Kinesin-3 UNC-104(KIF1A) is the major axonal transporter of synaptic vesicles. Employing yeast two-hybrid and co-immunoprecipitation (Co-IP) assays, we characterized a LIN-2(CASK) binding site overlapping with that of reported UNC-104 activator protein SYD-2(Liprin-α) on the motor's stalk domain. We identified the L27 and GUK domains of LIN-2 to be the most critical interaction domains for UNC-104. Further, we demonstrated that the L27 domain interacts with the sterile alpha motifs (SAM) domains of SYD-2, while the GUK domain is able to interact with both the coiled coils and SAM domains of SYD-2. LIN-2 and SYD-2 colocalize in Caenorhabditis elegans neurons and display interactions in bimolecular fluorescence complementation (BiFC) assays. UNC-104 motor motility and Synaptobrevin-1 (SNB-1) cargo transport are largely diminished in neurons of LIN-2 knockout worms, which cannot be compensated by overexpressing SYD-2. The absence of the motor-activating function of LIN-2 results in increased motor clustering along axons, thus retaining SNB-1 cargo in cell bodies. LIN-2 and SYD-2 both positively affect the velocity of UNC-104, however, only LIN-2 is able to efficiently elevate the motor's run lengths. From our study, we conclude that LIN-2 and SYD-2 act in a functional complex to regulate the motor with LIN-2 being the more prominent activator.

Bidirectional cargo transport in neurons can be explained by two models: the "tug-ofwar model" for short-range transport, in which several kinesin and dynein motors are bound to the same cargo but travel in opposing directions, and by the "motor coordination model" for long-range transport, in which small adaptors or the cargo itself activates or deactivates opposing motors. Direct interactions between the major axonal transporter kinesin-3 UNC-104(KIF1A) and the dynein/dynactin complex remains unknown. In this study, we dissected and evaluated the interaction sites between UNC-104 and dynein as well as between UNC-104 and dynactin using yeast two-hybrid assays. We found that the DYLT-1(Tctex) subunit of dynein binds near the coiled coil 3 (CC3) of UNC-104, and that the DYRB-1(Roadblock) subunit binds near the CC2 region of UNC-104. Regarding dynactin, we specifically revealed strong interactions between DNC-6(p27) and the FHA-CC3 stretch of UNC-104, as well as between the DNC-5(p25) and the CC2-CC3 region of UNC-104. Motility analysis of motors and cargo in the nervous system of Caenorhabditis elegans revealed impaired transport of UNC-104 and synaptic vesicles in dynein and dynactin mutants (or in RNAi knockdown animals). Further, in these mutants UNC-104 clustering along axons was diminished. Interestingly, when dynamic UNC-104 motors enter a stationary UNC-104 cluster their dwelling times are increased in dynein mutants (suggesting that dynein may act as an UNC-104 activator). In summary, we provide novel insights on how UNC-104 interacts with the dynein/dynactin complex and how UNC-104 driven axonal transport depends on dynein/dynactin in C. elegans neurons. K E Y W O R D Saxonal transport, Caenorhabditis elegans, KIF1A, kinesin, molecular motors, yeast two-hybrid

Background: Sepsis remains a common but fatal complication among patients with immune suppression. We aimed to investigate the performance of metagenomic next-generation sequencing (mNGS) compared with standard microbiological diagnostics in patients with hematologic malignancies. Methods: We performed a prospective study from June 2019 to December 2019. Adult patients with hematologic malignancies and a clinical diagnosis of sepsis were enrolled. Conventional diagnostic methods included blood cultures, serum galactomannan for Aspergillus, cryptococcal antigen and cytomegalovirus (CMV) viral loads. Blood samples for mNGS were collected within 24 h after hypotension developed. Results: Of 24 patients enrolled, mNGS and conventional diagnostic methods (blood cultures, serology testing and virus RT-PCR) reached comparable positive results in 9 cases. Of ten patients, mNGS was able to identify additional pathogens compared with conventional methods; most of the pathogens were virus. Conclusion: Our results show that mNGS may serve as adjunctive diagnostic tool for the identification of pathogens of hematologic patients with clinically sepsis.

Model organisms are increasingly used to study and understand how neurofilament (NF)‐based neurological diseases develop. However, whether a NF homolog exists in C. elegans remains unclear. We characterize TAG‐63 as a NF‐like protein with sequence homologies to human NEFH carrying various coiled coils as well as clustered phosphorylation sites. TAG‐63 also exhibits features of NFL such as a molecular weight of around 70 kD, the lack of KSP repeats and the ability to form 10 nm filamentous structures in transmission electron micrographs. An anti‐NEFH antibody detects a band at the predicted molecular weight of TAG‐63 in Western blots of whole worm lysates and this band cannot be detected in tag‐63 knockout worms. A transcriptional tag‐63 reporter expresses in a broad range of neurons, and various anti‐NFH antibodies stain worm neurons with an overlapping expression of axonal vesicle transporter UNC‐104(KIF1A). Cultured neurons grow shorter axons when incubating with drugs known to disintegrate the NF network and rhodamine‐labeled in vitro reconstituted TAG‐63 filaments disintegrate upon drug exposure. Speeds of UNC‐104 motors are diminished in tag‐63 mutant worms with visibly increased accumulations of motors along axons. UNC‐104/TAG‐63 and SNB‐1/TAG‐63 not only colocalize in neurons but also revealed positive BiFC (bimolecular fluorescence assay) signals. In summary, we identified and characterized TAG‐63 in C. elegans, and demonstrate that lack of this protein limits axonal transport efficiencies. Additionally, this study would aid in developing NF‐related disease models in the future.