlled trial of tai chi for tension headaches.

This study examined whether a traditional low-impact mind-body exercise, Tai Chi, affects health-related quality-of-life (HRQOL) and headache impact in an adult population suffering from tension-type headaches. Forty-seven participants were randomly assigned to either a 15 week intervention program of Tai Chi instruction or a wait-list control group. HRQOL (SF-36v2) and headache status (HIT-6trade mark) were obtained at baseline and at 5, 10 and 15 weeks post-baseline during the intervention period. Statistically significant (P < 0.05) improvements in favor of the intervention were present for the HIT score and the SF-36 pain, energy/fatigue, social functioning, emotional well-being and mental health summary scores. A 15 week intervention of Tai Chi practice was effective in reducing headache impact and also effective in improving perceptions of some aspects of physical and mental health.