EFFECTS OF HIV-1 TAT PROTEIN ON STRUCTURE, FUNCTION AND TRAFFICKING OF THE DOPAMINE TRANSPORTER

The alarming rise in HIV-1 associated neurocognitive disorder (HAND) is, at least in part, associated with HIV-1 viral proteins shed from infected macrophages, including transactivator of transcription (Tat) despite the success of anti-retroviral therapies. The dopamine (DA) system is greatly involved in the progression of HAND and is influenced by psychostimulants like cocaine. The DA transporter (DAT), a key regulator of neurocognitive functions, is a major molecular target for both Tat and cocaine. Our lab previously reported that exposure to Tat decreases DA uptake through allosteric regulation and alters cocaine binding sites in DAT. In this research project the hypothesis ‘HIV-1 Tat protein via allosteric modulation of DAT induces inhibition of DA transport, leading to dysfunction of the DA system’ was tested. Initially, it was shown that Tat protein directly interacts with DAT to impair DA translocation. Based on the predictions of computational modeling and simulations, Y470, Y88 and K92 residues of the human DAT (hDAT) are essential to stabilize the compact structure of DAT and potentially recognize Tat. Mutating these residues in hDAT – Y470H, Y88F, and K92M attenuated Tat-induced inhibition of DA uptake. Additional substitutions Y470A and Y470F at 470 displayed attenuated or no effect on Tat-induced inhibition of DA uptake respectively, indicating the significant role of aromatic ring of Y470 in DAT and Tat interaction. Pharmacological characterization showed that compared to wild type hDAT, Y470H and K92M but not Y88F reduce Vmax with no change in the Km values for DA uptake. Moreover, Y470H, K92M, and Y88F mutants exhibited no alterations in IC50 values of DA to inhibit [3H]DA uptake but increased [3H]DA uptake potency or [3H]WIN35,428 binding potency for cocaine and GBR12909, suggesting that these three Tat-recognition residues do not overlap with substrate DA binding but influence binding of small molecule inhibitors. Besides, all five mutants reversed zinc-induced increase of [3H]WIN35,428 binding and differentially altered basal DA efflux properties of the DAT, indicating that Tat protein through interaction at these recognition residues disrupts intermolecular interactions that are critical for maintenance of the outward-facing conformation of DAT. Another study was conducted to determine the effects of Tat on DAT phosphorylation, trafficking and its influence on sequestration of [3H]DA by vesicular monoamine transporter 2 (VMAT2). We found that protein kinase C (PKC) inhibitor, bisindolylmaleimide-I eliminates Tat effects on DA uptake and Tat increases intracellular DAT immunoreactivity. Moreover, Tat also produced inhibitory effects on VMAT2 function. Collectively, these findings revealed that Tat inhibits DAT function through PKC and trafficking- dependent mechanisms; besides, both DAT and VMAT2 proteins may involve in Tat-induced dysregulation of the DA system. In conclusion, Tat inhibits DA translocation process principally by altering the conformational states of the DAT through interaction at specific recognition residues. Furthermore, regulatory pathways that control the functional attributes of DAT may play a vital role in Tat-mediated impairment of the DA system. Future studies will be necessary to identify and characterize other recognition residues for Tat binding and these molecular insights will be helpful to develop adjunctive therapies to restore the impaired DA system in HIV-1 positive individuals

Focal adhesions are foci for tyrosine-based signal transduction via GIV/Girdin and G proteins.

GIV/Girdin is a multimodular signal transducer and a bona fide metastasis-related protein. As a guanidine exchange factor (GEF), GIV modulates signals initiated by growth factors (chemical signals) by activating the G protein Gαi. Here we report that mechanical signals triggered by the extracellular matrix (ECM) also converge on GIV-GEF via β1 integrins and that focal adhesions (FAs) serve as the major hubs for mechanochemical signaling via GIV. GIV interacts with focal adhesion kinase (FAK) and ligand-activated β1 integrins. Phosphorylation of GIV by FAK enhances PI3K-Akt signaling, the integrity of FAs, increases cell-ECM adhesion, and triggers ECM-induced cell motility. Activation of Gαi by GIV-GEF further potentiates FAK-GIV-PI3K-Akt signaling at the FAs. Spatially restricted signaling via tyrosine phosphorylated GIV at the FAs is enhanced during cancer metastasis. Thus GIV-GEF serves as a unifying platform for integration and amplification of adhesion (mechanical) and growth factor (chemical) signals during cancer progression

Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin.

