Structural and functional determinants in the S5-P region of HCN-encoded pacemaker channels revealed by cysteine-scanning substitutions

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are responsible for the membrane pacemaker current that underlies the spontaneous generation of bioelectrical rhythms. However, their structure-function relationship is poorly understood. Previously, we identified several pore residues that influence HCN gating properties and proposed a pore-to-gate mechanism. Here, we systematically introduced cysteine-scanning substitutions into the descending portion of the P loop (residues 339-345) of HCN1-R (where R is resistance to sulfhydryl-reactive agents) channels, in which all endogenous cysteines except C303 have been removed or replaced. F339C, K340C, A341C, M342C, S343C, and M345C did not produce functional currents. Interestingly, the loss of function phenotype of F339C could be rescued by the reducing agent dithiothreitol (DTT). H344C but not HCN1-R and DTT-treated F339C channels were sensitive to blockade by divalent Cd(2+) (current with 100 microM Cd(2+)/control current at -140 mV = 67.6 +/- 2.9%, 109.3 +/- 3.1%, and 103.8 +/- 1.7%, respectively). Externally applied methanethiosulfate ethylammonium, a covalent sulfhydryl-reactive compound, irreversibly modified H344C by reducing the current at -140 mV (to 43.7 +/- 6.5%), causing a hyperpolarizing steady-state activation shift (change in half-activation voltage: approximately 6 mV) and decelerated gating kinetics (by up to 3-fold). Based on these results, we conclude that pore residues 339-345 are important determinants of the structure-function properties of HCN channels and that the side chain of H344 is externally accessible.     

LUBAC regulates ciliogenesis by promoting CP110 removal from the mother centriole

Primary cilia transduce diverse signals in embryonic development and adult tissues. Defective ciliogenesis results in a series of human disorders collectively known as ciliopathies. The CP110–CEP97 complex removal from the mother centriole is an early critical step for ciliogenesis, but the underlying mechanism for this step remains largely obscure. Here, we reveal that the linear ubiquitin chain assembly complex (LUBAC) plays an essential role in ciliogenesis by targeting the CP110–CEP97 complex. LUBAC specifically generates linear ubiquitin chains on CP110, which is required for CP110 removal from the mother centriole in ciliogenesis. We further identify that a pre-mRNA splicing factor, PRPF8, at the distal end of the mother centriole acts as the receptor of the linear ubiquitin chains to facilitate CP110 removal at the initial stage of ciliogenesis. Thus, our study reveals a direct mechanism of regulating CP110 removal in ciliogenesis and implicates the E3 ligase LUBAC as a potential therapy target of cilia-associated diseases, including ciliopathies and cancers.     

Storage globulins pass through the Golgi apparatus and multivesicular bodies in the absence of dense vesicle formation during early stages of cotyledon development in mung bean

During seed development and maturation, large amounts of storage proteins are synthesized and deposited in protein storage vacuoles (PSVs). Multiple mechanisms have been proposed to be responsible for transporting storage proteins to PSVs in developing seeds. In this study, a specific antibody was raised against the mung bean (Vigna radiata) seed storage protein 8S globulin and its deposition was followed via immunogold electron microscopy in developing mung bean cotyledons. It is demonstrated that non-aggregated 8S globulins are present in multivesicular bodies (MVBs) in early stages of cotyledon development where neither dense vesicles (DVs) nor a PSV were recognizable. However, at later stages of cotyledon development, condensed globulins were visible in both DVs and distinct MVBs with a novel form of partitioning, with the internal vesicles being pushed to one sector of this organelle. These distinct MVBs were no longer sensitive to wortmannin. This study thus indicates a possible role for MVBs in transporting storage proteins to PSVs during the early stage of seed development prior to the involvement of DVs. In addition, wortmannin treatment is shown to induce DVs to form aggregates and to fuse with the plasma membrane.     

