AC6 regulates the microtubule-depolymerizing kinesin KIF19A to control ciliary length in mammals.

Motile cilia are hairlike structures that line the respiratory and reproductive tracts and the middle ear and generate fluid flow in these organs via synchronized beating. Cilium growth is a highly regulated process that is assumed to be important for flow generation. Recently, Kif19a, a kinesin residing at the cilia tip, was identified to be essential for ciliary length control through its microtubule depolymerization function. However, there is a lack of information on the nature of proteins and the integrated signaling mechanism regulating growth of motile cilia. Here, we report that adenylate cyclase 6 (AC6), a highly abundant AC isoform in airway epithelial cells, inhibits degradation of Kif19a by inhibiting autophagy, a cellular recycling mechanism for damaged proteins and organelles. Using epithelium-specific knockout mice of AC6, we demonstrated that AC6 knockout airway epithelial cells have longer cilia compared with the WT cells because of decreased Kif19a protein levels in the cilia. We demonstrated in vitro that AC6 inhibits AMP-activated kinase (AMPK), an important modulator of cellular energy-conserving mechanisms, and uncouples its binding with ciliary kinesin Kif19a. In the absence of AC6, activation of AMPK mobilizes Kif19a into autophagosomes for degradation in airway epithelial cells. Lower Kif19a levels upon pharmacological activation of AMPK in airway epithelial cells correlated with elongated cilia and vice versa. In all, the AC6-AMPK pathway, which is tunable to cellular cues, could potentially serve as one of the crucial ciliary growth checkpoints and could be channeled to develop therapeutic interventions for cilia-associated disorders.

Muscle and Tendon Contributions to Reduced Rate of Torque Development in Healthy Older Males.

Background: The ability to rapidly generate and transfer muscle force is essential for effective corrective movements in order to prevent a fall. The aim of this study was to establish the muscle and tendon contributions to differences in rate of torque development (RTD) between younger (YM) and older males (OM). Method: Twenty-eight young males (23.9 years ± 1.1) and 22 old males (68.5 years ± 0.5) were recruited for assessment of Quadriceps Anatomical CSA (ACSA), maximal voluntary contraction (MVC), rate of torque development (RTD), and tendon biomechanical properties. Activation capacity (AC), maximal muscle twitch df/dt) and time to peak EMG amplitude (TTPE) were also assessed. Results: Absolute RTD (aRTD) was lower in OM (577.5 ± 34.6 Nm/s vs 881.7 ± 45.6 Nm/s, p < .0001). RTD remained lower in OM following normalization (nRTD) for muscle ACSA (9.93 ± 0.7 Nm/s/cm2 vs 11.9 ± 0.6 Nm/s/cm2, p < .05). Maximal muscle twitch df/dt (1,086 Nm∙s-1 vs 2,209 Nm∙s-1, p < .0001), TTPE (109.2 ± 8.6ms vs 154.6 ± 16.6 ms, p < .05), and AC (75.8 ± 1.5% vs 80.1 ± 0.9%, p < .01) were all affected in OM. Tendon stiffness was found to be lower in OM (1,222 ± 78.4 N/mm vs 1,771 ± 154.1 N/mm, p < .004). nRTD was significantly correlated with tendon stiffness (R2 = .15). Conclusion: These observations provide evidence that in absolute terms, a lower RTD in the elderly adults is caused by slower muscle contraction speeds, slower TTPE, reduced ACSA, reduced MVC, and a decrease in tendon stiffness. Once the RTD is normalized to quadriceps ACSA, only MVC and tendon stiffness remain influential. This strongly reinforces the importance of both muscle and tendon characteristics when considering RTD.

Sarcopenia, dynapenia, and the impact of advancing age on human skeletal muscle size and strength; a quantitative review.

Changing demographics make it ever more important to understand the modifiable risk factors for disability and loss of independence with advancing age. For more than two decades there has been increasing interest in the role of sarcopenia, the age-related loss of muscle or lean mass, in curtailing active and healthy aging. There is now evidence to suggest that lack of strength, or dynapenia, is a more constant factor in compromised wellbeing in old age and it is apparent that the decline in muscle mass and the decline in strength can take quite different trajectories. This demands recognition of the concept of muscle quality; that is the force generating per capacity per unit cross-sectional area (CSA). An understanding of the impact of aging on skeletal muscle will require attention to both the changes in muscle size and the changes in muscle quality. The aim of this review is to present current knowledge of the decline in human muscle mass and strength with advancing age and the associated risk to health and survival and to review the underlying changes in muscle characteristics and the etiology of sarcopenia. Cross-sectional studies comparing young (18-45 years) and old (>65 years) samples show dramatic variation based on the technique used and population studied. The median of values of rate of loss reported across studies is 0.47% per year in men and 0.37% per year in women. Longitudinal studies show that in people aged 75 years, muscle mass is lost at a rate of 0.64-0.70% per year in women and 0.80-00.98% per year in men. Strength is lost more rapidly. Longitudinal studies show that at age 75 years, strength is lost at a rate of 3-4% per year in men and 2.5-3% per year in women. Studies that assessed changes in mass and strength in the same sample report a loss of strength 2-5 times faster than loss of mass. Loss of strength is a more consistent risk for disability and death than is loss of muscle mass.

