Biology subjects

Kramer, S.

Publications and source records attributed to Kramer, S..

At least 19 recordsLinked to original sources

bioRxiv2026

RNA trans-splicing treatment for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a fatal disorder caused by dystrophin mutations, leading to progressive muscle degeneration and cardiac failure. Although AAV-based DMD therapies with innovative designs to overcome the cargo limits of the virus are effective in animal models, these current systems may face clinical translation challenges related to efficiency, off-target effects, and immunogenicity. We developed a multi-vector RNA End Joining (REJ) system to split large genes into multiple co-delivered AAVs that engage the cell's intrinsic spliceosome to precisely reassemble RNA segments that encode very large scar-free proteins. We show the system is efficient and has negligible off-target interactions. In vivo, REJ vectors expressing the adenine base editor Abe8e, mini-dystrophin Dp253, or native full-length dystrophin Dp427 each prevented muscle degeneration. Machine-learning histopathology of more than 120,000 myofibers revealed robust reductions in nuclear infiltration, improved centronucleation, and normalized hypertrophy, supported by functional, transcriptomic, and behavioral analyses. These findings establish REJ as a clinically viable AAV-based strategy for DMD that enables expression of large therapeutic proteins while avoiding potentially antigenic bacterial protein- or DNA-recombinases.

molecular biology↗

bioRxiv2026

An intersectional expression platform for gene complementation using RNA-fragment end joining (REJ)

Gene complementation is a powerful tool for genetic selection and protein functional studies but typically requires extensive screening for complementary components. Here we engineered a platform for gene complementation based on RNA end-joining (REJ) that precisely and efficiently splices separate RNA units into functional coding mRNAs. REJ is mediated by short structured modular RNA segments of ~250bp that: (a) promote RNA::RNA interaction, (b) coopt the intrinsic cell splicing machinery to facilitate RNA trans-splicing, (c) minimizes translation of protein fragments from un-spliced RNA segments, and (d) encodes scar-free protein. We demonstrate that REJ is a broadly applicable system that can reliably split nearly any gene into complementary segments regardless of protein structure. To enable the use of this system we provide a toolbox of reporters and a web-based design tool to facilitate vector design. Demonstrated REJ applications include genetic complementation, intersectional labeling of cell types, and efficient expression of large proteins.

molecular biology↗

bioRxiv2026

Expanding the palette of trehalose-based fluorophores for live mycobacterial detection

Tuberculosis (TB) remains the worlds leading infectious cause of death. Trehalose-based fluorogenic probes have emerged as powerful tools for labeling and studying mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. However, existing probes occupy a limited spectral range and require compromise between brightness, specificity, and functional readouts. Here, we report the design and characterization of two trehalose conjugates derived from Janelia Fluor(R) dyes, JF635-Tre and JF646-Tre, which extend the trehalose-based platform into the far-red region. Following NHS ester-mediated synthesis, the trehalose-conjugated analogs displayed strong far-red fluorescence, with excitation/emission maxima at 638/654 nm and 648/663 nm for JF635-Tre and JF646-Tre, respectively. Both probes exhibited concentration- and time-dependent labeling of Mycobacterium smegmatis (Msmeg) and Mtb with minimal background fluorescence from the corresponding unconjugated dyes. Furthermore, we observed reduced labeling in heat-killed cells compared to live Msmeg, particularly for JF646-Tre, consistent with sensitivity to metabolic activity. Both JF-Tre derivatives produced significant cellular labeling and JF635-Tre distinguished untreated from INH-treated samples in drug susceptible Mtb, demonstrating the potential of the JF-Tre probes to report on INH susceptibility and resistance. Together, these findings expand the toolkit of trehalose-based probes and highlight how fluorophore identity influences probe performance in mycobacterial fluorescence imaging and drug susceptibility testing.

biochemistry↗

bioRxiv2026

A luminal proteome of the endoplasmic reticulum and Golgi apparatus reveals a novel modulator of ER stress tolerance in African trypanosomes

