Inwardly rectifying potassium channels (Kir) are a special subset of potassium selective ion channels which pass potassium more easily into rather than out of the cell. These channels mediate a variety of cellular functions, including control of membrane resting potential, maintenance of potassium homeostasis and regulation of cellular metabolism. Given the existence of fifteen Kir genes in mammals, current genetic studies using mutant animals that lack a single channel may have missed many important physiological functions of these channels due to gene redundancy. This issue can be circumvented by using a simple model organism like Drosophila, whose genome encodes only 3 Kir proteins. The sophisticated genetic approaches of Drosophila may also provide powerful tools to identify additional regulation mechanisms of Kir channels. Here we provide an overview of the progress made in elucidating the function of Drosophila Kir channels. The knowledge of Drosophila Kir channels may lead us to uncover novel functions and regulation mechanisms of human Kir channels and help on pathological studies of related diseases.
Combining comparative proteomics and molecular genetics uncovers regulators of synaptic and axonal stability and degeneration in vivo
Degeneration of synaptic and axonal compartments of neurons is an early event contributing to the pathogenesis of many neurodegenerative diseases, but the underlying molecular mechanisms remain unclear. Here, we demonstrate the effectiveness of a novel "top-down" approach for identifying proteins and functional pathways regulating neurodegeneration in distal compartments of neurons. A series of comparative quantitative proteomic screens on synapse-enriched fractions isolated from the mouse brain following injury identified dynamic perturbations occurring within the proteome during both initiation and onset phases of degeneration. In silico analyses highlighted significant clustering of proteins contributing to functional pathways regulating synaptic transmission and neurite development. Molecular markers of degeneration were conserved in injury and disease, with comparable responses observed in synapse-enriched fractions isolated from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. An initial screen targeting thirteen degeneration-associated proteins using mutant Drosophila lines revealed six potential regulators of synaptic and axonal degeneration in vivo. Mutations in CALB2, ROCK2, DNAJC5/CSP, and HIBCH partially delayed injury-induced neurodegeneration. Conversely, mutations in DNAJC6 and ALDHA1 led to spontaneous degeneration of distal axons and synapses. A more detailed genetic analysis of DNAJC5/CSP mutants confirmed that loss of DNAJC5/CSP was neuroprotective, robustly delaying degeneration in axonal and synaptic compartments. Our study has identified conserved molecular responses occurring within synapse-enriched fractions of the mouse brain during the early stages of neurodegeneration, focused on functional networks modulating synaptic transmission and incorporating molecular chaperones, cytoskeletal modifiers, and calcium-binding proteins. We propose that the proteins and functional pathways identified in the current study represent attractive targets for developing therapeutics aimed at modulating synaptic and axonal stability and neurodegeneration in vivo.
A daily body temperature rhythm (BTR) is critical for the maintenance of homeostasis in mammals. Whereas mammals use internal energy to regulate body temperature, ectotherms typically regulate body temperature behaviorally . Some ectotherms maintain homeostasis via a daily temperature preference rhythm (TPR) , but the underlying mechanisms are largely unknown. Here, we show that Drosophila exhibit a daily circadian clock-dependent TPR that resembles mammalian BTR. Pacemaker neurons critical for locomotor activity are not necessary for TPR; instead, the dorsal neuron 2 s (DN2s), whose function was previously unknown, is sufficient. This indicates that TPR, like BTR, is controlled independently from locomotor activity. Therefore, the mechanisms controlling temperature fluctuations in fly TPR and mammalian BTR may share parallel features. Taken together, our results reveal the existence of a novel DN2-based circadian neural circuit that specifically regulates TPR; thus, understanding the mechanisms of TPR will shed new light on the function and neural control of circadian rhythms.
The animal circadian pacemaker is composed of two transcriptional feedback loops, which regulate electrical activity in circadian neurons. Surprisingly, a new study reports that electrical activity can reprogram circadian transcription, and identifies CREB proteins as candidates for this reprograming.
