Utilization of a deoxynucleoside diphosphate substrate by HIV reverse transcriptase

Background: Deoxynucleoside triphosphates (dNTPs) are the normal substrates for DNA sysnthesis is catalyzed by polymerases such as HIV-1 reverse transcriptase (RT). However, substantial amounts of deoxynucleoside diphosphates (dNDPs) are also present in the cell. Use of dNDPs in HIV-1 DNA sysnthesis could have significant implications for the efficacy of nucleoside RT inhibitors such as AZT which are first line therapeutics fro treatment of HIV infection. Our earlier work on HIV-1 reverse transcriptase (RT) suggested that the interaction between the γ phosphate of the incoming dNTP and RT residue K65 in the active site is not essential for dNTP insertion, implying that this polymerase may be able to insert dNPs in addition to dNTPs. Methodology/Principal Findings: We examined the ability of recombinant wild type (wt) and mutant RTs with substitutions at residue K65 to utilize a dNDP substrate in primer extension reactions. We found that wild type HIV-1 RT indeed catalyzes incorporation of dNDP substrates whereas RT with mutations of residue K645 were unable to catalyze this reaction. Wild type HIV-1 RT also catalyzed the reverse reaction, inorganic phosphate-dependent phosphorolysis. Nucleotide-mediated phosphorolytic removal of chain-terminating 3′-terminal nucleoside inhibitors such as AZT forms the basis of HIV-1 resistance to such drugs, and this removal is enhanced by thymidine analog mutations (TAMs). We found that both wt and TAM-containing RTs were able to catalyze Pi-mediated phosphorolysis of 3′-terminal AZT at physiological levels of Pi with an efficacy similar to that for ATP-dependent AZT-excision. Conclusion: We have identified two new catalytic function of HIV-1 RT, the use of dNDPs as substrates for DNA synthesis, and the use of Pi as substrate for phosphorolytic removal of primer 3′-terminal nucleotides. The ability to insert dNDPs has been documented for only one other DNA polymerase The RB69 DNA polymerase and the reverse reaction employing inorganic phosphate has not been documented for any DNA polymerase. Importantly, our results show that Pi-mediated phosphorolysis can contribute to AZT resistance and indicates that factors that influence HIV resistance to AZT are more complex than previously appreciated. © 2008 Garforth et al

A Conserved Mitochondrial ATP-binding Cassette Transporter Exports Glutathione Polysulfide for Cytosolic Metal Cofactor Assembly

An ATP-binding cassette transporter located in the inner mitochondrial membrane is involved in iron-sulfur cluster and molybdenum cofactor assembly in the cytosol, but the transported substrate is unknown. ATM3 (ABCB25) from Arabidopsis thaliana and its functional orthologue Atm1 from Saccharomyces cerevisiae were expressed in Lactococcus lactis and studied in inside-out membrane vesicles and in purified form. Both proteins selectively transported glutathione disulfide (GSSG) but not reduced glutathione in agreement with a 3-fold stimulation of ATPase activity by GSSG. By contrast, Fe(2+) alone or in combination with glutathione did not stimulate ATPase activity. Arabidopsis atm3 mutants were hypersensitive to an inhibitor of glutathione biosynthesis and accumulated GSSG in the mitochondria. The growth phenotype of atm3-1 was strongly enhanced by depletion of the mitochondrion-localized, GSH-dependent persulfide oxygenase ETHE1, suggesting that the physiological substrate of ATM3 contains persulfide in addition to glutathione. Consistent with this idea, a transportomics approach using mass spectrometry showed that glutathione trisulfide (GS-S-SG) was transported by Atm1. We propose that mitochondria export glutathione polysulfide, containing glutathione and persulfide, for iron-sulfur cluster assembly in the cytosol.This work was supported in part by the Biotechnology and Biological Sciences Research Council Grant BB/H00288X/1

The pressure of strong coupling lattice QCD with heavy quarks, the hadron resonance gas model and the large N limit

In this paper we calculate the pressure of pure lattice Yang-Mills theories and lattice QCD with heavy quarks by means of strong coupling expansions. Dynamical fermions are introduced with a hopping parameter expansion, which also allows for the incorporation of finite quark chemical potential. We show that in leading orders the results are in full agreement with expectations from the hadron resonance gas model, thus validating it with a first principles calculation. For pure Yang-Mills theories we obtain the corresponding ideal glueball gas, in QCD with heavy quarks our result equals that of an ideal gas of mesons and baryons. Another finding is that the Yang-Mills pressure in the large N limit is of order $\sim N^0$ to the calculated orders, when the inverse 't Hooft coupling is used as expansion parameter. This property is expected in the confined phase, where our calculations take place.Comment: 12 pages, 4 figure

Substance P induces localization of MIF/α1-inhibitor-3 complexes to umbrella cells via paracellular transit through the urothelium in the rat bladder

