The Flagellar Motility of \u3cem\u3eChlamydomonas pf25\u3c/em\u3e Mutant Lacking an AKAP-binding Protein Is Overtly Sensitive to Medium Conditions

Radial spokes are a conserved axonemal structural complex postulated to regulate the motility of 9 + 2 cilia and flagella via a network of phosphoenzymes and regulatory proteins. Consistently, a Chlamydomonas radial spoke protein, RSP3, has been identified by RII overlays as an A-kinase anchoring protein (AKAP) that localizes the cAMP-dependent protein kinase (PKA) holoenzyme by binding to the RIIa domain of PKA RII subunit. However, the highly conserved docking domain of PKA is also found in the N termini of several AKAP-binding proteins unrelated to PKA as well as a 24-kDa novel spoke protein, RSP11. Here, we report that RSP11 binds to RSP3 directly in vitro and colocalizes with RSP3 toward the spoke base near outer doublets and dynein motors in axonemes. Importantly, RSP11 mutant pf25 displays a spectrum of motility, from paralysis with flaccid or twitching flagella as other spoke mutants to wild-typelike swimming. The wide range of motility changes reversibly depending on the condition of liquid media without replacing defective proteins. We postulate that radial spokes use the RIIa/AKAP module to regulate ciliary and flagellar beating; absence of the spoke RIIa protein exposes a medium-sensitive regulatory mechanism that is not obvious in wild-type Chlamydomonas

The Flagellar Motility of \u3cem\u3eChlamydomonas pf25\u3c/em\u3e Mutant Lacking an AKAP-binding Protein Is Overtly Sensitive to Medium Conditions

Radial spokes are a conserved axonemal structural complex postulated to regulate the motility of 9 + 2 cilia and flagella via a network of phosphoenzymes and regulatory proteins. Consistently, a Chlamydomonas radial spoke protein, RSP3, has been identified by RII overlays as an A-kinase anchoring protein (AKAP) that localizes the cAMP-dependent protein kinase (PKA) holoenzyme by binding to the RIIa domain of PKA RII subunit. However, the highly conserved docking domain of PKA is also found in the N termini of several AKAP-binding proteins unrelated to PKA as well as a 24-kDa novel spoke protein, RSP11. Here, we report that RSP11 binds to RSP3 directly in vitro and colocalizes with RSP3 toward the spoke base near outer doublets and dynein motors in axonemes. Importantly, RSP11 mutant pf25 displays a spectrum of motility, from paralysis with flaccid or twitching flagella as other spoke mutants to wild-typelike swimming. The wide range of motility changes reversibly depending on the condition of liquid media without replacing defective proteins. We postulate that radial spokes use the RIIa/AKAP module to regulate ciliary and flagellar beating; absence of the spoke RIIa protein exposes a medium-sensitive regulatory mechanism that is not obvious in wild-type Chlamydomonas

Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells

The trans-activation response (TAR) RNA stem–loop that occurs at the 5′ end of HIV RNA transcripts is an important antiviral target and is the site of interaction of the HIV-1 Tat protein together with host cellular factors. Oligonucleotides and their analogues targeted to TAR are potential antiviral candidates. We have investigated a range of cell penetrating peptide (CPP) conjugates of a 16mer peptide nucleic acid (PNA) analogue targeted to the apical stem–loop of TAR and show that disulfide-linked PNA conjugates of two types of CPP (Transportan or a novel chimeric peptide R(6)-Penetratin) exhibit dose-dependent inhibition of Tat-dependent trans-activation in a HeLa cell assay when incubated for 24 h. Activity is reached within 6 h if the lysosomotropic reagent chloroquine is co-administered. Fluorescein-labelled stably-linked conjugates of Tat, Transportan or Transportan TP10 with PNA were inactive when delivered alone, but attained trans-activation inhibition in the presence of chloroquine. Confocal microscopy showed that such fluorescently labelled CPP–PNA conjugates were sequestered in endosomal or membrane-bound compartments of HeLa cells, which varied in appearance depending on the CPP type. Co-administration of chloroquine was seen in some cases to release fluorescence from such compartments into the nucleus, but with different patterns depending on the CPP. The results show that CPP–PNA conjugates of different types can inhibit Tat-dependent trans-activation in HeLa cells and have potential for development as antiviral agents. Endosomal or membrane release is a major factor limiting nuclear delivery and trans-activation inhibition

On the danger of redundancies in some aerospace mechanisms

An attempt is made to show that redundancies in some aerospace mechanisms do not generally improve the odds for success. Some of these redundancies may even be the very cause for failure of the system. To illustrate this fallacy, two designs based on the Control of Flexible Structures I (COFS I) Mast deployer and retractor assembly (DRA) are presented together with novel designs to circumvent such design inadequacies, while improving system reliability

Trojan Horse nanotheranostics with dual transformability and multifunctionality for highly effective cancer treatment.