Circadian rhythms are generated by well-conserved interlocked transcriptional feedback loops in animals. In Drosophila, the dimeric transcription factor CLOCK/CYCLE (CLK/CYC) promotes period (per), timeless (tim), vrille (vri), and PAR-domain protein 1 (Pdp1) transcription. PER and TIM negatively feed back on CLK/CYC transcriptional activity, whereas VRI and PDP1 negatively and positively regulate Clk transcription, respectively. Here, we show that the alpha isoform of the Drosophila FOS homolog KAYAK (KAY) is required for normal circadian behavior. KAY-alpha downregulation in circadian pacemaker neurons increases period length by 1.5 h. This behavioral phenotype is correlated with decreased expression of several circadian proteins. The strongest effects are on CLK and the neuropeptide PIGMENT DISPERSING FACTOR, which are both under VRI and PDP1 control. Consistently, KAY-alpha can bind to VRI and inhibit its interaction with the Clk promoter. Interestingly, KAY-alpha can also repress CLK activity. Hence, in flies with low KAY-alpha levels, CLK derepression would partially compensate for increased VRI repression, thus attenuating the consequences of KAY-alpha downregulation on CLK targets. We propose that the double role of KAY-alpha in the two transcriptional loops controlling Drosophila circadian behavior brings precision and stability to their oscillations.
Glial cells are crucial regulators of synapse formation, elimination, and plasticity [1, 2]. In vitro studies have begun to identify glial-derived synaptogenic factors , but neuron-glia signaling events during synapse formation in vivo remain poorly defined. The coordinated development of pre- and postsynaptic compartments at the Drosophila neuromuscular junction (NMJ) depends on a muscle-secreted retrograde signal, the TGF-beta/BMP Glass bottom boat (Gbb) [3, 4]. Muscle-derived Gbb activates the TGF-beta receptors Wishful thinking (Wit) and either Saxophone (Sax) or Thick veins (Tkv) in motor neurons [3, 4]. This induces phosphorylation of Mad (P-Mad) in motor neurons, its translocation into the nucleus with a co-Smad, and activation of transcriptional programs controlling presynaptic bouton growth . Here we show that NMJ glia release the TGF-beta ligand Maverick (Mav), which likely activates the muscle activin-type receptor Punt to potently modulate Gbb-dependent retrograde signaling and synaptic growth. Loss of glial Mav results in strikingly reduced P-Mad at NMJs, decreased Gbb transcription in muscle, and in turn reduced muscle-to-motor neuron retrograde TGF-beta/BMP signaling. We propose that by controlling Gbb release from muscle, glial cells fine tune the ability of motor neurons to extend new synaptic boutons in correlation to muscle growth. Our work identifies a novel glia-derived synaptogenic factor by which glia modulate synapse formation in vivo.
Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.
Intercellular calcium signaling in a gap junction-coupled cell network establishes asymmetric neuronal fates in C. elegans
The C. elegans left and right AWC olfactory neurons specify asymmetric subtypes, one default AWC(OFF) and one induced AWC(ON), through a stochastic, coordinated cell signaling event. Intercellular communication between AWCs and non-AWC neurons via a NSY-5 gap junction network coordinates AWC asymmetry. However, the nature of intercellular signaling across the network and how individual non-AWC cells in the network influence AWC asymmetry is not known. Here, we demonstrate that intercellular calcium signaling through the NSY-5 gap junction neural network coordinates a precise 1AWC(ON)/1AWC(OFF) decision. We show that NSY-5 gap junctions in C. elegans cells mediate small molecule passage. We expressed vertebrate calcium-buffer proteins in groups of cells in the network to reduce intracellular calcium levels, thereby disrupting intercellular communication. We find that calcium in non-AWC cells of the network promotes the AWC(ON) fate, in contrast to the autonomous role of calcium in AWCs to promote the AWC(OFF) fate. In addition, calcium in specific non-AWCs promotes AWC(ON) side biases through NSY-5 gap junctions. Our results suggest a novel model in which calcium has dual roles within the NSY-5 network: autonomously promoting AWC(OFF) and non-autonomously promoting AWC(ON).
Beyond joint implant registries: a patient-centered research consortium for comparative effectiveness in total joint replacement
Discusses the Function and Outcomes Research for Comparative Effectiveness in TJR (FORCE-TJR) research program for total joint replacement funded by the Agency for Healthcare Research and Quality. Led by a team of researchers at the University of Massachusetts Medical School in cooperation with a national network of surgeons, FORCE-TJR assembled a consortium of orthopedic practices to serve as a research laboratory to generate comparative effectiveness research to guide surgeon and patient decisions. The FORCE-TJR has a national scope, is representative of US practices, includes longitudinal patient-reported outcomes, and has the ability to measure implant failure as well as important clinical outcomes and complications.
