The rockefeller university

G protein gated inward rectifier potassium (GIRK) channels are gated by direct binding of G protein beta-gamma subunits (Gb gamma), signaling lipids, and intracellular Na+. In cardiac pacemaker cells, hetero-tetramer GIRK1/4 channels and homo-tetramer GIRK4 channels play a central role in parasympathetic slowing of heart rate. It is known that the Na+ binding site of the GIRK1 subunit is defective, but the functional difference between GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers remains unclear. Here, using purified proteins and the lipid bilayer system, we characterize Gb gamma and Na+ regulation of GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers. We find in GIRK4 homo-tetramers that Na+ binding increases Gb gamma affinity and thereby increases the GIRK4 responsiveness to G protein stimulation. GIRK1/4 hetero-tetramers are not activated by Na+, but rather are in a permanent state of high responsiveness to Gb gamma, suggesting that the GIRK1 subunit functions like a GIRK4 subunit with Na+ permanently bound.

Group 2 innate lymphoid cells (ILC2) include IL-5- and IL-13-producing CRTh2(+)CD127(+) cells that are implicated in early protective immunity at mucosal surfaces. Whereas functional plasticity has been demonstrated for both human and mouse ILC3 subsets that can reversibly give rise to IFN-gamma-producing ILC1, plasticity of human or mouse ILC2 has not been shown. Here, we analyze the phenotypic and functional heterogeneity of human peripheral blood ILC2. Although subsets of human CRTh2(+) ILC2 differentially express CD117 (c-kit receptor), some ILC2 surface phenotypes are unstable and can be modulated in vitro. Surprisingly, human IL-13(+) ILC2 can acquire the capacity to produce IFN-gamma, thereby generating plastic ILC2. ILC2 cultures demonstrated that IFN-gamma(+) ILC2 clones could be derived and were stably associated with increased T-BET expression. The inductive mechanism for ILC2 plasticity was mapped to the IL-12-IL-12R signaling pathway and was confirmed through analysis of patients with Mendelian susceptibility to mycobacterial disease due to IL-12R beta 1 deficiencies that failed to generate plastic ILC2. We also detected IL-13(+)IFN-gamma(+) ILC2 ex vivo in intestinal samples from Crohn's disease patients. These results demonstrate cytokine production plasticity for human ILC2 and further suggest that environmental cues can dictate ILC phenotype and function for these tissue-resident innate effector cells.

G protein gated inward rectifier K+ (GIRK) channels open and thereby silence cellular electrical activity when inhibitory G protein coupled receptors (GPCRs) are stimulated. Here we describe an assay to measure neuronal GIRK2 activity as a function of membrane-anchored G protein concentration. Using this assay we show that four G beta gamma subunits bind cooperatively to open GIRK2, and that intracellular Na+ which enters neurons during action potentials further amplifies opening mostly by increasing G beta gamma affinity. A Na+ amplification function is characterized and used to estimate the concentration of G beta gamma subunits that appear in the membrane of mouse dopamine neurons when GABreceptors are stimulated. We conclude that GIRK2, through its dual responsiveness to G beta gamma and Na+, mediates a form of neuronal inhibition that is amplifiable in the setting of excess electrical activity.

Fatty acylation of cysteine residues provides spatial and temporal control of protein function in cells and regulates important biological pathways in eukaryotes. Although recent methods have improved the detection and proteomic analysis of cysteine fatty (S-fatty) acylated proteins, understanding how specific sites and quantitative levels of this posttranslational modification modulate cellular pathways are still challenging. To analyze the endogenous levels of protein S-fatty acylation in cells, we developed amass-tag labelingmethod based on hydroxylamine-sensitivity of thioesters and selective maleimide-modification of cysteines, termed acyl-PEG exchange (APE). We demonstrate that APE enables sensitive detection of protein S-acylation levels and is broadly applicable to different classes of S-palmitoylated membrane proteins. Using APE, we show that endogenous interferon-induced transmembrane protein 3 is S-fatty acylated on three cysteine residues and site-specific modification of highly conserved cysteines are crucial for the antiviral activity of this IFN-stimulated immune effector. APE therefore provides a general and sensitive method for analyzing the endogenous levels of protein S-fatty acylation and should facilitate quantitative studies of this regulated and dynamic lipid modification in biological systems.