BACKGROUND: Macrophage migration inhibitory factor (MIF) is released into the intraluminal fluid during bladder inflammation in the rat complexed to α1-inhibitor-3 (A1-I3; a rodent proteinase inhibitor in the α-macroglobulin family). The location of A1-I3 in the bladder had not been investigated. Therefore, we examined the location of A1-I3 and MIF/A1-I3 complexes in the bladder and changes due to experimental inflammation. METHODS: Anesthetized male rats had bladders removed with no treatment (intact) or were injected with Substance P (SP; s.c.; saline vehicle). After one hour intraluminal fluid was removed, bladder was excised and MIF and A1-I3 levels were determined using ELISA and/or western-blotting. MIF co-immunoprecipitation determined MIF/A1-I3 complexes in the bladder. Bladder sections were immunostained for A1-I3 and MIF/A1-I3. RESULTS: A1-I3 immunostaining was observed in interstitial spaces throughout the bladder (including submucosa) but not urothelium in intact and saline-treated rats. RT-PCR showed that the bladder does not synthesize A1-I3, therefore, A1-I3 in the interstitial space of the bladder must be plasma derived. In SP-treated rats, A1-I3 in the bladder increased and A1-I3 was observed traversing through the urothelium. Umbrella cells that do not show MIF and/or A1-I3 immunostaining in intact or saline-treated rats, showed co-localization of MIF and A1-I3 after SP-treatment. Western blotting demonstrated that in the bladder MIF formed non-covalent interactions and also binds covalently to A1-I3 to form high molecular weight MIF/A1-I3 complexes (170, 130 and 75-kDa, respectively, verified by co-immunoprecipitation). SP-induced inflammation selectively reduced 170-kDa MIF/A1-I3 in the bladder while increasing 170 and 130-kDa MIF/A1-I3 in the intraluminal fluid. CONCLUSION: A1-I3 and MIF/A1-I3 complexes are resident in bladder interstitium. During SP-induced inflammation, MIF/A1-I3 complexes are released from the bladder into the lumen. Binding of MIF/A1-I3 complexes to urothelial cells during inflammation suggests these complexes participate in the inflammatory reaction through activation of receptors for MIF and/or for A1-I3

Racial Bias Beliefs Related to COVID-19 Among Asian Americans, Native Hawaiians, and Pacific Islanders: Findings From the COVID-19 Effects on the Mental and Physical Health of Asian Americans and Pacific Islanders Survey Study (COMPASS)

Background: During the COVID-19 pandemic, there have been increased reports of racial biases against Asian American and Native Hawaiian and Pacific Islander individuals. However, the extent to which different Asian American and Native Hawaiian and Pacific Islander groups perceive and experience (firsthand or as a witness to such experiences) how COVID-19 has negatively affected people of their race has not received much attention. Objective: This study used data from the COVID-19 Effects on the Mental and Physical Health of Asian Americans and Pacific Islanders Survey Study (COMPASS), a nationwide, multilingual survey, to empirically examine COVID-19-related racial bias beliefs among Asian American and Native Hawaiian and Pacific Islander individuals and the factors associated with these beliefs. Methods: COMPASS participants were Asian American and Native Hawaiian and Pacific Islander adults who were able to speak English, Chinese (Cantonese or Mandarin), Korean, Samoan, or Vietnamese and who resided in the United States during the time of the survey (October 2020 to May 2021). Participants completed the survey on the web, via phone, or in person. The Coronavirus Racial Bias Scale (CRBS) was used to assess COVID-19-related racial bias beliefs toward Asian American and Native Hawaiian and Pacific Islander individuals. Participants were asked to rate the degree to which they agreed with 9 statements on a 5-point Likert scale (ie, 1=strongly disagree to 5=strongly agree). Multivariable linear regression was used to examine the associations between demographic, health, and COVID-19-related characteristics and perceived racial bias. Results: A total of 5068 participants completed the survey (mean age 45.4, SD 16.4 years; range 18-97 years). Overall, 73.97% (3749/5068) agreed or strongly agreed with ≥1 COVID-19-related racial bias belief in the past 6 months (during the COVID-19 pandemic). Across the 9 racial bias beliefs, participants scored an average of 2.59 (SD 0.96, range 1-5). Adjusted analyses revealed that compared with Asian Indians, those who were ethnic Chinese, Filipino, Hmong, Japanese, Korean, Vietnamese, and other or multicultural had significantly higher mean CRBS scores, whereas no significant differences were found among Native Hawaiian and Pacific Islander individuals. Nonheterosexual participants had statistically significant and higher mean CRBS scores than heterosexual participants. Compared with participants aged ≥60 years, those who were younger (aged \u3c30, 30-39, 40-49, and 50-59 years) had significantly higher mean CRBS scores. US-born participants had significantly higher mean CRBS scores than foreign-born participants, whereas those with limited English proficiency (relative to those reporting no limitation) had lower mean CRBS scores. Conclusions: Many COMPASS participants reported racial bias beliefs because of the COVID-19 pandemic. Relevant sociodemographic contexts and pre-existing and COVID-19-specific factors across individual, community, and society levels were associated with the perceived racial bias of being Asian during the pandemic. The findings underscore the importance of addressing the burden of racial bias on Asian American and Native Hawaiian and Pacific Islander communities among other COVID-19-related sequelae