African trypanosomes employ specialised mechanisms of membrane trafficking as a key strategy to persist in both the mammalian host and insect vector. Their survival and pathogenicity rely on the continuous synthesis and surface delivery of extremely abundant surface coat proteins, imposing an extraordinary biosynthetic burden on the secretory pathway. Despite this, the luminal proteome of the T. brucei endoplasmic reticulum (ER) and Golgi apparatus remains incompletely characterised. Here, we exploit TurboID proximity biotinylation, using the abundant ER chaperone BiP (Binding-immunoglobulin protein) as luminal bait to map the ER proteome in bloodstream and procyclic lifecycle stages of Trypanosoma brucei. Comparison with BiPN, a truncated secretory form of BiP that transits the Golgi, provides differential compartmental labelling, together identifying 366 (BiP) and 428 (BiPN) proximity partners respectively and encompassing established ER quality control machinery, secretory cargo, and Golgi proteins. Quantitative ranking of BiP labelling intensity identifies a cohort of candidate BiP interactors: the most strongly enriched is Tb927.5.1160, a protein sharing structural homology with the mammalian BiP nucleotide exchange inhibitor MANF (mesencephalic astrocyte-derived neurotrophic factor). Endogenous mNeonGreen tagging confirms ER localisation of TbMANF in both life cycle stages, and reciprocal manipulation of its abundance by RNAi and inducible expression produces opposing shifts in cellular sensitivity to ER stress. These data are consistent with a role in regulating BiP ATPase cycling in an organism that, unlike yeast and mammals, lacks a canonical unfolded protein response, making TbMANF the first candidate regulator of BiP activity identified in kinetoplastids. Finally, TurboID proximity labelling anchored at the inner face of the nuclear pore via NUP65 extends our endomembrane map to the inner nuclear membrane, identifying candidate proteins of this specialised ER-continuous domain. AUTHOR SUMMARYAfrican sleeping sickness is caused by Trypanosoma brucei, a parasite that survives in the mammalian bloodstream by constantly renewing its protective protein coat. To synthesise and export this surface coat, the parasite relies on two intracellular compartments, the endoplasmic reticulum (ER) and the Golgi apparatus, which function as a quality control and sorting factory for proteins entering the secretory pathway. However, the identity of the proteins that populate these compartments in blood-stage parasites, and that maintain functioning under stress conditions, has remained poorly mapped. Here, we used an enzyme-based proximity labelling strategy that identifies neighbouring proteins in live cells without disturbing their targeting signals, generating a comprehensive protein inventory of both compartments across the two main T. brucei lifecycle stages. Among the most strongly labelled proteins was Tb927.5.1160, a protein structurally related to a mammalian regulator of the master ER chaperone BiP. Reducing or increasing the abundance of Tb927.5.1160 in parasites produced opposite changes in ER stress tolerance, identifying it as a candidate modulator of ER homeostasis in a lineage that regulates protein quality control through mechanisms distinct from those operating in yeast or human cells. Together, our findings provide a new molecular resource for understanding how T. brucei sustains secretory pathway function under the biosynthetic demands of mammalian and insect host infection.

biochemistry↗

bioRxiv2026

The changing contributions of weakness and the flexor synergy to post-stroke arm function over time: A kinematic re-examination of Twitchell

