Cytoplasmic switch of ARS2 isoforms promotes nonsense-mediated mRNA decay and arsenic sensitivity.

The life of RNA polymerase II (RNAPII) transcripts is shaped by the dynamic formation of mutually exclusive ribonucleoprotein complexes (RNPs) that direct transcript biogenesis and turnover. A key regulator of RNA metabolism in the nucleus is the scaffold protein ARS2 (arsenic resistance protein 2), bound to the cap binding complex (CBC). We report here that alternative splicing of ARS2's intron 5, generates cytoplasmic isoforms that lack 270 amino acids from the N-terminal of the protein and are functionally distinct from nuclear ARS2. Switching of ARS2 isoforms within the CBC in the cytoplasm has dramatic functional consequences, changing ARS2 from a NMD inhibitor to a NMD promoter that enhances the binding of UPF1 to NCBP1 and ERF1, favouring SURF complex formation, SMG7 recruitment and transcript degradation. ARS2 isoform exchange is also relevant during arsenic stress, where cytoplasmic ARS2 promotes a global response to arsenic in a CBC-independent manner. We propose that ARS2 isoform switching promotes the proper recruitment of RNP complexes during NMD and the cellular response to arsenic stress. The existence of non-redundant ARS2 isoforms is relevant for cell homeostasis, and stress response.

A Survey Study of Surgeons and Anesthesiologists Regarding Perioperative Multimodal Analgesia for Opioid-Tolerant Patients.

PURPOSE: Perioperative pain management of opioid-tolerant patients can be challenging. Although regional anesthesia and multimodal analgesics may be beneficial, these modalities are often underused. It is unclear whether practice patterns for perioperative pain management are determined by the knowledge, attitudes, and beliefs of surgeons and anesthesiologists. DESIGN: Descriptive survey. METHODS: Using a Qualtrics survey, we polled a randomly selected group of 25 surgeons and 25 anesthesiologists regarding their knowledge, attitudes, beliefs, and practices for pain management in an opioid-tolerant patient. FINDINGS: Of 25, 23 anesthesiologists and 18/25 surgeons responded to the survey. Demographics were similar between the 2 groups. Most of the participant surgeons and anesthesiologists believed that pain management may be challenging in an opioid-tolerant patient. However, only 56% of surgeons would recommend a preoperative pain consultation. Most surgeons and anesthesiologists believed in the efficacy of regional anesthetics. However, 43% of surgeons would not advocate for a regional block, perhaps due to their perception of the added perioperative time. Multimodal analgesics were widely accepted by both surgeons and anesthesiologists. CONCLUSIONS: There is an urgent need to reinforce the importance of patient-centered care, with a specific focus on addressing knowledge gaps and improving perceptions for all the members of the team, including surgeons, anesthesiologists, and perioperative nursing teams, if optimal outcomes are to be achieved for our patients.

Peripheral Nerve Stimulation for Pain Management: A Review.

PURPOSE OF REVIEW: Peripheral nerve stimulation has seen a recent upsurge in utilization for various chronic pain conditions, specifically from a neuropathic etiology, where a single peripheral nerve can be pinpointed as a culprit for pain. RECENT FINDINGS: There is conflicting evidence about the efficacy and long-term outcomes of peripheral nerve stimulation for chronic pain, with most studies being small sized. The focus of this article is to review available evidence for the utilization of peripheral nerve stimulation for chronic pain syndromes as well as upcoming evidence in the immediate postoperative realm. The indications for the use of PNS have expanded from neuropathic pain such as occipital neuralgia and post-amputation pain, to more widespread disease processes such as chronic low back pain. Percutaneous PNS delivered over a 60-day period may provide significant carry-over effects including pain relief, potentially avoiding the need for a permanently implanted system while enabling improved function in patients with chronic pain.

Enhanced Recovery After Surgery: Opioid Sparing Strategies After Discharge: A Review.

