FLASH-seq protocol (V1) v1

The single-cell RNA-sequencing (scRNA-seq) field has evolved tremendously since the first paper was published back in 2009. While the first methods analysed just a handful of cells, the throughput and performance rapidly increased over a very short timespan. However, it was not until the introduction of emulsion droplets methods, that the robust and reproducible analysis of thousands of cells became feasible. Despite generating data at a speed and a cost per cell that remains unmatched by full-length protocols like Smart-seq, scRNA-seq in droplets still comes with the drawback of addressing only the terminal portion of the transcripts, thus lacking the required sensitivity for comprehensively analyzing the transcriptome of individual cells. Building upon the existing Smart-seq2/3 workflows, we developed FLASH-seq (FS), a new full-length scRNA-seq method capable of detecting a significantly higher number of genes than both previous versions, requiring limited hands-on time and with a great potential for customization.

The ASCC2 CUE domain contacts adjacent ubiquitins to recognize K63-linked polyubiquitin

Alkylation of DNA and RNA is a potentially toxic lesion that can result in mutations and cell death. In response to alkylation damage, K63-linked polyubiquitin chains are assembled that localize the ALKBH3-ASCC repair complex to damage sites in the nucleus. The protein ASCC2, a subunit of the ASCC complex, selectively binds K63-linked polyubiquitin chains using its CUE domain, a type of ubiquitin-binding domain that typically binds monoubiquitin and does not discriminate among different polyubiquitin linkage types. We report here that the ASCC2 CUE domain selectively binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. Whereas the ASCC2 CUE domain binds the distal ubiquitin in a manner similar to that reported for other CUE domains bound to a single ubiquitin, the contacts with the proximal ubiquitin are unique to ASCC2. The N-terminal portion of the ASCC2 α1 helix, including residues E467 and S470, contributes to the binding interaction with the proximal ubiquitin of K63-linked diubiquitin. Mutation of residues within the N-terminal portion of the ASCC2 α1 helix decreases ASCC2 recruitment in response to DNA alkylation, supporting the functional significance of these interactions during the alkylation damage response.

Design and validation of plasmid vectors for characterizing protein-protein interactions in Spodoptera frugiperda insect cells

There are limited molecular biology resources for interrogating protein-protein interactions (PPI) in insect cells. To address this deficiency, we developed plasmid vectors for localization, bi-molecular fluorescence complementation (BiFC), and co-immunoprecipitation (co-IP) assays in Sf9 insect cells. Plasmids were designed to express a protein of interest as a fusion with epitope tags and autofluorescent proteins using the Gateway cloning system. Two robust interactors were utilized to validate this system, the nucleoprotein (N) and the phosphoprotein (P) of maize mosaic virus. The viral N was fused with the carboxy-terminal portion of eYFP and a FLAG epitope tag, and P was fused with the amino-terminal portion of eYFP and a c-myc epitope tag. The two expression plasmids were co-transfected into Sf9 cells, and fluorescence microscopy was used to visualize BiFC and co-IP was performed to confirm that this system was sensitive enough to detect PPI between the two proteins. BiFC was seen in cells co-transfected with N and P and co-IP validated the interaction. This plasmid-based system can be used to investigate a variety of PPI that occur in insects. We validated viral protein interactions that occur in the insect vector which provides further insights into the biology of rhabdoviruses that are transmitted by insects. The ability to express viral and insect proteins in insect cells for studying PPI with this streamlined system represents an advancement for protein research in insects. Future work will focus on identifying interacting viral and host proteins and discovery of targets for control of viruses and insect vectors.