In 1951, the neurologist Thomas Twitchell published a seminal paper in Brain describing the time-course of recovery from hemiplegia after stroke in 25 participants from hospitalization to when they reached what he deemed steady state. His main emphasis was on the evolution of voluntary movements at the shoulder, elbow and hand, first within an obligatory flexor synergy, and then independently out-of-synergy. We thought that 75 years later, an update using modern motion capture technology should be attempted as it would allow for finer granularity in the characterization of the time courses of both functional recovery and of the flexor synergy, and then relate them to each other, to weakness and to well-established clinical scales. To this end, we used marker-less 3D kinematics to assess task performance and intrusion of synergies in thirty-three stroke participants longitudinally, from the early sub-acute stage (1 - 8 weeks post-stroke) to the chronic stage (24 - 64 weeks post-stroke). Participants performed an out-of-flexor synergy (shoulder flexion and elbow extension) reaching task. We assessed the time course of recovery of obligatory intrusion of pathological synergies based on measures derived from the angular velocity profiles of the shoulder and the elbow joints. Task-related kinematic measures were obtained and compared to sixteen healthy controls. Grip strength, Motor impairment (FMA), and function (ARAT) scores were also collected. Task kinematics were different from controls in the early, late sub-acute, and chronic stages, but showed gradual recovery over time. Weakness in the hand remained impaired at all time points. Flexor-synergy intrusion was maximal in the early sub-acute stage and then began to subside. Regression analysis with functional kinematic and clinical (FMA, ARAT) measures indicated that flexor-synergy intrusion was a significant predictor in the early and late sub-acute stages, but not in the chronic stage, while weakness remained a significant predictor at all stages of recovery. To better address the relationship between synergies, weakness, and function, we analyzed the more severe cases (ARAT<21) separately. In the sub-acute stage, most of them (11/13) suffered from intrusion of synergies, whereas in the chronic phase, only a minority (2/8) did. Weakness seemed to be the main contributor to poor outcome in the chronic phase. We conclude that weakness and synergy intrusion evolve separately from the subacute to the chronic phase, perhaps more so when neurorehabilitation is given at a dose higher than standard of care.

neuroscience↗

bioRxiv2026

The trypanosome mRNA decapping enzyme ALPH1 prefers caps without m7G methylation and produces diphosphate-RNA

5'ends of eukaryotic mRNAs are protected by the m7G cap, connected to the mRNA via a three-phosphate-bridge. In mRNA decay, the pyrophosphate bond between the and {beta} phosphate is cleaved by the nudix hydrolase DCP2. Uniquely among eukaryotes, Kinetoplastida lack DCP2 and instead employ the ApaH-like phosphatase ALPH1 for mRNA decapping. ALPH1 consists of an unstructured N-terminus, a catalytic domain and a structured C-terminus that mediates ALPH1 dimerisation. Here, we have analysed Trypanosoma brucei ALPH1 in greater detail. We find that the enzyme has broad substrate specificity and accepts different cap types and even cap analogues. Strikingly, cap-analogues and RNAs without the m7G-methyl group are turned over significantly faster than m7G methylated substrates. Moreover, all methylated and non-methylated cap analogues tested, with at least one additional nucleotide 3' to the NpppN moiety are cleaved at the {beta}-{gamma} pyrophosphate bond, producing the equivalent to a 5 diphosphate-RNA. While the presence of the ALPH1 C-terminal domain is essential for cell viability and increases enzyme activity in vitro, substrate preferences are determined solely by the catalytic domain. Altogether, these ALPH1 enzymatic properties exhibit intriguing differences to the canonical eukaryotic decapping enzyme DCP2, which we critically discuss and which potentially have biotechnological applications.

molecular biology↗

bioRxiv2025

A handheld microfluidic manifold for massively multiplexed CRISPR-based nucleic acid detection

Multiplexed methods for nucleic acid detection are immensely challenging to deploy outside of laboratory settings. Conversely, field-deployable methods are limited to low levels of multiplexing. Here, we introduce Scalable On-site Nucleic Acid Testing Architecture (SONATA), enabling >100-chamber reaction partitioning for multiplexed nucleic acid amplification and detection with a portable microfluidic manifold. The manifold directs a diluted sample into individual reaction chambers, each of which contains lyophilized reagents and a small stir bar or bead for mixing. Samples can be loaded using a syringe by hand, greatly simplifying the testing process. We demonstrate the integration of the platform with Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a sensitive and deployable CRISPR-based detection technology. We show that deployed with SONATA, SHINE retains its sensitivity, enabling highly multiplexed pathogen detection in [≤] 1 hour. In addition, we demonstrate the detection of single-nucleotide variants, including mutations associated with drug susceptibility.