PURPOSE OF REVIEW: Many surgical subspecialties have developed enhanced recovery after surgery (ERAS) protocols that focus on multimodal analgesia to limit opioid use during a hospital stay and improve patient recovery. Unfortunately, ERAS protocols do not extend to post-discharge patient care, and opioids continue to be over prescribed. The primary reason seems to be a lack of good quality research evaluating extended use of a multimodal analgesic approach. This review was undertaken to evaluate available evidence for non-opioid analgesics in the postoperative period after discharge, utilizing Pubmed, Scopus, and Google Scholar. RECENT FINDINGS: Several studies have explored strategies to reduce the overprescribing of opioids after surgery without worsening postoperative pain scores or complications. However, these studies do not necessarily reflect on situations where an ultra-restrictive protocol may fail, leading to breakthrough pain. Ultra-restrictive opioid protocols, therefore, could risk undertreatment of acute pain and the development of persistent post-surgical pain, highlighting the need for a review of non-opioid strategies. Our findings show that little research has been conducted on the efficacy of non-opioid therapies post-discharge including acetaminophen, NSAIDs, gabapentin, duloxetine, venlafaxine, tizanidine, valium, and oral ketamine. Further studies are warranted to more precisely evaluate the utility of these agents, specifically for their side effect profile and efficacy in improving pain-control and function while limiting opioid use.

Vsx1 and Chx10 paralogs sequentially secure V2 interneuron identity during spinal cord development.

Paralog factors are usually described as consolidating biological systems by displaying redundant functionality in the same cells. Here, we report that paralogs can also cooperate in distinct cell populations at successive stages of differentiation. In mouse embryonic spinal cord, motor neurons and V2 interneurons differentiate from adjacent progenitor domains that share identical developmental determinants. Therefore, additional strategies secure respective cell fate. In particular, Hb9 promotes motor neuron identity while inhibiting V2 differentiation, whereas Chx10 stimulates V2a differentiation while repressing motor neuron fate. However, Chx10 is not present at the onset of V2 differentiation and in other V2 populations. In the present study, we show that Vsx1, the single paralog of Chx10, which is produced earlier than Chx10 in V2 precursors, can inhibit motor neuron differentiation and promote V2 interneuron production. However, the single absence of Vsx1 does not impact on V2 fate consolidation, suggesting that lack of Vsx1 may be compensated by other factors. Nevertheless, Vsx1 cooperates with Chx10 to prevent motor neuron differentiation in early V2 precursors although these two paralog factors are not produced in the same cells. Hence, this study uncovers an original situation, namely labor division, wherein paralog genes cooperate at successive steps of neuronal development.

The COVID-19 vaccine and interventional procedures: Exploring the relationship between steroid administration and subsequent vaccine efficacy.

OBJECTIVE: Collate available evidence and provide guidance on whether to delay steroid injections after receiving a vaccine, and whether to delay vaccination if a recent steroid injection has been administered, leaving formal recommendations to various national societies. METHODS: A literature search was performed to identify information pertinent to steroid administration and the subsequent downstream effects on vaccine efficacy. The search was initiated on December 20, 2020, and the terms used were (steroid OR cortisone OR dexamethasone) AND (vaccine). The studies were limited to articles in the English language. RESULTS: Six studies specifically addressed the effect of steroids on vaccine efficacy. Three of the 6 studies indicated that steroids could be used during the peri-vaccine period without significant suppression of the immune response. One study associated intra-articular steroid injections with an increased risk of developing influenza even when vaccinated. The remaining 2 studies had mixed findings. One study showed that patients who received dexamethasone, but not prednisolone were able to mount an immune response resulting in increased IgG. Another study showed that vaccine efficacy was maintained if patients were on continuous steroids or steroids after vaccination, but not if they stopped steroids prior to vaccination. CONCLUSIONS: Although there is no shared consensus in the studies reviewed, all but one study noted scenarios in which patients receiving steroids can still be successfully vaccinated.

ARS2 is required for retinal progenitor cell S-phase progression and Müller glial cell fate specification.

During a developmental period that extends postnatally in the mouse, proliferating multipotent retinal progenitor cells produce one of 7 major cell types (rod, cone, bipolar, horizontal, amacrine, ganglion, and Müller glial cells) as they exit the cell cycle in consecutive waves. Cell production in the retina is tightly regulated by intrinsic, extrinsic, spatial, and temporal cues, and is coupled to the timing of cell cycle exit. Arsenic-resistance protein 2 (ARS2, also known as SRRT) is a component of the nuclear cap-binding complex involved in RNA Polymerase II transcription, and is required for cell cycle progression. We show that postnatal retinal progenitor cells (RPCs) require ARS2 for proper progression through S phase, and ARS2 disruption leads to early exit from the cell cycle. Furthermore, we observe an increase in the proportion of cells expressing a rod photoreceptor marker, and a loss of Müller glia marker expression, indicating a role for ARS2 in regulating cell fate specification or differentiation. Knockdown of Flice Associated Huge protein (FLASH), which interacts with ARS2 and is required for cell cycle progression and 3'-end processing of replication-dependent histone transcripts, phenocopies ARS2 knockdown. These data implicate ARS2-FLASH-mediated histone mRNA processing in regulating RPC cell cycle kinetics and neuroglial cell fate specification during postnatal retinal development.