The annual incidence of traumatic spinal cord injury worldwide is estimated to be 35 patients per million. Nonmissile penetrating spinal injuries most commonly occur in the thoracic region, and the majority has neurologic deficits on admission. The management of patients who lack neurologic deficits is controversial due to the risk of neurologic status alteration intraoperatively. However, failure to intervene increases the risk of infection, delayed onset of neurologic deficits, and worsening functional outcome.A 17-year-old boy presented with an intradural T7-T8 knife penetration injury to the spinal cord with no neurologic deficit. Rapid surgical intervention was critical because the knife was lodged between the 2 hemispheres of the spinal cord. The patient was intubated in the lateral position, transferred to the prone position on a Jackson table, and underwent surgical decompression with laminectomy 1 level above and below the injury site, removal of the knife blade in the original path of trajectory, and repair of the dural tear with a collagen matrix. The patient sustained no neurologic sequelae from the penetrating knife injury. He was able to ambulate at discharge and had no complications. To our knowledge, this is the only report of a patient with intradural spinal cord penetration by a foreign object (knife blade) presenting with a normal neurologic preoperative examination that persisted throughout the course of postoperative care.
A variant of a type V lateral clavicle fracture involving a posteriorly displaced medial segment. A case report
The clavicle connects the shoulder girdle to the axial skeleton, providing support and mobility for optimal upper extremity function. Fractures of the clavicle account for up to 4% of all fractures and comprise up to 44% of all injuries to the shoulder girdle. We present a 63-year-old female patient who suffered what appeared to be a minimally displaced Type V lateral clavicle fracture after a fall as evidenced by an anteroposterior shoulder radiograph. However, an axillary projection demonstrated the proximal segment to be posteriorly displaced and buttonholed through the trapezius musculature with tenting of the skin. The patient underwent an open reduction and Kirschner wire fixation of the fracture with complete healing, subsequent removal of the hardware and return to her previous level of function six months following surgery. After an extensive literature search, we believe this is the first case report documenting a variant of a Type V lateral clavicle fracture, specifically with significant posterior displacement of the proximal segment, mimicking a Type IV AC separation. This fracture pattern is unstable and represents a double disruption of the superior shoulder suspensory complex. Surgical management was successful in returning our patient back to her previous activity of daily living.
Osteoarthritis and lesions to cartilage tissue are diseases that frequently result in impaired joint function and patient disability. The treatment of osteoarthritis, along with local bone defects and systemic skeletal diseases, remains a significant clinical challenge for orthopaedic surgeons. Several bone morphogenetic proteins (BMPs) are known to have osteoinductive effects, whereof BMP-2 and BMP-7 are already approved for clinical applications. There is growing evidence that the metabolism of bone as well as the cartilage damage associated with the above disease processes are strongly inter-related with the interactions of the inflammation-related pathways (in particular prostaglandin E(2) (PGE(2))) and osteogenesis (in particular bone morphogenetic protein-2 (BMP-2)). There is strong evidence that the pathways of prostaglandins and bone morphogenetic proteins are intertwined, and they have recently come into focus in several experimental and clinical studies. This paper focuses on PGE(2) and BMP-2 intertwining pathways in bone and cartilage metabolism, and summarizes the recent experimental and clinical data.
MRI Study of Associated Shoulder Pathology in Patients With Full-thickness Subscapularis Tendon Tears
Subscapularis tendon tears are a well-established cause of shoulder pain. The objective of the current study was to evaluate the associated shoulder pathology in patients with full-thickness subscapularis tendon tears using magnetic resonance imaging. Forty-seven magnetic resonance imaging studies taken between 2008 and 2009 with a diagnosis of full-thickness subscapularis tendon tears were reviewed. The size of the subscapularis tendon tear, amount of muscle volume loss, Goutallier grade, biceps tendon pathology, coracohumeral distance, and associated rotator cuff tears were recorded. Statistical analysis was performed. Patients 55 years and older vs those 54 years and younger had an average subscapularis tear size of 35 vs 19 mm, an average Goutallier grade of 2.7 vs 0.8, and a total muscle volume loss of 25% vs 5%, respectively. Patients with a dislocated vs normal biceps tendons had an average subscapularis tear size of 37 vs 23 mm, an average Goutallier grade of 3 vs 0.9, and a total muscle volume loss of 28% vs 7%, respectively. Patients with vs without concomitant rotator cuff tears had an average subscapularis tear size of 32 vs 17 mm, an average Goutallier grade of 2.3 vs 0.6, and a total muscle volume loss of 21% vs 3%, respectively. Overall average coracohumeral distance measured in the axial plane was 10.8+/-4.6 mm. Average coracaohumeral distance was 14.8 vs 8.1 mm in patients with a Goutallier grade of 0 vs 3 or 4, resepectively, and 13.6 vs 8.5 mm in patients with no rotator cuff tear vs those with a supra- and infraspinatus tear, respectively.Increased age, dislocated biceps tendons, and concomitant rotator cuff tears in patients with full-thickness subscapularis tendon tears are associated with larger subscapularis tendon tear size, higher Goutallier grades, and increased subscapularis muscle volume loss. Decreased coracohumeral distance is associated with a higher Goutallier grade and rotator cuff tears.
