Clinical Outcomes of Adults With Diagnosed HIV Living in Ending the HIV Epidemic Priority Areas, Medical Monitoring Project, 2018.

OBJECTIVES: The Ending the HIV Epidemic (EHE) initiative prioritizes treatment and prevention efforts in counties where most new HIV diagnoses occur and states with substantial incidence of new HIV diagnoses in rural areas. Understanding the characteristics of adults with HIV living in EHE priority areas, and how these characteristics compare with adults with HIV living in non-EHE priority areas, can inform EHE efforts. METHODS: We analyzed data from the 2018 Medical Monitoring Project (MMP) to understand the characteristics of adults with HIV living in 36 of 48 EHE priority counties; San Juan, Puerto Rico; and 1 of 7 EHE priority states. We calculated weighted percentages of sociodemographic characteristics, behaviors, and clinical outcomes of adults with diagnosed HIV living in MMP EHE priority areas and compared them with characteristics of adults who did not live in MMP EHE priority areas using prevalence ratios (PRs) with predicted marginal means. RESULTS: Living in an MMP EHE priority area was more common among adults who were non-Hispanic Black or Hispanic, experienced homelessness, or were food insecure compared with adults who were non-Hispanic White (59.3% and 58.4% vs 41.0%), not experiencing homelessness (60.9% vs 51.9%), or not food insecure (59.8% vs 51.0%). Adults who lived in MMP EHE priority areas were significantly less likely to be adherent to their HIV medications (PR = 0.95; 95% CI, 0.91-0.99) and durably virally suppressed (PR = 0.94; 95% CI, 0.91-0.97), and more likely to miss scheduled appointments for HIV care (PR = 1.31; 95% CI, 1.10-1.56) than adults who did not live in MMP EHE priority areas. CONCLUSION: To increase viral suppression and reduce HIV transmission, it is essential to strengthen public health efforts to improve medication and appointment adherence in this population.

Encapsulating genetically modified Saccharomyces cerevisiae cells in a flow-through device towards the detection of diclofenac in wastewater.

Recently it has been proposed to use sensors based on genetically engineered reporter cells to perform continuous online water monitoring. Here we describe the design, assembly and performance of a novel flow-through device with immobilized genetically modified yeast cells that produce a fluorescent protein upon stimulation with diclofenac whose intensity is then detected by fluorescence microscopy. Although other devices employing immobilized cells for the detection of various analytes have already been described before, as novelty our system allows safe enclosure of the sensor cells, and thus, to obtain fluorescent signals that are not falsified by a loss of cells. Furthermore, the yeast cells are prevented from being released into the environment. Despite the safe containment, the immobilized reporter cells are accessible to nutrients and analytes. They thus have both the ability to grow and respond to the analyte. Both in cell culture medium and standardized synthetic wastewater, we are able to differentiate between diclofenac concentrations in a range from 10 to 100 µM. As particularly interesting feature, we show that only the biologically active fraction of diclofenac is detected. Nowadays, contamination of wastewater with diclofenac and other pharmaceutical residues is becoming a severe problem. Our investigations may pave the way for an easy-to-use and cost-efficient wastewater monitoring method.

Induction of apoptosis in human cancer cells by targeting mitochondria with gold nanoparticles.

A major challenge in designing cancer therapies is the induction of cancer cell apoptosis, although activation of intrinsic apoptotic pathways by targeting gold nanoparticles to mitochondria is promising. We report an in vitro procedure targeting mitochondria with conjugated gold nanoparticles and investigating effects on apoptosis induction in the human breast cancer cell line Jimt-1. Gold nanoparticles were conjugated to a variant of turbo green fluorescent protein (mitoTGFP) harbouring an amino-terminal mitochondrial localization signal. Au nanoparticle conjugates were further complexed with cationic maltotriose-modified poly(propylene imine) third generation dendrimers. Fluorescence and transmission electron microscopy revealed that Au nanoparticle conjugates were directed to mitochondria upon transfection, causing partial rupture of the outer mitochondrial membrane, triggering cell death. The ability to target Au nanoparticles into mitochondria of breast cancer cells and induce apoptosis reveals an alternative application of Au nanoparticles in photothermal therapy of cancer.

Assessment of factors influencing retention in the Philippine National Rural Physician Deployment Program.