A long-standing issue in the field of signal transduction is to understand the cross-talk between receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major and distinct signaling hubs that control eukaryotic cell behavior. Although stimulation of many RTKs leads to activation of trimeric G proteins, the molecular mechanisms behind this phenomenon remain elusive. We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins. Using a combination of homology modeling, protein-protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands. Using protein-protein interaction assays, we demonstrated that both SH2 and GEF domains of GIV are required for the formation of a ligand-activated ternary complex between GIV, Gαi, and growth factor receptors and for activation of Gαi after growth factor stimulation. Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV-Akt enhancement, actin remodeling, and cell migration. The mechanistic and structural insights gained here shed light on the long-standing questions surrounding RTK/G protein cross-talk, set a novel paradigm, and characterize a unique pharmacological target for uncoupling GIV-dependent signaling downstream of multiple oncogenic RTKs

PENERAPAN TEKNIK VOKAL BELTING PADA LAGU KISAH SEMPURNA KARYA MAHALINI RAHARJA

Teknik vokal merupakan suatu cara untuk memproduksi suara yang baik dan efisien, sehingga suara yang keluar dapat terdengar dengan jelas, indah, merdu, nyaring dan tentu memiliki nilai teknik dalam bernyanyi. Beberapa macam teknik dasar vokal meliputi intonasi, resonansi, pernapasan, interpretasi, serta teknik pengembangan seperti head, cheest, mix voice, interpretasi, dinamika dan salah satunya teknik vokal belting. Teknik belting adalah cara memproduksi suara saat bernyanyi dengan resonansi yang tepat dan menghasilkan suara seperti berteriak dengan tegas dan lantang. Penerapan teknik belting pada lagu Kisah Sempurna termasuk cara yang tepat dan efektif dalam proses belajar atau mengembangkan teknik vokal yang baik. Keberhasilan lagu Kisah Sempurna sangat banyak diminati publik, terbukti dengan jumlah penayangan jutaan lebih di media sosial dan Mahalini berhasil mengeksekusi dengan teknik vokal yang tepat yang menjadi arahan bagi para penyanyi untuk dipelajari.Teori ini dapat menjadi landasan untuk mengetahui dan membentuk teknik vokal belting yang benar. Proses eksplorasi yang dilakukan dengan mencari referensi tentang vokal belting, melatih pernapasan hal yang paling utama, melatih resonansi yang diantaranya head, chest, dan mix voice juga mengetahui ciri dan karakterisitik belting untuk mendapatkan hasil power vokal yang maksimal. Kesimpulan yang dihasilkan, penerapan teknik vokal belting memiliki ketertarikan yang sangat efektif untuk menambah wawasan dalam bernyanyi, berbagai pengetahuan seperti latihan yang diperlukan, menguasai konsep teknik vokal belting, tahapan-tahapan yang dilakukan sebelum melakukan teknik belting, dan memahami konsekuensi ketika tidak melakukan teknik vokal belting dengan baik dan benar.Kata Kunci: teknik vokal, belting, Kisah SempurnaApplication Of Vocal Belting Techniques To Songs Mahalini Raharja "Kisah Sempurna".Vocal technique is a way to produce  a good and efficient sound, so that the sound  that comes out  can  be heard  clearly, beautifully, melodiously, loudly and of course has the value of technique in singing. Some basic vocal techniques include intonation, resonance, breathing, interpretation, as well as development techniques such as head, cheest, mix voice, interpretation, dynamics and false the only vocal belting technique. The belting technique   is a way of producing sounds when singing with  the right  resonance and   producing  sounds such as shouting firmly and loudly. The application of   belting techniques to Kisah Sempurna songs is an appropriate  and effective  way  in  the process of learning  or  developing good  vocal    techniques.    The success of the   song  Kisah Sempurna  is in great   demand by the   public, as evidenced  by the   number  of views of  millions  more  on social media   and Mahalini  successfully  executing  with  the  technique of  proper   vocals  that become a   direction  for  the singers  to  learn.    This theory    can  be the   basis  for  knowing  and shaping  the correct  vocal  belting  technique.  Proses   exploration is done  by  finding  references  to    vocal belting, training   breathing the most important  thing, training  resonance  which includes head,  chest, and mix voice  also  knows   the characteristics   and characteristics of  belting  to  get maximum  vocal power results. In conclusion produced, the application of  vocal  belting  techniques  has  a very effective  interest in adding insight in singing, various knowledge  such as  necessary exercises, mastering  concepts  Vocal   belting technique, the stages performed  before performing the belting technique,  and understand the   consequences  when  not  performing    the vocal belting  technique properly and correctly.  Keywords: vocal technique, belting, Kisah Sempurn

Activation of G proteins by GIV-GEF is a pivot point for insulin resistance and sensitivity.