Evaluation of sterilization methods following contamination of hamstring autograft during anterior cruciate ligament reconstruction
PURPOSE: Inadvertent contamination of the hamstring autograft during ACL reconstruction is infrequent, but can result in significant complications. The purpose of this study is to evaluate bacterial contamination of hamstring autografts dropped onto the operating room floor and methods of graft decontamination.
METHODS: Hamstring tendons were harvested from patients. Excess tendon not used in the ACL procedure was divided into 6 segments. Segments were assigned to 6 groups (A through F, N = 30 in each group): group A: uncontaminated graft immediately postharvest (control), group B: graft dropped onto the floor (5 s), group C: graft dropped onto the floor (15 s). grafts in groups D to F were dropped onto floor for 15 s then rinsed with saline (group D), bacitracin solution (group E) or chlorhexidine 4 % solution (group F) for 3 min. All grafts were sent to the microbiology laboratory for anaerobic and aerobic cultures.
RESULTS: Cultures were positive in 23 % of graft segments from group A (7/30), 33 % of grafts from group B (10/30), 23 % from group C (7/30), 30 % from group D (9/30) and 3 % from both group E (1/30) and group F (1/30). Sixteen unique organisms were identified, with Staphylococcus aureus as the most common isolate. Grafts rinsed in either bacitracin solution or 4 % chlorhexidine solutions were significantly less likely to be culture positive when compared to control graft segments (p < 0.05). However, there was no significant difference between uncontaminated grafts retrieved in
CONCLUSION: This study supports the practice of decontaminating a dropped ACL hamstring autograft using either 4 % chlorhexidine or bacitracin solution. Specimens should be retrieved sterilely and washed for at least 3 min. This study also demonstrates no advantage in retrieval time of less than 5 s as compared to 15 s for uncontaminated graft. Hamstring harvest in ACL reconstruction may result in positive cultures, thus routine soaking of the hamstring autograft in either bacitracin or 4 % chlorhexidine solution is recommended. In addition, dropped hamstring autograft can be effectively sterilized with bacitracin or 4 % chlorhexidine solution.
LEVEL OF EVIDENCE: II.
pHEMA-nHA Encapsulation and Delivery of Vancomycin and rhBMP-2 Enhances its Role as a Bone Graft Substitute
BACKGROUND: Bone grafts are widely used in orthopaedic procedures. Autografts are limited by donor site morbidity while allografts are known for considerable infection and failure rates. A synthetic composite bone graft substitute poly(2-hydroxyethyl methacrylate)-nanocrystalline hydroxyapatite (pHEMA-nHA) was previously developed to stably press-fit in and functionally repair critical-sized rat femoral segmental defects when it was preabsorbed with a single low dose of 300 ng recombinant human bone morphogenetic protein-2/7 (rhBMP-2/7).
QUESTIONS/PURPOSES: To facilitate clinical translation of pHEMA-nHA as a synthetic structural bone graft substitute, we examined its ability to encapsulate and release rhBMP-2 and the antibiotic vancomycin.
METHODS: We analyzed the compressive behavior and microstructure of pHEMA-nHA as a function of vancomycin incorporation doses using a dynamic mechanical analyzer and a scanning electron microscope. In vitro release of vancomycin was monitored by ultraviolet-visible spectroscopy. Release of rhBMP-2 from pHEMA-nHA-vancomycin was determined by ELISA. Bioactivity of the released vancomycin and rhBMP-2 was examined by bacterial inhibition and osteogenic transdifferentiation capabilities in cell culture, respectively.