Driving Under the Influence of Cannabis: A Framework for Future Policy.

Marijuana is the most widely consumed illicit substance in the United States, and an increasing number of states have legalized it for both medicinal and recreational purposes. As it becomes more readily available, there will be a concurrent rise in the number of users and, consequently, the number of motor vehicle operators driving under the influence. This article examines the cognitive and psychomotor effects of cannabis, as well as current policy concerning driving under the influence of drugs. The authors performed a MEDLINE search on the epidemiology of cannabis use, its cognitive and psychomotor effects, and policies regarding driving under the influence of drugs. Twenty-eight epidemiological studies, 16 acute cognitive and psychomotor studies, 8 chronic cognitive and psychomotor studies, and pertinent state and federal laws and policies were reviewed. These search results revealed that marijuana use is associated with significant cognitive and psychomotor effects. In addition, the legalization of marijuana varies from state to state, as do the laws pertaining to driving under the influence of drugs. Marijuana is a commonly found illicit substance in motor vehicle operators driving under the influence of drugs. Current evidence shows that blood levels of tetrahydrocannabinol do not correlate well with the level of impairment. In addition, although acute infrequent use of cannabis typically leads to cognitive and psychomotor impairment, this is not consistently the case for chronic heavy use. To establish the framework for driving under the influence of cannabis policy, we must review the current published evidence and examine existing policy at state and federal levels.

Mapping the Pax6 3' untranslated region microRNA regulatory landscape.

BACKGROUND: PAX6 is a homeodomain transcription factor that acts in a highly dosage-sensitive manner to regulate the development and function of the eyes, nose, central nervous system, gut, and endocrine pancreas. Several individual microRNAs (miRNA) have been implicated in regulating PAX6 in different cellular contexts, but a more general view of how they contribute to the fine-tuning and homeostasis of PAX6 is poorly understood. RESULTS: Here, a comprehensive analysis of the Pax6 3' untranslated region was performed to map potential miRNA recognition elements and served as a backdrop for miRNA expression profiling experiments to identify potential cell/tissue-specific miRNA codes. Pax6 3'UTR pull-down studies identified a cohort of miRNA interactors in pancreatic αTC1-6 cells that, based on the spacing of their recognition sites in the Pax6 3'UTR, revealed 3 clusters where cooperative miRNA regulation may occur. Some of these interacting miRNAs have been implicated in α cell function but have not previously been linked to Pax6 function and may therefore represent novel PAX6 regulators. CONCLUSIONS: These findings reveal a regulatory landscape upon which miRNAs may participate in the developmental control, fine-tuning and/or homeostasis of PAX6 levels.

Alveolar epithelial cell processing of nanoparticles activates autophagy and lysosomal exocytosis.

Using confocal microscopy, we quantitatively assessed uptake, processing, and egress of near-infrared (NIR)-labeled carboxylated polystyrene nanoparticles (PNP) in live alveolar epithelial cells (AEC) during interactions with primary rat AEC monolayers (RAECM). PNP fluorescence intensity (content) and colocalization with intracellular vesicles in a cell were determined over the entire cell volume via z stacking. Isotropic cuvette-based microfluorimetry was used to determine PNP concentration ([PNP]) from anisotropic measurements of PNP content assessed by confocal microscopy. Results showed that PNP uptake kinetics and steady-state intracellular content decreased as diameter increased from 20 to 200 nm. For 20-nm PNP, uptake rate and steady-state intracellular content increased with increased apical [PNP] but were unaffected by inhibition of endocytic pathways. Intracellular PNP increasingly colocalized with autophagosomes and/or lysosomes over time. PNP egress exhibited fast Ca2+ concentration-dependent release and a slower diffusion-like process. Inhibition of microtubule polymerization curtailed rapid PNP egress, resulting in elevated vesicular and intracellular PNP content. Interference with autophagosome formation led to slower PNP uptake and markedly decreased steady-state intracellular content. At steady state, cytosolic [PNP] was higher than apical [PNP], and vesicular [PNP] (~80% of intracellular PNP content) exceeded both cytosolic and intracellular [PNP]. These data are consistent with the following hypotheses: 1) autophagic processing of nanoparticles is essential for maintenance of AEC integrity; 2) altered autophagy and/or lysosomal exocytosis may lead to AEC injury; and 3) intracellular [PNP] in AEC can be regulated, suggesting strategies for enhancement of nanoparticle-driven AEC gene/drug delivery and/or amelioration of AEC nanoparticle-related cellular toxicity.