bioengineering↗

bioRxiv2024

An updated map of the trypanosome nuclear pore and its associated proteins

Nuclear export of mRNAs requires loading the mRNP to the transporter Mex67/Mtr2 in the nucleoplasm, controlled access to the pore by the basket-localized TREX2 complex and mRNA release at the cytoplasmic site by the DEAD-box RNA helicase Dbp5. Asymmetric localisation of nucleoporins (NUPs) and transport components as well as the ATP dependency of Dbp5 ensure unidirectionality of transport. Trypanosomes possess homologues of the mRNA transporter Mex67/Mtr2, but not of TREX2 or Dbp5. Instead, nuclear export is likely fuelled by the GTP/GDP gradient created by the Ran GTPase. However, it remains unclear, how directionality is achieved since the current model of the trypanosomatid pore is mostly symmetric. We have revisited the architecture of the trypanosome nuclear pore complex using a novel combination of expansion microscopy, proximity labelling and streptavidin imaging. We could confidently assign the NUP76 complex, a known Mex67 interaction platform, to the cytoplasmic site of the pore. The resulting availability of reference proteins for basket, inner ring and cytoplasmic site allowed mapping of all 75 trypanosome proteins with known nuclear pore localisation to a sub-region of the pore based on mass spectrometry data from proximity labelling. This approach defined many further asymmetrically localised nuclear pore components. At the nuclear site, we identified several trypanosome-unique proteins, for instance the FG-NUPs NUP64/NUP98, but also proteins with structural homology to TREX-2 components. We mapped the components of the Ran-based nuclear export system and confirm the absence of a Dbp5 homologue. Lastly, we demonstrate, by deploying an auxin degron system, that NUP76 holds an essential role in mRNA export consistent with a functional orthology to NUP82/88. Altogether, the combination of proximity labelling with expansion microscopy revealed an asymmetric architecture of the trypanosome nuclear pore supporting inherent roles fort directed transport. Our approach delivered novel nuclear pore associated components inclusive positional information, which can now be interrogated for functional roles to explore trypanosome specific adaptions of the nuclear basket, export control and mRNP remodelling.

cell biology↗

bioRxiv2024

A single-nucleus census of immune and non-immune cell types for the major immune organ systems of chicken.

In the avian host, comprehensively cataloging immune cell types, their transcriptome profiles, and varying molecular responses to pathogen challenges are necessary steps toward a better understanding of the interplay between genetics and disease resilience. We present a first nuclei atlas of immune cell types derived from the three main immune organs of layer chickens, including spleen, bursa, and thymus. In bursa we also present, an accounting of cell type activation with the bacterial toxin lipopolysaccharide (LPS). Our analysis includes 36,370 total nuclei and 16, 12, and 12 transcriptionally distinct clusters for spleen, bursa, and thymus, respectively. We discover nuclei molecular profiles that uniquely distinguish states of the transcriptome within cell type that could serve as new means to characterize avian immune subtypes. We further subcluster refined immune cell type classifications, specifically highlighting the transcriptomic diversity of B and T cell subtypes. In the bursa, inferred intercellular communication and signaling pathway enrichment analyses across immune and non-immune cell types demonstrate the unappreciated complexity of the B cell repertoire in a model mimicking systemic bacterial infection. This census of all cell types in both primary and one major secondary avian immune organ system, although preliminary, provides a first review of how nuclei transcribe numerous genes, known and unknown, a critical prerequisite for the study avian immunogenetics by cell type.

immunology↗

bioRxiv2024

TAM receptors mediate the Fpr2-driven pain resolution and fibrinolysis after nerve injury

Nerve injury causes neuropathic pain and multilevel nerve barrier disruption. Nerve barriers consist of perineurial, endothelial, and myelin barriers. So far, it is unclear whether resealing nerve barriers fosters pain resolution and recovery. To this end, we analysed the nerve barrier property portfolio, pain behaviour battery, and lipidomics for precursors of specialized pro-resolving meditators (SPMs) and their receptors in chronic constriction injury of rat sciatic nerve to identify targets for pain resolution by resealing the selected nerve barriers. Of the three nerve barriers - perineurium, capillaries, and myelin - only capillary tightness specifically against larger molecules, such as fibrinogen, recuperated with pain resolution. Fibrinogen immunoreactivity was not only elevated in rats at the time of neuropathic pain but also in nerve biopsies from patients with (but not without) painful polyneuropathy indicating that sealing of the vascular barrier might be novel approach in pain treatment. 15R-HETE (hydroxyeicosatetraenoic acid), a precursor of aspirin-triggered lipoxin A4, were specifically upregulated at the beginning of pain resolution. Repeated local application of resolvin D1-laden nanoparticles or Fpr2 agonists sex-independently resulted in accelerated pain resolution and fibrinogen removal. Clearing macrophages (Cd206) and fibrinolytic pathways (Plat) were also induced while inflammation (Tnf) and inflammasomes (Nlrp3) were unaffected by this treatment. Blocking TAM receptors (Tyro3, Axl, and Mer) and tyrosine kinase receptors linking haemostasis and inflammation completely inhibited all the effects. In summary, nanoparticles can be used as transporters for fleeting lipids, such as SPMs, and therefore expand the array of possible therapeutic agents. Thus, the Fpr2-Cd206-TAM receptor axis may be a suitable target for strengthening the capillary barrier, removing endoneurial fibrinogen, and boosting pain resolution in patients with chronic neuropathic pain.