A Novel Experimental and Analytical Approach to the Multimodal Neural Decoding of Intent During Social Interaction in Freely-behaving Human Infants

Understanding typical and atypical development remains one of the fundamental questions in developmental human neuroscience. Traditionally, experimental paradigms and analysis tools have been limited to constrained laboratory tasks and contexts due to technical limitations imposed by the available set of measuring and analysis techniques and the age of the subjects. These limitations severely limit the study of developmental neural dynamics and associated neural networks engaged in cognition, perception and action in infants performing “in action and in context”. This protocol presents a novel approach to study infants and young children as they freely organize their own behavior, and its consequences in a complex, partly unpredictable and highly dynamic environment. The proposed methodology integrates synchronized high-density active scalp electroencephalography (EEG), inertial measurement units (IMUs), video recording and behavioral analysis to capture brain activity and movement non-invasively in freely-behaving infants. This setup allows for the study of neural network dynamics in the developing brain, in action and context, as these networks are recruited during goal-oriented, exploration and social interaction tasks.     

Using an Adenosine Triphosphate Bioluminescent Assay to Determine Effective Antibiotic Combinations against Carbapenem-Resistant Gram Negative Bacteria within 24 Hours

Background

Current in vitro combination testing methods involve enumeration by bacterial plating, which is labor-intensive and time-consuming. Measurement of bioluminescence, released when bacterial adenosine triphosphate binds to firefly luciferin-luciferase, has been proposed as a surrogate for bacterial counts. We developed an ATP bioluminescent combination testing assay with a rapid turnaround time of 24h to determine effective antibiotic combinations.

Methods

100 strains of carbapenem-resistant (CR) GNB [30 Acinetobacter baumannii (AB), 30 Pseudomonas aeruginosa (PA) and 40 Klebsiella pneumoniae (KP)] were used. Bacterial suspensions (10⁵ CFU/ml) were added to 96-well plates containing clinically achievable concentrations of multiple single and two-antibiotic combinations. At 24h, the luminescence intensity of each well was measured. Receiver operator characteristic curves were plotted to determine optimal luminescence threshold (T_RLU) to discriminate between inhibitory/non-inhibitory combinations when compared to viable plating. The unweighted accuracy (UA) [(sensitivity + specificity)/2] of T_RLU values was determined. External validation was further done using 50 additional CR-GNB.

Results

Predictive accuracies of T_RLU were high for when all antibiotic combinations and species were collectively analyzed (T_RLU = 0.81, UA = 89%). When individual thresholds for each species were determined, UA remained high. Predictive accuracy was highest for KP (T_RLU = 0.81, UA = 91%), and lowest for AB (T_RLU = 0.83, UA = 87%). Upon external validation, high overall accuracy (91%) was observed. The assay distinguished inhibitory/non-inhibitory combinations with UA of 80%, 94% and 93% for AB, PA and KP respectively.

Conclusion

We developed an assay that is robust at identifying useful combinations with a rapid turn-around time of 24h, and may be employed to guide the timely selection of effective antibiotic combinations.     

A Mucormycosis Case in a Cirrhotic Patient Successfully Treated with Posaconazole and Review of Published Literature

Mucormycosis is an invasive fungal infection associated with a high mortality rate, especially in immunocompromised hosts. Mucormycosis rarely occurs in cirrhotic patients. Here, we report a case of mucormycosis with underlying liver cirrhosis and diabetes mellitus. The patient suffered from maxillary sinusitis and osteomyelitis, and the infection was successfully treated with antifungal agents, surgical debridement, and hyperbaric oxygen therapy. The antifungal treatments used were liposomal amphotericin B, itraconazole, and posaconazole. Although our patient had liver cirrhosis (Child-Pugh classification B), no hepatic decompensation was developed during the treatment course of posaconazole. This is the first report of the safe and effective use of posaconazole for the treatment of mucormycosis in a cirrhotic patient.     