Estradiol Protects White Matter of Male C57BL6J Mice against Experimental Chronic Cerebral Hypoperfusion.

BACKGROUND AND PURPOSE: Estradiol is a sex steroid hormone known to protect the brain against damage related to transient and global cerebral ischemia. In the present study, we leverage an experimental murine model of bilateral carotid artery stenosis (BCAS) to examine the putative effects of estradiol therapy on chronic cerebral hypoperfusion. We hypothesize that long-term estradiol therapy protects against white matter injury and declarative memory deficits associated with chronic cerebral hypoperfusion. METHODS: Adult male C57BL/6J mice underwent either surgical BCAS or sham procedures. Two days after surgery, the mice were given oral estradiol (Sham+E, BCAS+E) or placebo (Sham+P, BCAS+P) treatments daily for 31-34 days. All mice underwent Novel Object Recognition (NOR) testing 31-34 days after the start of oral treatments. Following sacrifice, blood was collected and brains fixed, sliced, and prepared for histological examination of white matter injury and extracellular signal-regulated kinase (ERK) expression. RESULTS: Animals receiving long-term oral estradiol therapy (BCAS-E2 and Sham-E2) had higher plasma estradiol levels than those receiving placebo treatment (BCAS-P and Sham-P). BCAS-E2 mice demonstrated less white matter injury (Klüver-Barrera staining) and performed better on the NOR task compared to BCAS-P mice. ERK expression in the brain was increased in the BCAS compared to sham cohorts. Among the BCAS mice, the BCAS-E2 cohort had a greater number of ERK + cells. CONCLUSION: This study demonstrates a potentially protective role for oral estradiol therapy in the setting of white matter injury and declarative memory deficits secondary to murine chronic cerebral hypoperfusion.

Method to remove photoreceptors from whole mount retina in vitro.

Patch clamp recordings of neurons in the inner nuclear layer of the retina are difficult to conduct in a whole mount retina preparation because surrounding neurons block the path of the patch pipette. Vertical slice preparations or dissociated retinal cells provide access to bipolar cells at the cost of severing the lateral connection between neurons. We have developed a technique to remove photoreceptors from the rodent retina that exposes inner nuclear layer neurons, allowing access for patch clamp recording. Repeated application to and removal of filter paper from the photoreceptor side of an isolated retina effectively and efficiently removes photoreceptor cells and, in degenerate retina, hypertrophied Müller cell end feet. Live-dead assays applied to neurons remaining after photoreceptor removal demonstrated mostly viable cells. Patch clamp recordings from bipolar cells reveal responses similar to those recorded in traditional slice and dissociated cell preparations. An advantage of the photoreceptor peel technique is that it exposes inner retinal neurons in a whole mount retina preparation for investigation of signal processing. A disadvantage is that photoreceptor removal alters input to remaining retinal neurons. The technique may be useful for investigations of extracellular electrical stimulation, photoreceptor DNA analysis, and nonpharmacological removal of light input.NEW & NOTEWORTHY This study reports a method for removing photoreceptors from rodent whole mount retina while preserving the architecture of the inner retina. The method enables easier access to the inner retina for studies of neural processing, such as by patch clamp recording.

ImiRP: a computational approach to microRNA target site mutation.