BACKGROUND: The 'Doctors to the Barrios' (DTTB) Program was launched in 1993 in response to the shortage of doctors in remote communities in the Philippines. While the Program has attracted physicians to work in such areas for the prescribed 2-year period, ongoing monitoring shows that very few chose to remain there for longer and be absorbed by their Local Government Unit (LGU). This assessment was carried out to explore the reasons for the low retention rates and to propose possible strategies to reverse the trend. METHODS: A mixed methods approach was used comprising a self-administered questionnaire for members of the current cohort of DTTBs, and oral interviews with former DTTBs. RESULTS: Among former DTTBs, the wish to serve rural populations was the most widely cited motivation. By comparison, among the current cohort of DTTBs, more than half joined the Program due to return of service obligations; a quarter to help rural populations, and some out of an interest in public health. Those who joined the Program to return service experienced significantly less satisfaction, whilst those who joined out of an interest in public health were significantly more satisfied with their rural work. Those who graduated from medical schools in the National Capital Region were significantly more critical about their compensation and perceived there to be fewer options for leisure in rural areas. With regard to the factors impeding retention, lack of support from the LGU was most frequently mentioned, followed by concerns about changes in compensation upon absorption by the LGU, family issues and career advancement. CONCLUSIONS: Through improved collaboration with the Department of Health, LGUs need to strengthen the support provided to DTTBs. Priority could be given to those acting out of a desire to help rural populations or having an interest in public health, and those who have trained outside of the National Capital Region. Whether physicians should be able to use the Program to fulfil return service obligations should be critically assessed.

Application of the yeast pheromone system for controlled cell-cell communication and signal amplification.

AIMS: The aim of the work is to exploit the yeast pheromone system for controlled cell-cell communication and as an amplification circuit in technical applications, e.g. biosensors or sensor-actor systems. METHODS AND RESULTS: As a proof of principle, we developed recombinant Saccharomyces cerevisiae cells that express enhanced green fluorescent protein (EGFP) in response to different concentrations of the alpha(α)-factor mating pheromone. A respective reporter construct allowing the pheromone-driven expression of EGFP was transformed into the S. cerevisiae strains BY4741 and BY4741 bar1Δ. Upon addition of synthetic α-factor, the fluorescence strongly increases after 4 h. Furthermore, cells with constitutive α-factor expression were able to induce the expression of EGFP in co-cultivation with sensor cells only if both cell types were deleted for the gene BAR1, encoding α-factor protease. For technical applications, the immobilization of functionalized cells may be beneficial. We show that pheromone-induced expression of EGFP is effective in alginate-immobilized cells. CONCLUSIONS: Based on S. cerevisiae α-factor, we developed a controlled cell-cell communication system and amplification circuit for pheromone-driven expression of a target protein. The system is effective both in suspension and after cell immobilization. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed set of recombinant yeast strains is the basis to apply the yeast pheromone system for signal production and amplification in biosensors or sensor-actor systems.

Knockdown of human COX17 affects assembly and supramolecular organization of cytochrome c oxidase.

Assembly of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, requires a concerted activity of a number of chaperones and factors for the insertion of subunits, accessory proteins, cofactors and prosthetic groups. It is now well accepted that the multienzyme complexes of the respiratory chain are organized in vivo as supramolecular functional structures, so-called supercomplexes. Here, we investigate the role of COX17 in the biogenesis of the respiratory chain in HeLa cells. In accordance with its predicted function as a copper chaperone and its role in formation of the binuclear copper centre of cytochrome c oxidase, COX17 siRNA knockdown affects activity and assembly of cytochrome c oxidase. While the abundance of cytochrome c oxidase dimers seems to be unaffected, blue native gel electrophoresis reveals the disappearance of COX-containing supercomplexes as an early response. We observe the accumulation of a novel approximately 150 kDa complex that contains Cox1, but not Cox2. This observation may indicate that the absence of Cox17 interferes with copper delivery to Cox2, but not to Cox1. We suggest that supercomplex formation is not simply due to assembly of completely assembled complexes. An interdependent assembly scenario for the formation of supercomplexes that rather requires the coordinated synthesis and association of individual complexes, is proposed.

Germ cell-less expression in medaka (Oryzias latipes).

