A consensus platform for antibody characterization.

Antibody-based research applications are critical for biological discovery. Yet there are no industry standards for comparing the performance of antibodies in various applications. We describe a knockout cell line-based antibody characterization platform, developed and approved jointly by industry and academic researchers, that enables the systematic comparison of antibody performance in western blot, immunoprecipitation and immunofluorescence.

What Is the Role of a Periarticular Injection for Knee Arthroplasty Patients Receiving a Multimodal Analgesia Regimen Incorporating Adductor Canal and Infiltration Between the Popliteal Artery and Capsule of the Knee Blocks? A Randomized Blinded Placebo-Controlled Noninferiority Trial.

Background: Optimal analgesic protocols for total knee arthroplasty (TKA) patients remain controversial. Multimodal analgesia is advocated, often including peripheral nerve blocks and/or periarticular injections (PAIs). If 2 blocks (adductor canal block [ACB] plus infiltration between the popliteal artery and capsule of the knee [IPACK]) are used, also performing PAI may not be necessary.

The Arf-GEF GBF1 undergoes multi-domain structural shifts to activate Arf at the Golgi.

Golgi homeostasis require the activation of Arf GTPases by the guanine-nucleotide exchange factor requires GBF1, whose recruitment to the Golgi represents a rate limiting step in the process. GBF1 contains a conserved, catalytic, Sec7 domain (Sec7d) and five additional (DCB, HUS, HDS1-3) domains. Herein, we identify the HDS3 domain as essential for GBF1 membrane association in mammalian cells and document the critical role of HDS3 during the development of .

Phylogenetic profiling and cellular analyses of ARL16 reveal roles in traffic of IFT140 and INPP5E.

The ARF family of regulatory GTPases is ancient, with 16 members predicted to have been present in the last eukaryotic common ancestor. Our phylogenetic profiling of paralogues in diverse species identified four family members whose presence correlates with that of a cilium/flagellum: ARL3, ARL6, ARL13, and ARL16. No prior evidence links ARL16 to cilia or other cell functions, despite its presence throughout eukaryotes.

The ARF GAPs ELMOD1 and ELMOD3 act at the Golgi and cilia to regulate ciliogenesis and ciliary protein traffic.

ELMODs are a family of three mammalian paralogues that display GTPase-activating protein (GAP) activity toward a uniquely broad array of ADP-ribosylation factor (ARF) family GTPases that includes ARF-like (ARL) proteins. ELMODs are ubiquitously expressed in mammalian tissues, highly conserved across eukaryotes, and ancient in origin, being present in the last eukaryotic common ancestor. We described functions of ELMOD2 in immortalized mouse embryonic fibroblasts (MEFs) in the regulation of cell division, microtubules, ciliogenesis, and mitochondrial fusion.

A Eukaryote-Wide Perspective on the Diversity and Evolution of the ARF GTPase Protein Family.

The evolution of eukaryotic cellular complexity is interwoven with the extensive diversification of many protein families. One key family is the ARF GTPases that act in eukaryote-specific processes, including membrane traffic, tubulin assembly, actin dynamics, and cilia-related functions. Unfortunately, our understanding of the evolution of this family is limited.

A Comprehensive Enhanced Recovery Pathway for Rotator Cuff Surgery Reduces Pain, Opioid Use, and Side Effects.

Background: Patients often have moderate to severe pain after rotator cuff surgery, despite receiving analgesics and nerve blocks. There are many suggested ways to improve pain after rotator cuff surgery, but the effects of adopting a pathway that includes formal patient education, a long-acting nerve block, and extensive multimodal analgesia are unclear.

Questions/purposes: (1) Does adoption of a clinical pathway incorporating patient education, a long-acting nerve block, and preemptive multimodal analgesia reduce the worst pain during the first 48 hours after surgery compared with current standard institutional practices? (2) Does adoption of the pathway reduce opioid use? (3) Does adoption of the pathway reduce side effects and improve patient-oriented outcomes?

Methods: From September 2018 to January 2020, 281 patients scheduled for arthroscopic ambulatory rotator cuff surgery were identified for this paired sequential prospective cohort study.

Roles for ELMOD2 and Rootletin in ciliogenesis.

ELMOD2 is a GTPase-activating protein with uniquely broad specificity for ARF family GTPases. We previously showed that it acts with ARL2 in mitochondrial fusion and microtubule stability and with ARF6 during cytokinesis. Mouse embryonic fibroblasts deleted for ELMOD2 also displayed changes in cilia-related processes including increased ciliation, multiciliation, ciliary morphology, ciliary signaling, centrin accumulation inside cilia, and loss of rootlets at centrosomes with loss of centrosome cohesion.

Chloroprocaine Provides Safe, Effective, Short-Acting Spinal Anesthesia Ideal for Ambulatory Surgeries: A Retrospective Review.

Background: The ideal local anesthetic for use in ambulatory spinal anesthesia is safe, with minimal adverse effects, and of a duration that does not impede post-anesthesia care unit (PACU) discharge. Since its approval for use in spinal anesthesia in Europe in 2012, chloroprocaine has seen a resurgence. Recent studies have investigated the safety and efficacy of preservative-free chloroprocaine for use in spinal anesthesia, but few provide the incidence of adverse events such as urinary retention and transient neurologic symptoms.