Insulin resistance (IR) is a metabolic disorder characterized by impaired insulin signaling and cellular glucose uptake. The current paradigm for insulin signaling centers upon the insulin receptor (InsR) and its substrate IRS1; the latter is believed to be the sole conduit for postreceptor signaling. Here we challenge that paradigm and show that GIV/Girdin, a guanidine exchange factor (GEF) for the trimeric G protein Gαi, is another major hierarchical conduit for the metabolic insulin response. By virtue of its ability to directly bind InsR, IRS1, and phosphoinositide 3-kinase, GIV serves as a key hub in the immediate postreceptor level, which coordinately enhances the metabolic insulin response and glucose uptake in myotubes via its GEF function. Site-directed mutagenesis or phosphoinhibition of GIV-GEF by the fatty acid/protein kinase C-theta pathway triggers IR. Insulin sensitizers reverse phosphoinhibition of GIV and reinstate insulin sensitivity. We also provide evidence for such reversible regulation of GIV-GEF in skeletal muscles from patients with IR. Thus GIV is an essential upstream component that couples InsR to G-protein signaling to enhance the metabolic insulin response, and impairment of such coupling triggers IR. We also provide evidence that GIV-GEF serves as therapeutic target for exogenous manipulation of physiological insulin response and reversal of IR in skeletal muscles

Entry, Retention, and Virological Suppression in an HIV Cohort Study in India: Description of the Cascade of Care and Implications for Reducing HIV-Related Mortality in Low- and Middle-Income Countries

HIV treatment, care, and support programmes in low- and middle-income countries have traditionally focused more on patients remaining in care after the initiation of antiretroviral therapy (ART) than on earlier stages of care. This study describes the cumulative retention from HIV diagnosis to the achievement of virological suppression after ART initiation in an HIV cohort study in India. Of all patients diagnosed with HIV, 70% entered into care within three months. 65% of patients ineligible for ART at the first assessment were retained in pre-ART care. 67% of those eligible for ART initiated treatment within three months. 30% of patients who initiated ART died or were lost to followup, and 82% achieved virological suppression in the last viral load determination. Most attrition occurred the in pre-ART stages of care, and it was estimated that only 31% of patients diagnosed with HIV engaged in care and achieved virological suppression after ART initiation. The total mortality attributable to pre-ART attrition was considerably higher than the mortality for not achieving virological suppression. This study indicates that early entry into pre-ART care along with timely initiation of ART is more likely to reduce HIV-related mortality compared to achieving virological suppression

Penerapan Teknik Vokal Belting Pada Lagu "Kisah Sempurna" Karya Mahalini Raharja

Teknik vokal merupakan suatu cara untuk memproduksi suara yang baik dan efisien, sehingga suara yang keluar dapat terdengar dengan jelas, indah, merdu, nyaring dan tentu memiliki nilai teknik dalam bernyanyi. Beberapa macam teknik dasar vokal meliputi intonasi, resonansi, pernapasan, interpretasi, serta teknik pengembangan seperti head, cheest, mix voice, interpretasi, dinamika dan salah satunya teknik vokal belting. Teknik belting adalah cara memproduksi suara saat bernyanyi dengan resonansi yang tepat dan menghasilkan suara seperti berteriak dengan tegas dan lantang. Penerapan teknik belting pada lagu Kisah Sempurna termasuk cara yang tepat dan efektif dalam proses belajar atau mengembangkan teknik vokal yang baik. Keberhasilan lagu Kisah Sempurna sangat banyak diminati publik, terbukti dengan jumlah penayangan jutaan lebih di media sosial dan Mahalini berhasil mengeksekusi dengan teknik vokal yang tepat yang menjadi arahan bagi para penyanyi untuk dipelajari. Teori ini dapat menjadi landasan untuk mengetahui dan membentuk teknik vokal belting yang benar. Proses eksplorasi yang dilakukan dengan mencari referensi tentang vokal belting, melatih pernapasan hal yang paling utama, melatih resonansi yang diantaranya head, chest, dan mix voice juga mengetahui ciri dan karakterisitik belting untuk mendapatkan hasil power vokal yang maksimal. Kesimpulan yang dihasilkan, penerapan teknik vokal belting memiliki ketertarikan yang sangat efektif untuk menambah wawasan dalam bernyanyi, berbagai pengetahuan seperti latihan yang diperlukan, menguasai konsep teknik vokal belting, tahapan-tahapan yang dilakukan sebelum melakukan teknik belting, dan memahami konsekuensi ketika tidak melakukan teknik vokal belting dengan baik dan benar. Kata Kunci : Teknik Vokal, Belting, Kisah Sempurna