The gene germ cell-less (gcl) plays an important role in the early differentiation of germ cells in Drosophila. We isolated the gcl homolog of the model teleost medaka (Oryzias latipes) using degenerated primers and an ovary cDNA bank. The predicted amino acid sequence of medaka gcl showed 92, 68 and 31% overall identity to mouse, human and Drosophila gcl respectively. RT-PCR revealed stronger expression in the ovary and weaker expression in testis, brain, heart, liver and muscle tissue. Expression in early embryos indicates the presence of maternal mRNA. By in situ hybridisation (ISH), gcl could not be detected in embryos. In contrast to vasa, ISH revealed expression of gcl in the ovary but not in the testis. Mol. Reprod. Dev. 67: 15-18, 2004.

Cytochrome c oxidase deficiency due to mutations in SCO2, encoding a mitochondrial copper-binding protein, is rescued by copper in human myoblasts.

Mutations in SCO2, a cytochrome c oxidase (COX) assembly gene, have been reported in nine infants with early onset fatal cardioencephalomyopathy and a severe COX deficiency in striated muscle. Studies on a yeast homolog have suggested that human Sco2 acts as a copper chaperone, transporting copper to the Cu(A) site on the Cox II subunit, but the mechanism of action remains unclear. To investigate the molecular basis of pathogenesis of Sco2 defects in humans we performed genetic and biochemical studies on tissues, myoblasts and fibroblasts from affected patients, as well as on a recombinant human C-terminal Sco2 segment (22 kDa), bearing the putative CxxxC metal-binding motif. Recombinant Sco2 was shown to bind copper with a 1:1 stoichiometry and to form homomeric complexes in vitro, independent of the metal-binding motif. Immunohistochemistry using antibodies directed against different COX subunits showed a marked tissue-specific decrease in the Cox II/III subunits that form part of the catalytic core, consistent with the differential tissue involvement, but a more uniform distribution of Cox Vab, a nuclear-encoded subunit. Sco2 was severely reduced in patient fibroblasts and myoblasts by immunoblot analysis. Patient fibroblasts showed increased (64)Cu uptake but normal retention values and, consistent with this, the copper concentration was four times higher in Sco2-deficient myoblasts than in controls. COX activity in patient myoblasts was completely rescued by transduction with a retroviral vector expressing the human SCO2 coding sequence, and more interestingly by addition of copper-histidine (300 microM) to the culture medium. Whether the latter is accomplished by the very low residual levels of Sco2 in the patient cells, direct addition of copper to the Cu(A) site, or by another copper-binding protein remains unknown. Whatever the mechanism, this result suggests a possible therapy for the early treatment of this fatal infantile disease.

Expression of a VEGF-like protein from Parapoxvirus ovis in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe.

We report on the expression of a VEGF-like protein encoded by Parapoxvirus ovis in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. We show that a lysine residue at amino acid position 2 (K2) is an important determinant for the stability of this protein in S. cerevisiae. Replacement of K2 by an arginine results in stabilization of the protein. This observation suggests that this lysine may be a target for ubiquitinylation, which is a prerequisite for proteasome-mediated protein degradation. Interestingly, in S. pombe the lysine (K2) has no influence on the stability of the protein. This result indicates that the two yeast species exhibit significant differences in their protein degradation pathways.

The P(174)L mutation in the human hSCO1 gene affects the assembly of cytochrome c oxidase.

Mutations of the yeast SCO1 gene result in impaired COX assembly. Recently, heterozygous mutations in the human homologue hSCO1 have been reported in infants suffering from neonatal ketoacidotic coma and isolated COX deficiency (Valnot et al., 2000). One of the hSCO1 alleles harboured a frame shift mutation resulting in a premature stop codon, the other a missense mutation leading to a substitution of proline(174) by leucine. This position is next to the essential CXXXC motif, which is conserved in all Sco1p homologues. We used chimeric proteins with the amino-terminal portion derived from yeast Sco1p and carboxy-terminal portion including the CXXXC motif from the human hSco1p to provide experimental evidence for the pathogenic nature of the P(174)L mutation. These chimeras are able to complement yeast sco1 null mutants. Introduction of the P(174)L mutation affects the function of these chimeric proteins severely, as shown by impaired COX assembly and loss of COX activity.

Mitochondrial copper metabolism in yeast: interaction between Sco1p and Cox2p.