BACKGROUND: MicroRNAs (miRNAs) are small ~22 nucleotide non-coding RNAs that function as post-transcriptional regulators of messenger RNA (mRNA) through base-pairing to 6-8 nucleotide long target sites, usually located within the mRNA 3' untranslated region. A common approach to validate and probe microRNA-mRNA interactions is to mutate predicted target sites within the mRNA and determine whether it affects miRNA-mediated activity. The introduction of miRNA target site mutations, however, is potentially problematic as it may generate new, "illegitimate sites" target sites for other miRNAs, which may affect the experimental outcome. While it is possible to manually generate and check single miRNA target site mutations, this process can be time consuming, and becomes particularly onerous and error prone when multiple sites are to be mutated simultaneously. We have developed a modular Java-based system called ImiRP (Illegitimate miRNA Predictor) to solve this problem and to facilitate miRNA target site mutagenesis. RESULTS: The ImiRP interface allows users to input a sequence of interest, specify the locations of multiple predicted target sites to mutate, and set parameters such as species, mutation strategy, and disallowed illegitimate target site types. As mutant sequences are generated, ImiRP utilizes the miRBase high confidence miRNA dataset to identify illegitimate target sites in each mutant sequence by comparing target site predictions between input and mutant sequences. ImiRP then assembles a final mutant sequence in which all specified target sites have been mutated. CONCLUSIONS: ImiRP is a mutation generator program that enables selective disruption of specified miRNA target sites while ensuring predicted target sites for other miRNAs are not inadvertently created. ImiRP supports mutagenesis of single and multiple miRNA target sites within a given sequence, including sites that overlap. This software will be particularly useful for studies looking at microRNA cooperativity, where mutagenesis of multiple microRNA target sites may be desired. The software is available at imirp.org and is available open source for download through GitHub ( https://github.com/imirp ).

Assessing characteristics of RNA amplification methods for single cell RNA sequencing.

BACKGROUND: Recently, measurement of RNA at single cell resolution has yielded surprising insights. Methods for single-cell RNA sequencing (scRNA-seq) have received considerable attention, but the broad reliability of single cell methods and the factors governing their performance are still poorly known. RESULTS: Here, we conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. All three methods detected greater than 70% of the expected number of genes and had a 50% probability of detecting genes with abundance greater than 2 to 4 molecules. Despite the small number of molecules, sequencing depth significantly affected gene detection. While biases in detection and quantification were qualitatively similar across methods, the degree of bias differed, consistent with differences in molecular protocol. Measurement reliability increased with expression level for all methods and we conservatively estimate measurements to be quantitative at an expression level greater than ~5-10 molecules. CONCLUSIONS: Based on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information.

Regulation of retinal interneuron subtype identity by the Iroquois homeobox gene Irx6.

Interneuronal subtype diversity lies at the heart of the distinct molecular properties and synaptic connections that shape the formation of the neuronal circuits that are necessary for the complex spatial and temporal processing of sensory information. Here, we investigate the role of Irx6, a member of the Iroquois homeodomain transcription factor family, in regulating the development of retinal bipolar interneurons. Using a knock-in reporter approach, we show that, in the mouse retina, Irx6 is expressed in type 2 and 3a OFF bipolar interneurons and is required for the expression of cell type-specific markers in these cells, likely through direct transcriptional regulation. In Irx6 mutant mice, presumptive type 3a bipolar cells exhibit an expansion of their axonal projection domain to the entire OFF region of the inner plexiform layer, and adopt molecular features of both type 2 and 3a bipolar cells, highlighted by the ectopic upregulation of neurokinin 3 receptor (Nk3r) and Vsx1. These findings reveal Irx6 as a key regulator of type 3a bipolar cell identity that prevents these cells from adopting characteristic features of type 2 bipolar cells. Analysis of the Irx6;Vsx1 double null retina suggests that the terminal differentiation of type 2 bipolar cells is dependent on the combined expression of the transcription factors Irx6 and Vsx1, but also points to the existence of Irx6;Vsx1-independent mechanisms in regulating OFF bipolar subtype-specific gene expression. This work provides insight into the generation of neuronal subtypes by revealing a mechanism in which opposing, yet interdependent, transcription factors regulate subtype identity.

Relationship of pain relief with catastrophizing following interventional pain procedures for low back pain.

