Enzymatic synthesis of polymers containing nicotinamide mononucleotide

Nicotinamide mononucleoside 5'-diphosphate in its reduced form is an excellent substrate for polynucleotide phosphorylase from Micrococcus luteus both in de novo polymerization reactions and in primer extension reactions. The oxidized form of the diphosphate is a much less efficient substrate; it can be used to extend primers but does not oligomerize in the absence of a primer. The cyanide adduct of the oxidized substrate, like the reduced substrate, polymerizes efficiently. Loss of cyanide yields high molecular weight polymers of the oxidized form. Terminal transferase from calf thymus accepts nicotinamide mononucleoside 5'-triphosphate as a substrate and efficiently adds one residue to the 3'-end of an oligodeoxynucleotide. T4 polynucleotide kinase accepts oligomers of nicotinamide mononucleotide as substrates. However, RNA polymerases do not incorporate nicotinamide mononucleoside 5'-triphosphate into products on any of the templates that we used

Useful Tools for Biomolecule Isolation, Detection, and Identification: Acylhydrazone-Based Cleavable Linkers

SummaryProteomic searches using affinity-based chromatography (e.g., biotin-[strept]avidin) have been severely hampered by low protein recovery yields, protein destruction and denaturation, and the release of background proteins from the support. These limitations confound protein identification. A new acylhydrazone-based cleavable linker was developed to permit the efficient isolation of proteins with a traceable tag allowing detection and identification under mild conditions. The utility of the acylhydrazone linker was validated in a proteomic search wherein aldehyde dehydrogenase-1 was selectively captured and isolated from the mouse soluble liver proteome without interfering background proteins. The use of acylhydrazone linkers is expected to be generalized, allowing for the selective release of tagged molecules from noncovalent and covalently tagged supports

Analisis Sikap Multiatribut Fishbein Dalam Pengambilan Keputusan Mahasiswa Memilih Kampus IBI Kesatuan

Penelitian ini bertujuan untuk menganalisis keputusan calon mahasiswa dalam memutuskan pemilihan Perguruan Tinggi di Institut Bisnis dan Informatika Kesatuan, Model analisis yang digunakan adalah Analisis sikap Multiatribut  Fishbein. Terdapat 10 aribut yang dianalisis, yakni (1) kualitas akreditasi, (2) fasilitas, (3) kualitas dosen, (4) biaya kuliah, (5) lokasi, (6) faktor teman/keluarga, (7) suasana kampus, (8) beasiswa, (9) pelayanan dan (10) mudah bekerja. Analisis sikap mahasiswa dilihat dari dua variabel yakni tingkat kepercayaan (belief) mahasiswa atas atribut Perguruan Tinggi dan evaluasi mahasiswa atas atribut tersebut dan sekaligus mengukur score kinerjanya. Teknisk pengambilan sampel adalah proportional random sampling, dengan sampel 288 responden dari populasi 1025 orang mahasiswa. Berdasarkan nilai kepercayaan sikap mahasiswa terhadap pemilihan perguruan tinggi di Kota Bogor diperoleh rata-rata score dengan nilai 3.841, dan berada pada kategori baik, sedangkan nilai evaluasi sikap mahasiswa terhadap perguruan tinggi IBI Kesatuan diperoleh rata-rata score 4.009 berada pada kategori baik. Terdapat dua atribut yang paling tinggi dan masuk dalam kategori sangat penting, yakni Mudah bekerja (4.316) dan Kualitas Akreditasi (4.313), dan Akreditasi yang mencerminkan kualitas IBI Kesatuan. Nilai sikap dengan model Fishbein dalam pemilihan Perguruan Tinggi IBI Kesatuan didapat score sikap sebesar 159,94 berada pada interval “baik”.   Kata Kunci : Fishbein, belief, evaluation, performance, importance

Prediksi Daya Tampung Kapasitas Ruang Kelas Dan Rasio Dosen Pada Perguruan Tinggi Sebagai Bagian Dari Target Promosi Penerimaan Mahasiswa Baru