Yeast mitochondrial Sco1p is required for the formation of a functional cytochrome c oxidase (COX). It was suggested that Sco1p aids copper delivery to the catalytic center of COX. Here we show by affinity chromatography and coimmunoprecipitation that Sco1p interacts with subunit Cox2p. In addition we provide evidence that Sco1p can form homomeric complexes. Both homomer formation and binding of Cox2p are neither dependent on the presence of copper nor affected by mutations of His-239, Cys-148 or Cys-152. These amino acids, which are conserved among the members of the Sco1p family, have been suggested to act in the reduction of the cysteines in the copper binding center of Cox2p and are discussed as ligands for copper.

Identification of functionally important regions of the Saccharomyces cerevisiae mitochondrial translational activator Cbs1p.

Translation of cytochrome b mRNA in yeast mitochondria requires activation by the nuclear-encoded Cbs1p. According to the current model, Cbs1p tethers cytochrome b mRNA to the inner mitochondrial membrane via interaction with the 5'-untranslated leader. Cbs1p is predicted to be a hydrophilic protein with two hydrophobic segments near the carboxyl-terminal end, which are both too short to span the membrane. Nevertheless Cbs1p is tightly associated with the mitochondrial membrane, as shown by its behaviour in extraction experiments with taurodeoxycholate. In an attempt to define functionally important regions of Cbs1p, we created a number of mutant alleles by random and directed mutagenesis. We report that a Cbs1p mutant protein lacking the mitochondrial presequence is still able to complement a Deltacbs1 strain, suggesting that the presequence does not contain essential mitochondrial targeting information. Mutations in a cluster of positively charged amino acids at the extremeC-terminus have no effect on Cbs1p function, but removal of this segment severely impairs Cbs1p function. Truncation of 12 or more amino acids from the C-terminus results in a completely defective protein. We further show that both short hydrophobic regions are essential for Cbs1p function, although membrane association is observed even in the absence of these regions.

Yeast translational activator Cbs2p: mitochondrial targeting and effect of overexpression.

The yeast translational activator protein Cbs2p is imported into mitochondria without obvious proteolytic processing. To test the importance of amino-terminal amino acids for mitochondrial targeting we fused varying portions of the N-terminus with green fluorescent protein and examined the intracellular distribution of the reporter protein. We show that the 25 N-terminal amino acids are sufficient to direct the majority of the fusion protein into mitochondria. Cbs2p derivatives lacking 9 to 35 amino acids from the N-terminus fail to complement the respiratory deficiency of a deltacbs2 strain, but are still imported into mitochondria. Therefore Cbs2p contains at least one independent mitochondrial targeting information in addition to the N-terminal signal. We further analyzed the effect of over-expression of Cbs2p on mitochondrial function. Elevated concentrations of Cbs2p lead to slightly impaired mitochondrial gene expression, probably as the result of the formation of inactive Cbs2p aggregates.

Mutational analysis of yeast mitochondrial translational activator Cbs2p and of YHR063Cp, a protein with similarity to Cbs2p.

Translation of mitochondrial cytochrome b in Saccharomyces cerevisiae requires the nuclearly encoded proteins Cbs1p, Cbs2p and Cbp6p. So far no homologs have been identified, except for the product of the S. cerevisiae orf YHR063C, which has some similarity to Cbs2p. Here we analyze the effect of a null mutation of YHR063C and show that it is not required for mitochondrial respiration. In addition, we report on the importance of the carboxyl-terminus of Cbs2p for its function. We show that truncations and some directed mutations in the carboxyl-terminal region of Cbs2p render the protein non-functional. The importance of the COOH-terminus is further underscored by the finding that mutational alteration of the cbs2-1 allele results in the substitution of Ile(372) by Lys.

Human members of the SCO1 gene family: complementation analysis in yeast and intracellular localization.

Cytochrome c oxidase is a multiprotein complex in the mitochondrial membrane whose biogenesis requires a number of proteins besides the structural subunits. Several yeast proteins as well as a human disease-related protein have been reported which are involved in cytochrome c oxidase assembly. The S. cerevisiae Sco1p protein has been implicated in the transfer of copper to cytochrome c oxidase subunits Cox1p and/or Cox2p. Here we report on the complementation behavior in yeast of two recently identified ScSco1p homologs of chromosome 17 and chromosome 22 from human. When allotropically expressed in yeast, both genes fail to complement the lack of the ScSCO1 gene. However, a chimera of the N-terminal half of ScSco1p and the C-terminal half of the chromosome 17 homolog does substitute for the ScSco1p function. Interestingly, the respective chimera with the human homolog of chromosome 22 is not able to complement. Expression of EGFP fusions in HeLa cells shows that both human ScSco1p homologs are located in the mitochondria of human cells.