RESULTS: Up to 4.8 wt% of vancomycin was incorporated into pHEMA-nHA without compromising its structural integrity and compressive modulus. Encapsulated vancomycin was released in a dose-dependent and sustained manner in phosphate-buffered saline over 2 weeks, and the released vancomycin inhibited Escherichia coli culture. The pHEMA-nHA-vancomycin composite released preabsorbed rhBMP-2 in a sustained manner over 8 days and locally induced osteogenic transdifferentiation of C2C12 cells in culture.
CONCLUSIONS: pHEMA-nHA can encapsulate and deliver vancomycin and rhBMP-2 in a sustained and localized manner with reduced loading doses.
CLINICAL RELEVANCE: The elasticity, osteoconductivity, and rhBMP-2/vancomycin delivery characteristics of pHEMA-nHA may benefit orthopaedic reconstructions or fusions with enhanced safety and efficiency and reduced infection risk.
Dermatophagoides pteronyssinus Major Allergen 1 Activates the Innate Immune Response of the Fruit Fly Drosophila melanogaster
Some allergens with relevant protease activity have the potential to directly interact with host structures. It remains to be elucidated whether this activity is relevant for developing their allergenic properties. The major goal of this study was to elucidate whether allergens with a strong protease activity directly interact with modules of the innate immune system, thereby inducing an immune response. We chose Drosophila melanogaster for our experiments to prevent the results from being influenced by the adaptive immune system and used the armamentarium of methods available for the fly to study the underlying mechanisms. We show that Dermatophagoides pteronyssinus major allergen 1 (Der p 1), the major allergen of the house dust mite, efficiently activates various facets of the Drosophila innate-immune system, including both epithelial and systemic responses. These responses depend on the immune deficiency (IMD) pathway via activation of the NF-κB transcription factor Relish. In addition, the major pathogen associated molecular pattern recognizing receptor of the IMD pathway, peptidoglycan recognition protein-LC, was necessary for this response. We showed that Der p 1, which has cysteine protease activity, cleaves the ectodomain of peptidoglycan recognition protein-LC and, thus, activates the IMD pathway to induce a profound immune response. We conclude that the innate immune response to this allergen-mediated proteolytic cleavage represents an ancient type of danger signaling that may be highly relevant for the primary allergenicity of compounds such as Der p 1.
Pathogen-derived effectors trigger protective immunity via activation of the Rac2 enzyme and the IMD or Rip kinase signaling pathway
Although infections with virulent pathogens often induce a strong inflammatory reaction, what drives the increased immune response to pathogens compared to nonpathogenic microbes is poorly understood. One possibility is that the immune system senses the level of threat from a microorganism and augments the response accordingly. Here, focusing on cytotoxic necrotizing factor 1 (CNF1), an Escherichia coli-derived effector molecule, we showed the host indirectly sensed the pathogen by monitoring for the effector that modified RhoGTPases. CNF1 modified Rac2, which then interacted with the innate immune adaptors IMD and Rip1-Rip2 in flies and mammalian cells, respectively, to drive an immune response. This response was protective and increased the ability of the host to restrict pathogen growth, thus defining a mechanism of effector-triggered immunity that contributes to how metazoans defend against microbes with pathogenic potential.
The mechanisms by which epithelial cells distinguish pathogens from commensal microbes have long puzzled us. Now, McEwan et al. (2012) and Dunbar et al. (2012), in this issue of Cell Host and Microbe, demonstrate that in C. elegans, microbial toxin-induced inhibition of host cellular functions, especially blockade of protein translation, activates the effector-triggered immune response dependent on the transcription factor ZIP-2.
A common defining characteristic of pathogenic bacteria is the expression of a repertoire of effector molecules that have been named virulence factors. These bacterial factors include a -variety of proteins, such as toxins that are internalized by receptors and translocate across endosomal membranes to reach the cytosol, as well as others that are introduced directly into the cell by means of bacterial secretory apparatuses. Given the importance of these effectors for understanding bacterial pathogenicity, significant effort has been made to dissect their molecular mechanisms of action and their respective roles during infection. Herein we will discuss how Drosophila have been used as a model system to study these important microbial effectors, and to understand their contribution to pathogenicity.