Nanotheranostics with integrated diagnostic and therapeutic functions show exciting potentials towards precision nanomedicine. However, targeted delivery of nanotheranostics is hindered by several biological barriers. Here, we report the development of a dual size/charge- transformable, Trojan-Horse nanoparticle (pPhD NP) for delivery of ultra-small, full active pharmaceutical ingredients (API) nanotheranostics with integrated dual-modal imaging and trimodal therapeutic functions. pPhD NPs exhibit ideal size and charge for drug transportation. In tumour microenvironment, pPhD NPs responsively transform to full API nanotheranostics with ultra-small size and higher surface charge, which dramatically facilitate the tumour penetration and cell internalisation. pPhD NPs enable visualisation of biodistribution by near-infrared fluorescence imaging, tumour accumulation and therapeutic effect by magnetic resonance imaging. Moreover, the synergistic photothermal-, photodynamic- and chemo-therapies achieve a 100% complete cure rate on both subcutaneous and orthotopic oral cancer models. This nanoplatform with powerful delivery efficiency and versatile theranostic functions shows enormous potentials to improve cancer treatment

Novel pGreen/pSoup dual-binary vector system in multiple T-DNA co-cultivation as a method of producing marker-free (clean gene) transgenic rice (Oriza sativa L) plant

The possibility of producing marker-free transgenic rice plants using a novel dual binary pGreen/pSoup vectors, in multiple T-DNA co-cultivation, was investigated and demonstrated to be feasible. The T-DNA in pSoup (pRT47) vector was engineered to contain the selection marker hygromycin phosphotransferase (aphIV) gene (plus intron in 5′ UTR), and the green fluorescent protein (gfp) as a reporter gene both driven by the CaMV35S promoter and the nopaline synthase terminator. T-DNA in the pGreen (pRT18) vector harboured the phosphinothricin acetyl transferase (bar), as selection marker gene, and the b- glucuronidase (gusA) plus intron as a reporter gene, both driven by the maize 5′ ubiquitin region and the nopaline synthase terminator. Both the pGreen and pSoup plasmids were transformed into E. coli strain DH5α using the PEG-transformation technique and into Agrobacterium strains AGL1using a freeze-thaw method. AGL1 was then used to transform embryogenic nodular units (ENU), derived from mature seeds of the model rice genotype Nipponbare. Selection on herbicide (PPT) or antibiotic (hygromycin) of co-cultured ENUs led to the production of numerous independently transformed callus clones containing both T-DNAs from the selected and unselected vector. While co- ransformation frequencies were 71% and 80% for the hygromycin only and herbicide (PPT) only selection, respectively, data showed that co-expression frequency is most useful for the production of marker free transgenic rice. About half (50%) of the independent transgenic plant lines contained at least one unlinked T-DNA integration. In this work, we showed for the first time, that the novel dual-binary pGreen/pSoup can efficiently produce marker-free transgenic rice.African Journal of Biotechnology Vol. 4 (6), pp. 531-540, 200

Exploiting macrophage autophagy-lysosomal biogenesis as a therapy for atherosclerosis

Macrophages specialize in removing lipids and debris present in the atherosclerotic plaque. However, plaque progression renders macrophages unable to degrade exogenous atherogenic material and endogenous cargo including dysfunctional proteins and organelles. Here we show that a decline in the autophagy-lysosome system contributes to this as evidenced by a derangement in key autophagy markers in both mouse and human atherosclerotic plaques. By augmenting macrophage TFEB, the master transcriptional regulator of autophagy-lysosomal biogenesis, we can reverse the autophagy dysfunction of plaques, enhance aggrephagy of p62-enriched protein aggregates and blunt macrophage apoptosis and pro-inflammatory IL-1β levels, leading to reduced atherosclerosis. In order to harness this degradative response therapeutically, we also describe a natural sugar called trehalose as an inducer of macrophage autophagy-lysosomal biogenesis and show trehalose's ability to recapitulate the atheroprotective properties of macrophage TFEB overexpression. Our data support this practical method of enhancing the degradative capacity of macrophages as a therapy for atherosclerotic vascular disease

Mechanical design engineering. NASA/university advanced design program: Lunar Bulk Material Transport Vehicle

The design of a Lunar Bulk Material Transport Vehicle (LBMTV) is discussed. Goals set in the project include a payload of 50 cubic feet of lunar soil with a lunar of approximately 800 moon-pounds, a speed of 15 mph, and the ability to handle a grade of 20 percent. Thermal control, an articulated steering mechanism, a dump mechanism, a self-righting mechanism, viable power sources, and a probable control panel are analyzed. The thermal control system involves the use of small strip heaters to heat the housing of electronic equipment in the absence of sufficient solar radiation and multi-layer insulation during periods of intense solar radiation. The entire system uses only 10 W and weighs about 60 pounds, or 10 moon-pounds. The steering mechanism is an articulated steering joint at the center of the vehicle. It utilizes two actuators and yields a turning radius of 10.3 feet. The dump mechanism rotates the bulk material container through an angle of 100 degree using one actuator. The self-righting mechanism consists of two four bar linkages, each of which is powered by the same size actuator as the other linkages. The LBMTV is powered by rechargeable batteries. A running time of at least two hours is attained under a worst case analysis. The weight of the batteries is 100 pounds. A control panel consisting of feedback and control instruments is described. The panel includes all critical information necessary to control the vehicle remotely. The LBMTV is capable of handling many types of cargo. It is able to interface with many types of removable bulk material containers. These containers are made to interface with the three-legged walker, SKITTER. The overall vehicle is about 15 feet in length and has a weight of about 1000 pounds, or 170 lunar pounds