INTRODUCTION: Catastrophizing is associated with worse pain outcomes after various procedures suggesting its utility in predicting response. However, the stability of pain catastrophizing as a static predictor has been challenged. We assess, among patients undergoing steroid injections for chronic low back pain (cLBP), whether catastrophizing changes with the clinical response to pain interventions. METHODS: This prospective study enrolled patients undergoing fluoroscopic-guided injections for cLBP. Patients filled out Brief Pain Inventory (BPI) and Pain Catastrophizing Scale (PCS) at baseline and 1-month follow-up. We assessed the change in PCS scores from pre-injection to post-injection and examined its predictors. We also examined the correlation of various domains of BPI, such as pain severity and effect on Relationships, Enjoyment, and Mood (REM), with PCS scores at baseline and follow-up. RESULTS: 128 patients were enrolled. Mean (SD) PCS and pain severity scores at baseline were 22.38 (±13.58) and 5.56 (±1.82), respectively. Follow-up PCS and pain severity scores were 19.76 (±15.25) and 4.42 (±2.38), respectively. The change in PCS pre-injection to post-injection was not significant (p=0.12). Multiple regression models revealed baseline PCS and REM domain of BPI as the most important predictors of change in PCS after injection. Pain severity, activity-related pain, age, sex, insurance status, depression, prior surgery, opioid use, or prior interventions did not predict change in PCS score. In correlation analysis, change in PCS was moderately correlated with change in pain (r=0.38), but weakly correlated with baseline pain in all pain domains. CONCLUSIONS: PCS showed non-significant improvement following steroid injections; the study was not powered for this outcome. Follow-up PCS scores were predicted by the REM domain of BPI, rather than pain severity. Larger studies are needed to evaluate a statistically significant and clinically meaningful change in catastrophizing scores following pain interventions.

The Ramus Intermedius: A Bridge to Survival in the Setting of Triple-Vessel Total Occlusion.

Coronary artery disease continues to remain the leading cause of mortality worldwide. Coronary blood supply is provided through the right and left main coronary arteries. The left main coronary artery (LMCA) in turn gives rise to the left anterior descending (LAD) and left circumflex (LCX) arteries. In some cases, LMCA may trifurcate into the ramus intermedius (RI) in addition to the LAD and LCX arteries. Atherosclerotic plaque formation and rupture with subsequent clot formation and occlusion of coronary arteries are the underlying mechanisms of myocardial infarction. Though the clinical implications of the presence of ramus intermedius (RI) are controversial some data suggest that the RI is associated with an increased risk of atherosclerotic plaque formation in the LMCA and the proximal LAD. Conversely, it has been proposed that the RI provides an additional collateral source of blood supply to the myocardium and may potentially contribute to improved survival. Case reports tout the benefits of RI, specifically in the setting of multivessel coronary artery occlusions. Whether it increases the risk of atherosclerotic plaque formation or whether it is protective has yet to be determined. We present a case of a 58-year-old male who presented with acute coronary syndrome and cardiogenic shock due to total ostial occlusion of LAD. The patient had also chronic total occlusions of the right coronary artery and LCX but a patent RI, which was the only source of blood supply to the myocardium and practically determined the patient's survival. Additionally, we performed a literature review to identify similar cases, to support RI's potentially protective role in enhancing survival.

Complexin facilitates exocytosis and synchronizes vesicle release in two secretory model systems.

Complexins (Cplxs) are small, SNARE-associated proteins believed to regulate fast, calcium-triggered exocytosis. However, studies have pointed to either an inhibitory and/or facilitatory role in exocytosis, and the role of Cplxs in synchronizing exocytosis is relatively unexplored. Here, we compare the function of two types of complexin, Cplx 1 and 2, in two model systems of calcium-dependent exocytosis. In mouse neuromuscular junctions (NMJs), we find that lack of Cplx 1 significantly reduces and desynchronizes calcium-triggered synaptic transmission; furthermore, high-frequency stimulation elicits synaptic facilitation, instead of normal synaptic depression, and the degree of facilitation is highly sensitive to the amount of cytoplasmic calcium buffering. In Cplx 2-null adrenal chromaffin cells, we also find decreased and desynchronized evoked release, and identify a significant reduction in the vesicle pool close to the calcium channels (immediately releasable pool, IRP). Viral transduction with either Cplx 1 or 2 rescues both the size of the evoked response and the synchronicity of release, and it restores the IRP size. Our findings in two model systems are mutually compatible and indicate a role of Cplx 1 and 2 in facilitating vesicle priming, and also lead to the new hypothesis that Cplxs may synchronize vesicle release by promoting coupling between secretory vesicles and calcium channels.