IBI Kesatuan adalah perguruan tinggi swasta di wilayah LLDIKTI IV dengan akreditasi institusi B. Pada semester ganjil 2021/2022 jumlah mahasiswa mencapai 4480 mahasiswa, yang terdiri dari tiga Fakultas, yang dalam operasional kegiatan belajar mengajar dengan jumlah 122 orang jumlah dosen. Dengan jumlah total ruangan 56 ruangan kelas yang mana kapasitas ruangan rata-rata adalah 35 mahasiswa per kelasnya. Untuk itu perlu dilakukan penelitian lebih lanjut. Dengan melakukan analisa jumlah dosen yang tersedia, jumlah kelas yang ada, jumlah mahasiswa aktif dan jumlah mahasiswa, maka dapat ditentukan berapa perkiraan sisa daya tampung menurut okupansi ruangan yang masih ada dan berapa perkiraan jumlah mahasiswa yang dapat diterima pada periode tahun ajar 2022/2023. Jumlah ini sebagai prediksi target tim marketing IBIK untuk merekrut calon mahasiswa baru. Hasil analisa ini diharapkan bisa menjadi acuan terkait berapa banyak target yang harus ditetapkan di tahun selanjutnya.   Kata Kunci : Prediksi,  Daya Tampung, Promos

Characterization of endogenous and recombinant human calpain-10

Calpain-10 is a novel ubiquitous calpain family member that has been implicated as a susceptibility gene for type 2 diabetes. One of the major challenges is that the biochemical function of calpain-10 is not yet known. To address the problem, we purified human calpain-10 from different sources, including the endogenous and the recombinant calpain-10 from HeLa S3 and 293F cells, respectively. Both endogenous and recombinant calpain-10 were present as two major forms with different origins. Interestingly, radiolabeled calpain-10 was found to be efficiently cleaved at the N-terminal region by calpain-2, but not by other proteases. None of these calpain-10 proteins has putative proteolytic activity under in vitro conditions when examined using different peptide substrates, including more than 70 in vitro translated, radiolabeled oligopeptides. Our results raise the possibility that calpain-10 requires special intracellular localization or interacting partner(s) to acquire proteolytic activity, or it functions by interacting with other proteins rather than through its proteolytic activity

Local and transient gene expression primes the liver to resist cancer metastasis

The liver is the primary site of metastasis for gastrointestinal cancers and is a location highly susceptible to the establishment of metastasis in numerous other primary cancers, including breast, lung, and pancreatic cancers. The current standard of care typically consists of primary tumor resection and systemic administration of potent but toxic chemotherapeutics, yielding a minimal improvement in the median survival rate. CXCL12, a chemokine, is a key factor for activating the migration/survival pathways of CXCR4+ cancer cells and for recruiting immunosuppressive cells to areas of inflammation. Therefore, reducing CXCL12 concentrations within the liver has the potential to decrease tumor and immunosuppressive cell activation/migration within the liver. However, because of off-target toxicities associated with systemic administration of anti-CXCL12 therapies, transient and liver-specific expression of a CXCL12 trap is necessary. To address this challenge, we developed a lipid calcium phosphate nanoparticle optimized for delivering plasmid DNA, encoding an engineered CXCL12 protein trap, to the nucleus of liver hepatocytes. This pCXCL12-trap formulation yielded transient (4 days) liver-specific expression, which greatly decreased the occurrence of liver metastasis in two aggressive liver metastasis models, including colorectal [CT-26(FL3)] and breast (4T1) cancers. Subsequent studies in an aggressive human colorectal liver metastasis model (HT-29) decreased the establishment of liver metastasis more effectively than did systemic administration of the CXCL12 protein trap and to a level comparable to a high-dose regimen of a potent CXCR4 antagonist (AMD3100)