neuroscience↗

bioRxiv2024

Two distinct Trypanosoma eIF4F complexes co-exist, bind different mRNAs and are regulated during nutritional stress

Many eIF4F subunits and PABP paralogues are found in trypanosomes: six eIF4E, five eIF4G, one eIF4A and two PABPs. They are expressed simultaneously and assemble into different complexes, contrasting the situation in metazoans that use distinct complexes in different cell types or developmental stages. Each eIF4F complex has its own proteins, mRNAs and, consequently, a distinct function. We set out to study the function and regulation of the two major eIF4F complexes of Trypanosoma cruzi and identified the associated proteins and mRNAs of eIF4E3 and eIF4E4 in cells in exponential growth and in nutritional stress. Upon stress, eIF4G/eIF4A and PABP remain associated to the eIF4E, but the associations with other 43S pre-initiation factors decrease, indicating that ribosome attachment is impaired. Most eIF4E3-associated mRNAs encode for metabolic proteins, while eIF4E4 associate to mRNAs encoding ribosomal proteins. Interestingly, for both eIF4E3/4, more mRNAs were associated in stressed cells than in non-stressed cells, even though these mRNAs have lower translational efficiencies in stress. In summary, trypanosomes have two co-existing eIF4F complexes involved in translation of distinct mRNA cohorts important for growth. Under stress conditions, both complexes exit translation but remain bound to their mRNA targets.

molecular biology↗

bioRxiv2024

Intron-loss in Kinetoplastea correlates with a non-functional EJC and loss of NMD factors

In metazoans, mRNA quality is tightly monitored from transcription to translation. A key role lies with the exon junction complex (EJC) that is placed upstream of the exon-exon junction after splicing. The EJC inner core is composed of Magoh, Y14, eIF4AIII and BTZ and the outer core of proteins involved in mRNA splicing (CWC22), export (Yra1), translation (PYM) and non-sense mediated decay (NMD, UPF1/2/3). The protozoan parasite Trypanosoma brucei encodes only two genes with introns, but all mRNAs are processed by trans-splicing. The presence of the three core EJC proteins and a potential BTZ homologue (Rbp25) in trypanosomes has been suggested as an adaptation of the EJC function to mark trans-spliced mRNAs. We analysed trypanosome EJC components and noticed major differences between eIF4AIII and Magoh/Y14: (i) whilst eIF4AIII is essential, knocking out both Magoh and Y14 elicits only a mild growth phenotype and does not affect processing, export or stability of the two cis-spliced mRNAs (ii) eIF4AIII localization is mostly nucleolar, with a minor proportion in the nucleoplasm and cytoplasm, while Magoh and Y14 are nucleolar and nucleoplasmic but excluded from the cytoplasm (iii) eIF4AIII associates with nucleolar proteins (including NOM1/Sgd1p) and the splicing factor CWC22, but not with Y14 or Magoh, while Magoh and Y14 associate with each other, but not with eIF4AIII, CWC22 or the nucleolar proteins. Our data argue against the presence of a functional EJC in trypanosomes, but rather indicate that eIF4AIII adopted non-EJC related, essential functions, while Magoh and Y14 became redundant. Trypanosomes also possess homologues to the NMD proteins UPF1 and UPF2. We found that depletion of UPF1 causes only a minor reduction in growth and detailed phylogenetic analyses show several independent losses of UPF1 and UPF2, as well as total loss of UPF3 in the Kinetoplastida group. The data indicate that UPF1-dependent NMD is not essential in trypanosomes consistent with the observation that the cells tolerate the presence of mRNAs with allele-specific, endogenous PTCs (premature termination codons). Altogether, our comprehensive analyses of conserved components provide no evidence for a canonical EJC or NMD pathway in (almost) intron-less trypanosomatids.