The ARF GAP ELMOD2 acts with different GTPases to regulate centrosomal microtubule nucleation and cytokinesis.

ELMOD2 is a ∼32 kDa protein first purified by its GTPase-activating protein (GAP) activity toward ARL2 and later shown to have uniquely broad specificity toward ARF family GTPases in in vitro assays. To begin the task of defining its functions in cells, we deleted ELMOD2 in immortalized mouse embryonic fibroblasts and discovered a number of cellular defects, which are reversed upon expression of ELMOD2-myc. We show that these defects, resulting from the loss of ELMOD2, are linked to two different pathways and two different GTPases: with ARL2 and TBCD to support microtubule nucleation from centrosomes and with ARF6 in cytokinesis.

ARF family GTPases with links to cilia.

The ADP-ribosylation factor (ARF) superfamily of regulatory GTPases, including both the ARF and ARF-like (ARL) proteins, control a multitude of cellular functions, including aspects of vesicular traffic, lipid metabolism, mitochondrial architecture, the assembly and dynamics of the microtubule and actin cytoskeletons, and other pathways in cell biology. Considering their general utility, it is perhaps not surprising that increasingly ARF/ARLs have been found in connection to primary cilia. Here, we critically evaluate the current knowledge of the roles four ARF/ARLs (ARF4, ARL3, ARL6, ARL13B) play in cilia and highlight key missing information that would help move our understanding forward.

ARL13B regulates Sonic hedgehog signaling from outside primary cilia.

ARL13B is a regulatory GTPase highly enriched in cilia. Complete loss of disrupts cilia architecture, protein trafficking and Sonic hedgehog signaling. To determine whether ARL13B is required within cilia, we knocked in a cilia-excluded variant of ARL13B (V358A) and showed it retains all known biochemical function.

Homozygous Variant in ARL3 Causes Autosomal Recessive Cone Rod Dystrophy.

Purpose: Cone rod dystrophy (CRD) is a group of inherited retinopathies characterized by the loss of cone and rod photoreceptor cells, which results in poor vision. This study aims to clinically and genetically characterize the segregating CRD phenotype in two large, consanguineous Pakistani families.

Methods: Funduscopy, optical coherence tomography (OCT), electroretinography (ERG), color vision, and visual acuity assessments were performed to evaluate the retinal structure and function of the affected individuals.

Cerebral Oxygenation in the Sitting Position Is Not Compromised During Spontaneous or Positive-Pressure Ventilation.

Background: General anesthesia with neuromuscular blockade may facilitate total shoulder arthroplasty but appears to increase risk of cerebral oxygen desaturation. Cerebral desaturation is undesirable and is a proxy for risk of stroke.

Purposes/questions: This study tested the hypothesis that cerebral oxygen desaturation occurs frequently during general anesthesia with neuromuscular blockade and positive-pressure ventilation but does not occur with spontaneous ventilation.

Ancient complement and lineage-specific evolution of the Sec7 ARF GEF proteins in eukaryotes.

Guanine nucleotide exchange factors (GEFs) are the initiators of signaling by every regulatory GTPase, which in turn act to regulate a wide array of essential cellular processes. To date, each family of GTPases is activated by distinct families of GEFs. Bidirectional membrane trafficking is regulated by ADP-ribosylation factor (ARF) GTPases and the development throughout eukaryotic evolution of increasingly complex systems of such traffic required the acquisition of a functionally diverse cohort of ARF GEFs to control it.

Compositional complexity of rods and rings.

Rods and rings (RRs) are large linear- or circular-shaped structures typically described as polymers of IMPDH (inosine monophosphate dehydrogenase). They have been observed across a wide variety of cell types and species and can be induced to form by inhibitors of IMPDH. RRs are thought to play a role in the regulation of de novo guanine nucleotide synthesis; however, the function and regulation of RRs is poorly understood.

The Incidence of Complications Is Low Following Foot and Ankle Surgery for Which Peripheral Nerve Blocks Are Used for Postoperative Pain Management.

Background: The incidence of neurologic complications from foot and ankle surgery utilizing regional anesthesia is not well established.

Questions/purposes: The purpose of this study was to prospectively determine the incidence of neurologic and peripheral nerve block (PNB) site complications on a busy foot and ankle service that utilizes ankle blocks (ABs) and popliteal blocks (POPs).

Patients And Methods: This prospective observational study included patients undergoing foot and ankle surgery with ABs or POPs.

Perineural Low-Dose Dexamethasone Prolongs Interscalene Block Analgesia With Bupivacaine Compared With Systemic Dexamethasone: A Randomized Trial.

Background And Objectives: Perineural dexamethasone and intravenous (IV) dexamethasone have been shown to prolong peripheral nerve block duration. The effects of perineural and IV dexamethasone have only been compared at doses of 4 mg or greater. This triple-blind, randomized trial examined the effect of 1 mg IV versus perineural dexamethasone on interscalene block (ISB) analgesia duration.

Readiness for Discharge After Foot and Ankle Surgery Using Peripheral Nerve Blocks: A Randomized Controlled Trial Comparing Spinal and General Anesthesia as Supplements to Nerve Blocks.

Background: Neuraxial anesthesia is often viewed as superior to general anesthesia but may delay discharge. Comparisons do not typically use multimodal analgesics and nerve blockade. Combining nerve blockade with general anesthesia may reduce pain, opioid consumption, and nausea.