HBO1-MLL interaction promotes AF4/ENL/P-TEFb-mediated leukemogenesis

Leukemic oncoproteins cause uncontrolled self-renewal of hematopoietic progenitors by aberrant gene activation, eventually causing leukemia. However, the molecular mechanism underlying aberrant gene activation remains elusive. Here, we showed that leukemic MLL fusion proteins associate with the HBO1 histone acetyltransferase (HAT) complex through their trithorax homology domain 2 (THD2) in various human cell lines. MLL proteins associated with the HBO1 complex through multiple contacts mediated mainly by the ING4/5 and PHF16 subunits in a chromatin-bound context where histone H3 lysine 4 tri-methylation marks were present. Of the many MLL fusions, MLL-ELL particularly depended on the THD2-mediated association with the HBO1 complex for leukemic transformation. The C-terminal portion of ELL provided a binding platform for multiple factors including AF4, EAF1, and p53. MLL-ELL activated gene expression in murine hematopoietic progenitors by loading an AF4/ENL/P-TEFb (AEP) complex onto the target promoters wherein the HBO1 complex promoted the association with AEP complex over EAF1 and p53. Moreover, the NUP98-HBO1 fusion protein exerted its oncogenic properties via interaction with MLL but not its intrinsic HAT activity. Thus, the interaction between the HBO1 complex and MLL is an important nexus in leukemic transformation, which may serve as a therapeutic target for drug development.

Quasi Moyamoya Disease: Case report and literature review

Quasi moyamoya disease is the unilateral form of typical moyamoya disease which is associated with acute or chronic stages of inflammatory diseases. Here, I present 21-year-old boy presented with sudden onset of weakness of right sided limbs following physical workout, had acute ischemic infarction of left internal capsule in diffusion restriction MR image and unilateral steno- occlusive changes of terminal portion of left internal carotid artery was noted in digital subtraction angiography.

The β-link motif in protein architecture

The β-link is a composite protein motif consisting of a G1β β-bulge and a type II β-turn, and is generally found at the end of two adjacent strands of antiparallel β-sheet. The 1,2-positions of the β-bulge are also the 3,4-positions of the β-turn, with the result that the N-terminal portion of the polypeptide chain is orientated at right angles to the β-sheet. Here, it is reported that the β-link is frequently found in certain protein folds of the SCOPe structural classification at specific locations where it connects a β-sheet to another area of a protein. It is found at locations where it connects one β-sheet to another in the β-sandwich and related structures, and in small (four-, five- or six-stranded) β-barrels, where it connects two β-strands through the polypeptide chain that crosses an open end of the barrel. It is not found in larger (eight-stranded or more) β-barrels that are straightforward β-meanders. In some cases it initiates a connection between a single β-sheet and an α-helix. The β-link also provides a framework for catalysis in serine proteases, where the catalytic serine is part of a conserved β-link, and in cysteine proteases, including Mpro of human SARS-CoV-2, in which two residues of the active site are located in a conserved β-link.

The C-Terminal Domain of LRRK2 with the G2019S Substitution Increases Mutant A53T α-Synuclein Toxicity in Dopaminergic Neurons In Vivo

Alpha-synuclein (α-syn) and leucine-rich repeat kinase 2 (LRRK2) play crucial roles in Parkinson’s disease (PD). They may functionally interact to induce the degeneration of dopaminergic (DA) neurons via mechanisms that are not yet fully understood. We previously showed that the C-terminal portion of LRRK2 (ΔLRRK2) with the G2019S mutation (ΔLRRK2G2019S) was sufficient to induce neurodegeneration of DA neurons in vivo, suggesting that mutated LRRK2 induces neurotoxicity through mechanisms that are (i) independent of the N-terminal domains and (ii) “cell-autonomous”. Here, we explored whether ΔLRRK2G2019S could modify α-syn toxicity through these two mechanisms. We used a co-transduction approach in rats with AAV vectors encoding ΔLRRK2G2019S or its “dead” kinase form, ΔLRRK2DK, and human α-syn with the A53T mutation (AAV-α-synA53T). Behavioral and histological evaluations were performed at 6- and 15-weeks post-injection. Results showed that neither form of ΔLRRK2 alone induced the degeneration of neurons at these post-injection time points. By contrast, injection of AAV-α-synA53T alone resulted in motor signs and degeneration of DA neurons. Co-injection of AAV-α-synA53T with AAV-ΔLRRK2G2019S induced DA neuron degeneration that was significantly higher than that induced by AAV-α-synA53T alone or with AAV-ΔLRRK2DK. Thus, mutated α-syn neurotoxicity can be enhanced by the C-terminal domain of LRRK2G2019 alone, through cell-autonomous mechanisms.