biochemistry↗

bioRxiv2023

Beyond BioID: Streptavidin outcompetes antibody fluorescence signals in protein localization and readily visualises targets evading immunofluorescence detection.

Immunofluorescence is a common method to localise proteins within their cellular context via fluorophore labelled antibodies and for some applications without alternative. However, some protein targets evade detection due to low protein abundance or accessibility issues. In addition, some imaging methods require a massive reduction in antigen density thus impeding detection of even medium-abundant proteins. Here, we show that the fusion of the target protein to TurboID, a biotin ligase labelling lysine residues in close proximity, and subsequent detection of biotinylation by fluorescent streptavidin offers an "all in one" solution to the above-mentioned restrictions. For a wide range of target proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the imaging sensitivity in expansion microscopy and correlative light and electron microscopy, with no loss in resolution. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus or RNA granules, were readily detected with streptavidin, while most antibodies fail to label proteins in these environments. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can be used to map antibody- accessibility to certain cellular regions. As a proof of principle, we mapped antibody access to all trypanosome nuclear pore proteins (NUPs) and found restricted antibody labelling of all FG NUPs of the central channel that are known to be phase-separated, while most non-FG Nups could be labelled. Lastly, we show that streptavidin imaging can resolve dynamic, temporally and spatially distinct sub-complexes and, in specific cases, reveal a history of dynamic protein interaction. In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, can provide information on protein interactions and biophysical environment.

cell biology↗

bioRxiv2023

High-content fluorescence bioassay to investigate pore formation, ion channel modulation and cell membrane lysis induced by venoms

Venoms comprise highly evolved bioactive molecules modulating ion channels, receptors, coagulation factors, and the cellular membrane. This array of targets and bioactivity requires high-content bioassays to aid the development of novel envenomation treatments and biotechnological and pharmacological agents. To address this gap in venoms research, we developed a fluorescence-based high-throughput and high-content cellular assay to simultaneously identify common cellular activities produced by venoms: membrane lysis, pore-formation, and ion channel modulation. By combining intracellular calcium with extracellular nucleic acid measurements, we distinguished these venom mechanisms using one cellular assay. We applied our high-content bioassay in three cell types exposed to venom components representing lytic, ion pore-forming or ion channel modulator toxins. Beyond the distinct profiles produced by these three types of action mechanisms, we found that the pore-forming latrotoxin -Lt1a prefers human neuroblastoma to kidney cells and cardiomyocytes, while the lytic bee peptide melittin is not selective. Furthermore, evaluation of snake venoms showed that Elapid species induced rapid membrane lysis, while Viper species showed variable to no activity on neuroblastoma cells. These findings demonstrate that our high-content bioassay distinguish clades and interspecific traits and capture the clinical observations at venom level and is capable of differentiate ion pore-forming from membrane lysis and ion channel modulation. We hope our research will accelerate the understanding of venom biology and the diversity of toxins inducing cytotoxic, cardiotoxic and neurotoxic effects and assist in identifying venom components whose properties could benefit humankind.

pharmacology and toxicology↗

bioRxiv2022

Intrusion of pathological synergies does not explain impaired 3D arm movements in subacute stroke