Increased asymmetric and multi-daughter cell division in mechanically confined microenvironments

As the microenvironment of a cell changes, associated mechanical cues may lead to changes in biochemical signaling and inherently mechanical processes such as mitosis. Here we explore the effects of confined mechanical environments on cellular responses during mitosis. Previously, effects of mechanical confinement have been difficult to optically observe in three-dimensional and in vivo systems. To address this challenge, we present a novel microfluidic perfusion culture system that allows controllable variation in the level of confinement in a single axis allowing observation of cell growth and division at the single-cell level. The device is capable of creating precise confinement conditions in the vertical direction varying from high (3 μm) to low (7 μm) confinement while also varying the substrate stiffness (E = 130 kPa and 1 MPa). The Human cervical carcinoma (HeLa) model with a known 3N+ karyotype was used for this study. For this cell line, we observe that mechanically confined cell cycles resulted in stressed cell divisions: (i) delayed mitosis, (ii) multi- daughter mitosis events (from 3 up to 5 daughter cells), (iii) unevenly sized daughter cells, and (iv) induction of cell death. In the highest confined conditions, the frequency of divisions producing more than two progeny was increased an astounding 50-fold from unconfined environments, representing about one half of all successful mitotic events. Notably, the majority of daughter cells resulting from multipolar divisions were viable after cytokinesis and, perhaps suggesting another regulatory checkpoint in the cell cycle, were in some cases observed to re-fuse with neighboring cells post-cytokinesis. The higher instances of abnormal mitosis that we report in confined mechanically stiff spaces, may lead to increased rates of abnormal, viable, cells in the population. This work provides support to a hypothesis that environmental mechanical cues influences structural mechanisms of mitosis such as geometric orientation of the mitotic plane or planes.     

Identification of major histocompatibility complex class I C molecule as an attachment factor that facilitates coronavirus HKU1 spike-mediated infection

Human coronavirus HKU1 (HCoV-HKU1) is a recently discovered human coronavirus associated with respiratory tract infections worldwide. In this study, we have identified the major histocompatibility complex class I C molecule (HLA-C) as an attachment factor in facilitating HCoV-HKU1 spike (S)-mediated infection. HCoV-HKU1 S pseudotyped virus was assembled using a human immunodeficiency virus type 1-derived reporter virus harboring the human codon-optimized spike of HCoV-HKU1. We identified human alveolar epithelial A549 cells as the most susceptible cell line among those tested to infection by HCoV-HKU1 S pseudotypes. A549 cells were shown to bind purified soluble HCoV-HKU1 S(1-600) glycopeptide. To search for the functional receptor for HCoV-HKU1, an A549 cDNA expression library was constructed and transduced into the nonpermissive, baby hamster kidney cells line BHK-21. Transduced cells that bind soluble HCoV-HKU1 S(1-600) glycoprotein with C-terminal FLAG were sorted. Sequencing of two independent clones revealed cDNA inserts encoding HLA-C. Inhibition of HLA-C expression or function by RNAi silencing and anti-HLA-C antibody decreased HCoV-HKU1 S pseudotyped virus infection of A549 cells by 62 to 65%, whereas pretreatment of cells with neuraminidase decreased such infection by only 13%. When HLA-C was constitutively expressed in another nonpermissive cell line, NIH-3T3, quantitative PCR showed that the binding of HCoV-HKU1 S pseudotyped virus to cell surfaces was increased by 200-fold, but the cells remained nonsusceptible to HCoV-HKU1 S pseudotyped virus infection. Our data suggest that HLA-C is involved in the attachment of HCoV-HKU1 to A549 cells and is a potential candidate to facilitate cell entry. However, other unknown surface proteins on A549 cells may be concomitantly utilized by S glycoprotein of HCoV-HKU1 during viral entry. Further studies are required to elucidate other putative receptors or coreceptors for HCoV-HKU1 and the mechanism of HCoV-HKU1 S-mediated cell entry.