A variant human IgG1-Fc mediates improved ADCC.

Ribosome display was applied to the Fc region of human immunoglobulin G (IgG1) to select for improved binding to human FcγRIIIa, the receptor expressed on human natural killer cells that mediates antibody-dependent cellular cytotoxicity (ADCC). A library of human Fcγ1 variants was generated using error-prone polymerase chain reaction, and subjected to multiple rounds of ribosome display selection against progressively decreasing concentrations of soluble human FcγRIIIa, to enrich for improved binders. Radioimmunoassay and alphascreen analyses of the aglycosylated IgG-Fc output revealed variants with improved binding to FcγRIIIa relative to wild-type IgG-Fc. Subsequent expression in human (HEK-EBNA) cells generated glycosylated IgGs with modified activity in ADCC assays. One particular variant, 125_B01 triggered enhanced ADCC (EC(50) up to four-fold reduced with increased maximal lysis) relative to wild-type antibody, having more equal levels of ADCC for each allotype (V158/F158) of FcγRIIIa. Deconvolution of individual replacements within the variant showed that improved function arose from the Phe243Leu replacement within the CH2 domain, rather than the CH3 domain replacements Thr393Ala or His433Pro. Surprisingly, the oligosaccharide profiles of 125_B01 indicated more oligosaccharide chains lacking fucose, or with bisecting N-acetylglucosamine relative to wild-type IgG1, which correlates with improved function and the replacement Phe243Leu that is a carbohydrate contact residue within the C(H)2 domain.

Molding single DNA molecules in metals and sample preparation for electronic sequencing.

We demonstrate the molding of single DNA molecules in 8 nm thin platinum molds. The molded structures have an apparent depth of 1 nm under STM imaging, and closely follow the contours of the DNA molecules. We have confirmed the presence of the embedded molecules and have verified the ability of this technique to scale down to single molecules. Additionally, we have utilized this method to perform electron tunneling analysis on embedded DNA molecules.

Scanning probe and nanopore DNA sequencing: core techniques and possibilities.

We provide an overview of the current state of research towards DNA sequencing using nanopore and scanning probe techniques. Additionally, we provide methods for the creation of two key experimental platforms for studies relating to nanopore and scanning probe DNA studies: a synthetic nanopore apparatus and an atomically flat conductive substrate with stretched DNA molecules.

Identification of cyclic peptides able to mimic the functional epitope of IgG1-Fc for human Fc gammaRI.

Identification of short, structured peptides able to mimic potently protein-protein interfaces remains a challenge in drug discovery. We report here the use of a naive cyclic peptide phage display library to identify peptide ligands able to recognize and mimic IgG1-Fc functions with Fc gammaRI. Selection by competing off binders to Fc gammaRI with IgG1 allowed the isolation of a family of peptides sharing the common consensus sequence TX(2)CXXthetaPXLLGCPhiXE (theta represents a hydrophobic residue, Phi is usually an acidic residue, and X is any residue) and able to inhibit IgG1 binding to Fc gammaRI. In soluble form, these peptides antagonize superoxide generation mediated by IgG1. In complexed form, they trigger phagocytosis and a superoxide burst. Unlike IgG, these peptides are strictly Fc gammaRI-specific among the Fc gammaRs. Molecular modeling studies suggest that these peptides can adopt 2 distinct and complementary conformers, each able to mimic the discontinuous interface contacts constituted by the Cgamma2-A and -B chains of Fc for Fc gammaRI. In addition, by covalent homodimerization, we engineered a synthetic bivalent 37-mer peptide that retains the ability to trigger effector functions. We demonstrate here that it is feasible to maintain IgG-Fc function within a small structured peptide. These peptides represent a new format for modulation of effector functions.

Toward nanoscale genome sequencing.

This article reports on the state-of-the-art technologies that sequence DNA using miniaturized devices. The article considers the miniaturization of existing technologies for sequencing DNA and the opportunities for cost reduction that 'on-chip' devices can deliver. The ability to construct nano-scale structures and perform measurements using novel nano-scale effects has provided new opportunities to identify nucleotides directly using physical, and not chemical, methods. The challenges that these technologies need to overcome to provide a US$1000-genome sequencing technology are also presented.

Label-free direct electronic detection of biomolecules with amorphous silicon nanostructures.