Solids are distinguished from fluids by their ability to resist shear. In equilibrium systems, the resistance to shear is associated with the emergence of broken translational symmetry as exhibited by a nonuniform density pattern that is persistent, which in turn results from minimizing the free energy. In this work, we focus on a class of systems where this paradigm is challenged. We show that shear-driven jamming in dry granular materials is a collective process controlled by the constraints of mechanical equilibrium. We argue that these constraints can lead to a persistent pattern in a dual space that encodes the statistics of contact forces and the topology of the contact network. The shear-jamming transition is marked by the appearance of this persistent pattern. We investigate the structure and behavior of patterns both in real space and the dual space as the system evolves through the rigidity transition for a range of packing fractions and in two different shear protocols. We show that, in the protocol that creates homogeneous jammed states without shear bands, measures of shear jamming do not depend on strain and packing fraction independently but obey a scaling form with a packing-fraction-dependent characteristic strain that goes to zero at the isotropic jamming point phi(J). We demonstrate that it is possible to define a protocol-independent order parameter in this dual space, which provides a quantitative measure of the rigidity of shear-jammed states.

Objectives/HypothesisTo describe the management and outcomes of Fanconi anemia (FA) patients with head and neck squamous cell carcinoma. Study DesignCohort study. MethodsDemographic information, prognostic factors, therapeutic management, and survival outcomes for FA patients enrolled in the International Fanconi Anemia Registry who developed head and neck squamous cell carcinoma (HNSCC) were analyzed. ResultsThirty-five FA patients were diagnosed with HNSCC at a mean age of 32 years. The most common site of primary cancer was the oral cavity (26 of 35, 74%). Thirty patients underwent surgical resection of the cancer. Sixteen patients received radiation therapy with an average radiation dose of 5,050 cGy. The most common toxicities were high-grade mucositis (9 of 16, 56%), hematologic abnormalities (8 of 16, 50%), and dysphagia (8 of 16, 50%). Three patients received conventional chemotherapy and had significant complications, whereas three patients who received targeted chemotherapy with cetuximab had fewer toxicities. The 5-year overall survival rate was 39%, with a cause-specific survival rate of 47%. ConclusionsFanconi anemia patients have a high risk of developing aggressive HNSCC at an early age. Fanconi anemia patients can tolerate complex ablative and reconstructive surgeries, but careful postoperative care is required to reduce morbidity. The treatment of FA-associated HNSCC is difficult secondary to the poor tolerance of radiation and chemotherapy. However, radiation should be used for high-risk cancers due to the poor survival in these patients. Level of Evidence4. Laryngoscope, 126:870-879, 2016

In the city that never sleeps, great science never takes a break. On 15 January 2016, the 'New York Symposium on Quantitative Biology of the Cell', a one-day local meeting of the American Society for Cell Biology (ASCB), took place at Columbia University Medical Center in upper Manhattan. Focusing on the quantitative understanding of cellular and multicellular systems, this meeting created an otherwise rare opportunity for interaction among scientists at various career levels with differing but complementary backgrounds. Highlighting cutting-edge experimental measurements and theoretical modeling, the symposium broke the barrier between disciplines and ignited a hopefully continuing regional dialogue on the emergent topic of quantitative biology of the cell.

Ingestion is a highly regulated behavior that integrates taste and hunger cues to balance food intake with metabolic needs. To study the dynamics of ingestion in the vinegar fly Drosophila melanogaster, we developed Expresso, an automated feeding assay that measures individual meal-bouts with high temporal resolution at nanoliter scale. Flies showed discrete, temporally precise ingestion that was regulated by hunger state and sucrose concentration. We identify 12 cholinergic local interneurons (IN1, for "ingestion neurons'') necessary for this behavior. Sucrose ingestion caused a rapid and persistent increase in IN1 interneuron activity in fasted flies that decreased proportionally in response to subsequent feeding bouts. Sucrose responses of IN1 interneurons in fed flies were significantly smaller and lacked persistent activity. We propose that IN1 neurons monitor ingestion by connecting sugar-sensitive taste neurons in the pharynx to neural circuits that control the drive to ingest. Similar mechanisms for monitoring and regulating ingestion may exist in vertebrates.

Background: Caregiver burden associated with caring for women with ovarian cancer has received limited focus. However, these patients often have complex needs, requiring a high level of care at home and imposing substantial burdens on caregivers. Objectives: This pilot study assessed the level of caregiver burden experienced by the primary caregivers of patients with end-stage ovarian cancer and identified variables associated with caregiver burden. Methods: Caregiver burden was assessed using the Caregiver Reaction Assessment. Fifty caregivers completed an anonymous and voluntary survey. Pearson correlations and independent samples t tests were used to analyze data. Findings: Most participants were Caucasian, married or living with a partner, and college graduates, with an annual household income of less than $90,000. Caregiver ages ranged from 29-81 years. Participants agreed most with the self-esteem scale, indicating they had pride in caring for their loved ones. Disrupted schedules and financial problems were the most burdensome factors in providing care. Because financial issues affected caregiver burden, nurses should facilitate interdisciplinary support. Future research is needed to determine the impact of nurse-led interventions to reduce caregiver burden.