It has long been of interest to characterize the components that generate the motor control abnormalities in the arm after stroke. One approach has been to decompose the hemiparesis phenotype into negative signs, such as weakness, and positive signs, such as intrusion of synergies. Here, we sought to identify the contributions of weakness and the flexor synergy to motor function and impairment, as defined by kinematic and clinical scales, respectively, in sub-acute stroke using two 3D arm tasks that differed in their requirement for elbow extension. Thirty-three sub-acute post-stroke participants and sixteen healthy controls performed a cup-to-mouth task, requiring shoulder and elbow flexion (within flexor synergy), and a reaching task, requiring shoulder flexion and elbow extension (out of flexor synergy). Using markerless 3D pose-estimation, we analyzed upper limb kinematics to assess overall task performance and intrusion of pathological synergies. Weakness was measured using a grip dynamometer. Performance in both tasks was impaired to a similar degree in the stroke participants compared to controls. Subsequent analysis of coordination patterns between the elbow and the shoulder joints revealed intrusion of synergies in the reaching task based on the time spent within a flexion-flexion pattern (flexor synergy proportion) and the correlation between shoulder and elbow angles when the shoulder was flexing (flexion synergy strength). Regression analysis indicated that the significant predictors of poor task performance were weakness and flexor synergy intrusion. Notably, the Fugl-Meyer Assessment was abnormal even when just weakness caused the impairment, which means that caution is required when using this scale to quantify synergies. We conclude that both weakness and synergy intrusion contribute to impaired coordination of the elbow and shoulder joints in the sub-acute post-stroke period. This study shows that careful kinematic analysis of naturalistic movements is required to better characterize the components of upper limb impairment after stroke.

neuroscience↗

bioRxiv2022

Multiple instance learning to predict immune checkpoint blockade efficacy using neoantigen candidates

A successful response to immune checkpoint blockade treatment (ICB) depends on the functional re-invigoration of neoantigen-specific T cells and their anti-tumoral activity. Previous studies showed that the patients neoantigen candidate load is an imperfect predictor of the response to ICB. Further studies provided evidence that the overall response to ICB is also affected by the qualitative properties of a few or even single candidates, limiting the predictive power based on candidate quantity alone. To our knowledge, this is the first study to predict the response to ICB therapy based on qualitative neoantigen candidate profiles in the context of the mutation type, using a multiple instance learning approach. Multiple instance learning is a special branch of machine learning which classifies labelled bags that are formed by a set of unlabeled instances. The multiple instance learning approach performed systematically better than random guessing and was independent of the neoantigen candidate load. Qualitative modeling performed better in comparison to the quantitative approach, in particular for modelling low-abundant fusion genes. Our findings suggest that multiple instance learning is an appropriate method to predict immunotherapy efficacy based on qualitative neoantigen candidate profiles without relying on direct T-cell response information and provide a foundation for future developments in the field.

bioinformatics↗

bioRxiv2021

OPEN leaf: an open-source cloud-based phenotyping system for tracking dynamic changes at leaf-specific resolution in Arabidopsis

The first draft of the Arabidopsis genome was released more than 20 years ago and despite intensive molecular research, more than 30% of Arabidopsis genes remained uncharacterized or without an assigned function. This is in part due to gene redundancy within gene families or the essential nature of genes, where their deletion results in lethality (i.e., the dark genome). High-throughput plant phenotyping (HTPP) offers an automated and unbiased approach to characterize subtle or transient phenotypes resulting from gene redundancy or inducible gene silencing; however, commercial HTPP platforms remain unaffordable. Here we describe the design and implementation of OPEN leaf, an open-source HTPP system with cloud connectivity and remote bilateral communication to facilitate data collection, sharing and processing. OPEN leaf, coupled with the SMART imaging processing package was able to consistently document and quantify dynamic morphological changes over time at the whole rosette level and also at leaf-specific resolution when plants experienced changes in nutrient availability. The modular design of OPEN leaf allows for additional sensor integration. Notably, our data demonstrate that VIS sensors remain underutilized and can be used in high-throughput screens to identify characterize previously unidentified phenotypes in a leaf-specific manner. Significance StatementMany bottlenecks exist in high-throughput phenotyping involving computing power for processing and a lack of focus on abiotic stresses that has prevented an advancement in phenotyping on par with genotyping. Therefore, we create an automated HTP system that performs nutrient studies on Arabidopsis thaliana with cloud-based image processing that quantifies plant traits at a whole and leaf-level.

plant biology↗

bioRxiv2021

The nucleolar DExD/H protein Hel66 is involved in ribosome biogenesis in Trypanosoma brucei

The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process.

microbiology↗