Condensation of pericentrin proteins in human cells illuminates phase separation in centrosome assembly

At the onset of mitosis, centrosomes expand the pericentriolar material (PCM) to maximize their microtubule-organizing activity. This step, termed centrosome maturation, ensures proper spindle organization and faithful chromosome segregation. However, as the centrosome expands, how PCM proteins are recruited and held together without membrane enclosure remains elusive. We found that endogenously expressed pericentrin (PCNT), a conserved PCM scaffold protein, condenses into dynamic granules during late G2/early mitosis before incorporating into mitotic centrosomes. Furthermore, the N-terminal portion of PCNT—enriched with conserved coiled-coils (CCs) and low-complexity regions (LCRs)—phase separates into dynamic condensates that selectively recruit PCM proteins and nucleate microtubules in cells. We propose that CCs and LCRs, two prevalent sequence features in the centrosomal proteome, are preserved under evolutionary pressure in part to mediate liquid-liquid phase separation, a process that bestows upon the centrosome distinct properties critical for its assembly and functions.

Lysosomal proteolysis of amyloid beta is impeded by fibrils grown in both acidic and neutral pH environments

Aggregation of amyloid-beta (A) into extracellular plaques is a well-known hallmark of Alzheimers disease (AD). Similarly, autophagic vacuoles, autophagosomes, and other residual bodies within dystrophic neurites, though more difficult to detect, are characteristic features of AD. To explore the potential intersection between these observations, we conducted experiments to assess whether A fibril formation disrupts lysosomal proteolysis. Fibrils constituted from either A 1-40 or A 1-42 were grown under both neutral and acidic pH. The extent of proteolysis by individual cathepsins (L, D, B, and H) was monitored by both thioflavin T fluorescence and liquid-chromatography combined with mass spectrometry. The results show that all A fibrils are resistant to cathepsin digestion, with significant amounts of undigested material remaining for samples of fibrils grown in both neutral and acidic pH. Further analysis revealed that the neutral-grown fibrils are proteolytically resistant throughout the sequence, while the acid-grown fibrils prevented digestion primarily in the C-terminal portion of the sequence. Fibrils grown from A 1-42 are generally more resistant to degradation compared to A 1-40. Overall, the results indicate that A fibrils formed in the neutral pH environments found in intracellular or extracellular spaces may pose the greatest difficulty for complete digestion by the lysosome, particularly when the fibrils are comprised of A 1-42.

A Novel Pathogenic Variant in the MN1 Gene in a Patient Presenting with Rhombencephalosynapsis and Craniofacial Anomalies, Expanding MN1 C-terminal Truncation Syndrome

Meningioma-1 is a transcription activator that regulates mammalian palate development and is required for appropriate osteoblast proliferation, motility, differentiation, and function. Microdeletions involving the MN1 gene have been linked to syndromes including craniofacial anomalies, such as Toriello–Carey syndrome. Recently, truncating variants in the C-terminal portion of the MN1 transcriptional factor have been linked to a characteristic and distinct phenotype presenting with craniofacial anomalies and partial rhombencephalosynapsis, a rare brain malformation characterized by midline fusion of the cerebellar hemispheres with partial or complete loss of the cerebellar vermis. It has been called MN1 C-terminal truncation (MCTT) syndrome or CEBALID (Craniofacial defects, dysmorphic Ears, Brain Abnormalities, Language delay, and Intellectual Disability) and suggested to be caused by dominantly acting truncated protein MN1 instead of haploinsufficiency. As a proto-oncogene, MN1 is also involved in familial meningioma. In this study, we present a novel case of MCTT syndrome in a female patient presenting with craniofacial anomalies and rhombencephalosynapsis, harboring a de novo pathogenic variant in the MN1 gene: c.3686_3698del, p.(Met1229Argfs*87).