Effect of Formula Feeding and Breastfeeding on Child Growth, Infant Mortality, and HIV Transmission in Children Born to HIV-Infected Pregnant Women Who Received Triple Antiretroviral Therapy in a Resource-Limited Setting: Data from an HIV Cohort Study in India

We describe a programme for the prevention of mother-to-child transmission (PMTCT) of HIV that provided universal antiretroviral therapy (ART) to all pregnant women regardless of the CD4 lymphocyte count and formula feeding for children with high risk of HIV transmission through breastfeeding in a district of India. The overall rate of HIV transmission was 3.7%. Although breastfeeding added a 3.1% additional risk of HIV acquisition, formula-fed infants had significantly higher risk of death compared to breastfed infants. The cumulative 12-month mortality was 9.6% for formula-fed infants versus 0.68% for breastfed infants. Anthropometric markers (weight, length/height, weight for length/height, body mass index, head circumference, mid-upper arm circumference, triceps skinfold, and subscapular skinfold) showed that formula-fed infants experience severe malnutrition during the first two months of life. We did not observe any death after rapid weaning at 5-6 months in breastfed infants. The higher-free-of HIV survival in breastfed infants and the low rate of HIV transmission found in this study support the implementation of PMTCT programmes with universal ART to all HIV-infected pregnant women and breastfeeding in order to reduce HIV transmission without increasing infant mortality in developing countries

Pengaruh Bubuk Daun Kenikir (Cosmos Caudatus) Terhadap Kadar Malondialdehyde Plasma Tikus Wistar Diabetes Diinduksi Streptozotocin

Latar Belakang: Komplikasi vaskular diabetes terjadi akibat meningkatnya pembentukan radikal bebas sehingga menyebabkan stress oksidatif. Parameter tingkat stress oksidatif paling stabil adalah malondialdehyde (MDA). Stress oksidatif dapat dikendalikan dengan meningkatkan konsumsi antioksidan nonenzimatik. Daun kenikir memiliki zat antioksidan nonenzimatik potensial golongan flavonoid yaitu kuersetin. Penelitian ini bertujuan menganalisis pengaruh bubuk daun kenikir terhadap kadar malondialdehyde plasma tikus Wistar diabetes diinduksi streptozotocin.Metode: Jenis penelitian ini adalah true experimental dengan post-test only randomized control group design. Subjek penelitian yaitu 21 ekor tikus Wistar jantan dibagi menjadi 3 kelompok, K+, P1, dan P2. Seluruh kelompok diinduksi streptozotocin 65 mg/kg dan nicotinamide 230 mg/kg, kelompok perlakuan diberi bubuk daun kenikir dosis 700 mg/200gBB/hari dan 1400 mg/200gBB/hari selama 21 hari. Pemeriksaan kadar MDA plasma dengan metode 2-Thiobarbituric Acid Reactive Substance (TBARS). Data dianalisis menggunakan uji One Way Anova dan Post-hoc LSD.Hasil: Dosis 700 mg (P1) dan 1400 mg (P2) bubuk daun kenikir mampu menurunkan kadar MDA plasma tikus Wistar diabetes diinduksi streptozotocin (p<0,05). Rerata kadar MDA plasma kelompok kontrol positif sebesar 7,7±0,61, perlakuan 1 sebesar 6,1±0,58 dan perlakuan 2 sebesar 2,8±0,50. Secara statistik terdapat perbedaan rerata kadar MDA plasma antar kelompok (p<0,05).Simpulan: Bubuk daun kenikir dosis 700 mg/200gBB/hari dan 1400 mg/200gBB/hari selama 21 hari mampu menurunkan kadar MDA plasma tikus Wistar diabetes diinduksi streptozotocin. Dosis 1400 mg/200gBB/hari bubuk daun kenikir lebih efektif menurunkan kadar MDA plasma