Mitochondrial copper metabolism in yeast: mutational analysis of Sco1p involved in the biogenesis of cytochrome c oxidase.

Saccharomyces cerevisiae Sco1p is believed to be involved in the transfer of copper from the carrier Cox17p to the mitochondrial cytochrome c oxidase subunits 1 and 2. We here report on the results of a mutational analysis of Sco1p. The two cysteine residues of a potential metal-binding motif (CxxxC) are essential for protein function as shown by their substitution by alanines. Chimeras consisting of Sco1p and its homolog S. cerevisiae Sco2p restrict the specificity of Sco1p function to the N-terminal half of the protein. A candidate region for conferring specificity on Sco1p is a stretch of hydrophobic amino acids, which act as a membrane anchor. In line with this suggestion is the result that alterations of individual amino acids within this region impair Sco1p function.

Disseminated neocortical and subcortical encephalopathy (DNSE) with widespread activation of brain macrophages: a new dementia disorder? Autopsy reports of two postmenopausal women from families with mitochondrial DNA mutations.

In this study we present 2 postmenopausal women who showed clinical symptoms that resembled those of a rather well-defined group of vascular dementia disorders, termed subcortical dementia (Binswanger disease, CADASIL). Patient 1 exhibited mitochondrial DNA (mtDNA) variants in the ND5 gene at position 13,708 and the Cytb gene at position 15,257. These DNA variants have been described in a number of neurologic disorders, but their pathogenetic potential is unclear. Patient 2 showed the same DNA alterations and an additional mtDNA variant at position 15,812 in the Cytb gene. The principal neurohistologic features of the 2 atrophic brains presented here include: subtotal selective neuronal cell loss in the cortex and, to a lesser degree, in the basal ganglia (claustrum, putamen, globus pallidus), sparing palaeocortex and periarchaeocortex, and a very characteristic and diagnostic feature was detachment of astrocytic processes from capillary walls resulting in pericapillary space formation. These pericapillary spaces were partially filled with macrophages. The spaces were not associated with total breakdown of the blood vessel walls as demonstrated by the absence of erythrocytes, lymphocytes, or polymorphonuclear leukocytes outside the vascular bed of the brain; progressive subcortical encephalopathy, as it is seen in subcortical dementia (Binswanger), but lacking arterial lipohyalinosis. The cerebral grey and white matter revealed cuffing of arteries and arterioles by adventitial macrophages. The neocortical and subcortical changes were accompanied by myriads of activated macrophages filled with lipids. The pathology of our 2 cases differs from that of other neurodegenerative disorders and we suggest the term of "disseminated neocortical and subcortical encephalopathy (DNSE) with widespread activation of brain macrophages".

A soluble 12-kDa protein of the mitochondrial intermembrane space, Mrs11p, is essential for mitochondrial biogenesis and viability of yeast cells.

We have isolated an essential yeast gene termed MRS11, which codes for a soluble protein of the mitochondrial intermembrane space. Interestingly, this new gene shares many similarities with the previously characterized MRS5 gene: when expressed from a multicopy plasmid, MRS11 like MRS5 restores respiration competence to yeast strains defective in the splicing of mitochondrial group II introns. Both genes are essential for viability of yeast cells, as the disruption of either of them is lethal. The proteins encoded by MRS5 and MRS11, which display 35%, sequence identity are both located in the mitochondrial intermembrane space. Depletion of Mrs11p results in a phenotype similar to that observed in Mrs5p-depleted cells: accumulation of the precursor form of mitochondrial hsp60, inability to form spectrophotometrically detectable amounts of cytochromes and changes in the mitochondrial morphology. Although similar in sequence and function, Mrs5p and Mrs11p are not functionally equivalent and neither can substitute for the other, even when overexpressed. Taken together, our data suggest a cooperative mode of action of Mrs11p and Mrs5p in mitochondrial protein import or other related essential mitochondrial processes.