We present the fabrication and characterization of a nano-scale sensor made of amorphous silicon for the label-free, electronic detection of three classes of biologically important molecules: ions, oligonucleotides, and proteins. The sensor structure has an active element which is a 50 nm wide amorphous silicon semicircle and has a total footprint of less than 4 microm2. We demonstrate the functionalization of the sensor with receptor molecules and the electronic detection of three targets: H(+) ions, short single-stranded DNAs, and streptavidin. The sensor is able to reliably distinguish single base-pair mismatches in 12 base long strands of DNA and monitor the introduction and identification of straptavidin in real-time. The versatile sensor structure can be readily functionalized with a wide range of receptor molecules and is suitable for integration with high-speed electronic circuits as a post-process on an integrated circuit chip.

Colorectal cancer: current care, future innovations and economic considerations.

For those involved in colorectal cancer management, the present day is an exciting time. There is a multitude of new techniques to be considered for early detection (screening). National population screening for 60-69-year olds in England is due to start this year. Also, minimally invasive surgical techniques and multimodal pathways of care are aiding faster recovery, and there are increasing options for both adjuvant and palliative therapies. This article summarizes how colorectal cancer is currently managed in the UK and discusses the developments that are in the early stages of clinical use or on the horizon. Current management is discussed in detail in the hope that innovators reading the article may identify areas for improvement and allow comparison of new interventions with what are currently the gold standards. As changes are moving so fast, this review will probably only relate to the next 10 years at most. It does not provide a detailed reference list to support all therapies but indicates the key publications that will enable more detailed reading.

Engineering high affinity superantigens by phage display.

Protein L (PpL) is a B-cell superantigen from Peptostreptococcus magnus known to bind to mammalian Vkappa light chains. PpL from P.magnus strain 312 comprises five homologous immunoglobulin (Ig) binding domains. We first analysed the binding of the individual domains (B1-B5) of PpL(312) to human Vkappa light chains (huVkappa) subtypes 1 (huVkappaI) and 3 (huVkappaIII). Using a combination of rational design and phage selection we isolated mutants of the N-terminal B1 domain with a 14-fold increased affinity for huVkappa1 (B1kappa1) and >tenfold increased affinity for huVkappaIII (B1kappa3). We investigated the potential of the selected domains, in particular the B1kappa1 domain, as reagents in immunochemistry and immunotherapy. B1kappa1 proved a superior reagent than the wild-type domain, allowing up to tenfold more sensitive detection of human Vkappa antibody fragments in ELISA. A fusion protein of B1kappa1 with a human Vlambda antibody scFv fragment promoted the efficient recruitment of antibody encoded effector functions including complement, mononuclear phagocyte respiratory burst and phagocytosis through retargeting of IgGkappa and IgMkappa. Our results suggest that superantigens with improved affinity and/or specificity are easily accessible through protein engineering. Such engineered superantigens should prove useful as reagents in immunochemistry and may have potential as agents in immunotherapy.

Site-specific glycosylation of an aglycosylated human IgG1-Fc antibody protein generates neoglycoproteins with enhanced function.

A range of well-defined IgG glycoforms was prepared by employing a combination of synthetic carbohydrate chemistry and genetic engineering. The key aspect of this methodology is the coupling of thioaldoses with cysteine-containing proteins to give disulfide-linked neoglycoproteins. This technology was applied to the synthesis of a series of synthetic N-glycan thioaldoses which were coupled to an aglycosylated IgG1-Fc fragment, engineered to have Cys-297 in place of glycan-linked Asn (Deltah-Fc N297C). Analysis of the resulting Fc neoglycoproteins by mass spectrometry and trypsin digestion showed that the saccharides were site-selectively incorporated at Cys-297 to full occupancy without affecting other Fc protein disulfides. The neoglycoproteins were tested for their ability to interact with human FcgammaRI by inhibiting superoxide production by gamma-interferon-stimulated U937 cells. The neoglycoproteins displayed enhanced superoxide inhibition relative to aglycosylated Deltah-Fc N297C, where increased glycan size correlated positively with increased inhibition.

Interaction sites on human IgG-Fc for FcgammaR: current models.

Recombinant monoclonal antibodies have entered the clinic as effective in vivo therapeutic. A majority of the therapeutics antibodies employed are intact IgG molecules. IgG-antibody/antigen complexes can activate a wide range of biological responses that result in elimination and destruction of immune complexes. Principle ligands for the activation of clearance (inflammatory) mechanisms are the three types of cellular Fc receptors (FcgammaR) expressed on leucocytes. The effector functions activated by FcgammaR in vivo can be 'orchestrated', in part, through choice of the IgG subclass employed, however, there is potential to customize antibody therapeutics for optimal biological efficacy, in a particular clinical setting, i.e. with respect to the specific disease and the patient response. In order to engineer IgG antibodies and customize their abilities to activate FcgammaR it is necessary to elucidate the molecular specificity of their mutual interactions. This mini-review summarizes our current understanding of interactions of FcgammaRI, FcgammaRII and FcgammaRIII with human IgG antibodies. Particular emphasis is given to the influence of IgG-Fc glycosylation.