Social Support and Technology Use and Their Association With Mental and Physical Health During the COVID-19 Pandemic Among Asian Americans: The COMPASS Cross-sectional Study

BACKGROUND: The global COVID-19 pandemic disproportionately affected Asian Americans and Pacific Islanders (AAPIs) and revealed significant health disparities with reports of increased discrimination and xenophobia. Among AAPIs, the pandemic exacerbated their social, linguistic, and geographic isolation. Social support may be especially important for AAPIs given the salience of collectivism as a cultural value. Another mechanism for support among AAPIs was technology use, as it is generally widespread among this population. However, older adults may not perceive the same benefits. OBJECTIVE: We examined social support and technology use and their relationships with mental and physical health outcomes through the COVID-19 pandemic among AAPIs. METHODS: Data were drawn from the COVID-19 Effects on the Mental and Physical Health of AAPI Survey Study (COMPASS) for the time period of October 2020 to February 2021. COMPASS was a cross-sectional, multilingual, national survey conducted online, by phone, and in person with AAPI adults who were ≥18 years of age, in collaboration with academic and community partners in the United States. Data were analyzed using multivariable linear regression using the outcome variables of mental and physical health with various predictors such as social support and technology use. We tested for interactions specific to age and ethnicity. RESULTS: Among 4631 AAPIs (mean age 45.9, SD 16.3 years; 2992/4631, 63.1% female), we found that (1) increased social support was associated with better physical health, (2) total social support was positively associated with better mental health, (3) higher technology use was associated with poorer mental health and inversely associated with poorer physical health, (4) the association of technology use with mental health was weaker among those with low social support (vs those with high social support), (5) adults younger than 60 years old (vs ≥60 years old) were more negatively affected with social support and mental health, and (6) Korean Americans appeared to be a high-risk group for poor physical health with increased technology use. CONCLUSIONS: Our paper identified mental and physical health needs along with supportive therapies observed among AAPIs during the pandemic. Future research on how social support can be leveraged, especially among AAPIs younger than 60 years old, and how various types of technology are being utilized are important to guide the recovery efforts to address both mental and physical disparities across communities as a result of the COVID-19 pandemic

An evaluation of enteral nutrition practices and nutritional provision in children during the entire length of stay in critical care

<b>Background</b> Provision of optimal nutrition in children in critical care is often challenging. This study evaluated exclusive enteral nutrition (EN) provision practices and explored predictors of energy intake and delay of EN advancement in critically ill children.<p></p> <b>Methods</b> Data on intake and EN practices were collected on a daily basis and compared against predefined targets and dietary reference values in a paediatric intensive care unit. Factors associated with intake and advancement of EN were explored.<p></p> <b>Results</b> Data were collected from 130 patients and 887 nutritional support days (NSDs). Delay to initiate EN was longer in patients from both the General Surgical and congenital heart defect (CHD) Surgical groups [Median (IQR); CHD Surgical group: 20.3 (16.4) vs General Surgical group: 11.4 (53.5) vs Medical group: 6.5 (10.9) hours; p <= 0.001]. Daily fasting time per patient was significantly longer in patients from the General Surgical and CHD Surgical groups than those from the Medical group [% of 24 h, Median (IQR); CHD Surgical group: 24.0 (29.2) vs General Surgical group: 41.7 (66.7) vs Medical group: 9.4 (21.9); p <= 0.001]. A lower proportion of fluids was delivered as EN per patient (45% vs 73%) or per NSD (56% vs 73%) in those from the CHD Surgical group compared with those with medical conditions. Protein and energy requirements were achieved in 38% and 33% of the NSDs. In a substantial proportion of NSDs, minimum micronutrient recommendations were not met particularly in those patients from the CHD Surgical group. A higher delivery of fluid requirements (p < 0.05) and a greater proportion of these delivered as EN (p < 0.001) were associated with median energy intake during stay and delay of EN advancement. Fasting (31%), fluid restriction (39%) for clinical reasons, procedures requiring feed cessation and establishing EN (22%) were the most common reasons why target energy requirements were not met.<p></p> <b>Conclusions</b> Provision of optimal EN support remains challenging and varies during hospitalisation and among patients. Delivery of EN should be prioritized over other "non-nutritional" fluids whenever this is possible.<p></p&gt