Tetraspecific ligand for tumor-targeted delivery of nanomaterials

The polygenetic nature of most cancers emphasizes the necessity of cancer therapies that target multiple essential signaling pathways. However, there is a significant paucity of targeting ligands with multi-specificities for targeted delivery of biomaterials. To address this unmet need, we generated a tetraspecific targeting ligand that recognizes four different cancer biomarkers, including VEGFR2, αvβ3 integrin, EGFR, and HER2 receptors, which have been implicated in numerous malignant tumors. The tetraspecific targeting ligand was constructed by sequentially connecting four targeting ligand subunits via flexible linkers, yielding a fusion protein that can be highly expressed in E. coli and readily purified to near homogeneity. Surface Plasmon Resonance (SPR), Bio-Layer Interferometry (BLI) studies and extensive cellular binding analyses indicated that all the targeting ligand subunits in the tetraspecific fusion protein recognized their target receptors proximately to the corresponding monospecific ligands. The resulting tetraspecific targeting ligand was applied for the delivery of nanomaterials such as gold nanoparticles (AuNPs) for targeted hyperthermic killing of various cancer cell lines with biomarkers of interest expressed. We demonstrate that the tetraspecific ligand can be facilely introduced on the surface of AuNPs and efficient target-dependent killing of cancer cells can be achieved only when the AuNPs are conjugated with the tetraspecific ligand. Significantly, the tetraspecific ligand simultaneously interacts with more than one receptors, such as EGFR and HER2 receptors, when they are expressed on the surface of the same cell, as demonstrated by in vitro binding assays and cell binding analyses. Our results demonstrate that the tetraspecific ligand, through multivalency and synergistic binding, can be readily used to generate various ‘smart’ biomaterials with greatly broadened tumor targeting range for simultaneous targeting of multiple signaling pathways on many different cancer types

Ca 2+ /Calmodulin Directly Interacts with the Pleckstrin Homology Domain of AKT1

AKT kinase, also known as protein kinase B, is a key regulator of cell growth, proliferation, and metabolism. The activation of the AKT signaling pathway is one of the most frequent molecular alterations in a wide variety of human cancers. Dickson and coworkers recently observed that Ca(2+).calmodulin (Ca(2+).CaM) may be a common regulator of AKT1 activation (Deb, T. B., Coticchia, C. M., and Dickson, R. B. (2004) J. Biol. Chem. 279, 38903-38911). In our efforts to scan the mRNA-displayed proteome libraries for Ca(2+).CaM-binding proteins, we found that both human and Caenorhabditis elegans AKT1 kinases bound to CaM in a Ca(2+)-dependent manner (Shen, X., Valencia, C. A., Szostak, J., Dong, B., and Liu, R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 5969-5974 and Shen, X., Valencia, C. A., Gao, W., Cotten, S. W., Dong, B., Chen, M., and Liu, R. (2007) submitted for publication). Here we demonstrate that Ca(2+).CaM and human AKT1 were efficiently co-immunoprecipitated, and their interaction was direct rather than mediated by other proteins. The binding is in part attributed to the first 42 residues of the pleckstrin homology (PH) domain, a region that is critical for the recognition of its lipid ligands. The PH domain of human AKT1 can disrupt the complex of the full-length AKT1 with Ca(2+).CaM. In addition, Ca(2+).CaM competes with phosphatidylinositol 3,4,5-trisphophate for interaction with the PH domain of human AKT1. Our findings suggest that Ca(2+).CaM is directly involved in regulating the functions of AKT1, presumably by releasing the activated AKT1 from the plasma membrane and/or prohibiting it from re-association with phosphoinositides on plasma membrane

The Smart Targeting of Nanoparticles

One major challenge in nanomedicine is how to selectively deliver nanoparticles to diseased tissues. Nanoparticle delivery system requires targeting for specific delivery to pathogenic sites when enhanced permeability and retention (EPR) is not suitable or inefficient. Functionalizing nanoparticles is a widely-used technique that allows for conjugation with targeting ligands, which possess inherent ability to direct selective binding to cell types or states and, therefore, confer “smartness” to nanoparticles. This review illustrates methods of ligand-nanoparticle functionalization, provides a cross-section of various ligand classes, including small molecules, peptides, antibodies, engineered proteins, or nucleic acid aptamers, and discusses some unconventional approaches currently under investigation