Single-cell multi-gene identification of somatic mutations and gene rearrangements in cancer.

In this proof-of-concept study, we developed a single-cell method that provides genotypes of somatic alterations found in coding regions of messenger RNAs and integrates these transcript-based variants with their matching cell transcriptomes. We used nanopore adaptive sampling on single-cell complementary DNA libraries to validate coding variants in target gene transcripts, and short-read sequencing to characterize cell types harboring the mutations. CRISPR edits for 16 targets were identified using a cancer cell line, and known variants in the cell line were validated using a 352-gene panel. Variants in primary cancer samples were validated using target gene panels ranging from 161 to 529 genes. A gene rearrangement was also identified in one patient, with the rearrangement occurring in two distinct tumor sites.

More Than an ERAS Pathway is Needed to Meet Target Length of Stay After Pancreaticoduodenectomy.

BACKGROUND: Enhanced Recovery After Surgery (ERAS) protocols have been successfully instituted for pancreaticoduodenectomy (PD). This study evaluates reasons patients fail to meet length of stay (LOS) and areas for pathway improvement. MATERIALS AND METHODS: A multidisciplinary team developed and implemented an ERAS protocol for open PD in 2017. The study includes a medical record review of all patients who were perioperatively managed with the ERAS protocol and failed to meet LOS after PD procedures. Target LOS was defined as 7 d. RESULTS: From 2017 to 2020, 44% (93 of 213) of patients using ERAS protocol after PD procedures failed to meet target LOS. The most common reason to fail target LOS was ileus or delayed gastric emptying (47 of 93, LOS 11). Additional reasons included work-up of leukocytosis or pancreatic leak (17 of 93, LOS 14), additional "night" of observation (14 of 93, LOS 8), and orthostatic hypotension (3 of 93, LOS 10). Of these additional 46 patients, 19 patients underwent computed tomography (on or after POD 7) and only four patients received additional inpatient intervention. CONCLUSIONS: The most common reason for PD pathway failure included slow return of gastrointestinal function, a known complication after PD. The remaining patients were often kept for observation without additional intervention. This group represents an actionable cohort to target for improving LOS through surgeon awareness rather than protocol modification.

Rab6 promotes insulin receptor and cathepsin trafficking to regulate autophagy induction and activity in Drosophila.

The self-degradative process of autophagy is important for energy homeostasis and cytoplasmic renewal. This lysosome-mediated pathway is negatively regulated by the target of rapamycin kinase (TOR) under basal conditions, and requires the vesicle trafficking machinery regulated by Rab GTPases. However, the interactions between autophagy, TOR and Rab proteins remain incompletely understood in vivo Here, we identify Rab6 as a critical regulator of the balance between TOR signaling and autolysosome function. Loss of Rab6 causes an accumulation of enlarged autophagic vesicles resulting in part from a failure to deliver lysosomal hydrolases, rendering autolysosomes with a reduced degradative capacity and impaired turnover. Additionally, Rab6-deficient cells are reduced in size and display defective insulin-TOR signaling as a result of mis-sorting and internalization of the insulin receptor. Our findings suggest that Rab6 acts to maintain the reciprocal regulation between autophagy and TOR activity during distinct nutrient states, thereby balancing autophagosome production and turnover to avoid autophagic stress.

Coordination of autophagosome-lysosome fusion and transport by a Klp98A-Rab14 complex in Drosophila.

Degradation of cellular material by autophagy is essential for cell survival and homeostasis, and requires intracellular transport of autophagosomes to encounter acidic lysosomes through unknown mechanisms. Here, we identify the PX-domain-containing kinesin Klp98A as a new regulator of autophagosome formation, transport and maturation in Drosophila. Depletion of Klp98A caused abnormal clustering of autophagosomes and lysosomes at the cell center and reduced the formation of starvation-induced autophagic vesicles. Reciprocally, overexpression of Klp98A redistributed autophagic vesicles towards the cell periphery. These effects were accompanied by reduced autophagosome-lysosome fusion and autophagic degradation. In contrast, depletion of the conventional kinesin heavy chain caused a similar mislocalization of autophagosomes without perturbing their fusion with lysosomes, indicating that vesicle fusion and localization are separable and independent events. Klp98A-mediated fusion required the endolysosomal GTPase Rab14, which interacted and colocalized with Klp98A, and required Klp98A for normal localization. Thus, Klp98A coordinates the movement and fusion of autophagic vesicles by regulating their positioning and interaction with the endolysosomal compartment.