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Background: The HIV-1 latent reservoir is a major barrier to developing an HIV cure. Gene therapy is a promising treatment, highlighted by the success of the Berlin and London patients. [...]

BackgroundResidual renal function (RRF) is a marker for a good index of health and is associated with improved survival for individuals with end stage renal disease on peritoneal dialysis. As RRF declines with time on dialysis, fluid balance is more difficult to achieve. Urine output plays a vital role in fluid removal and it has been postulated that loop diuretics improve diuresis in peritoneal dialysis (PD) patients. The aim of this study is to evaluate our use of furosemide and its effect on diuresis in a home peritoneal dialysis program.MethodsSixty-one patients met inclusion criteria of having been on PD continuously for one year from their start date with complete 24-hour urine kinetics. Twenty patients were on furosemide and 41 patients were in the control group. Data for urine volume (UV), serum creatinine (SCr), total and residual creatinine clearance (CrCl[total] and CrCl[residual), total and residual urea clearance (Kt/V[total] and Kt/V[residual]), and dry body weight were collected at baseline, six months and one year. The average change in UV, CrCl[total], and Kt/V[total] from baseline at six and 12 months and the proportion of patients who developed anuria at one year were determined.ResultsUV declined in the furosemide and control groups at six months by an average of 78.00 +/- 445.2 mL/day and 105.5 +/- 401.8 mL/day (p=0.8) and at 12 months by 85.00 +/- 481.7 mL/day and 110.7 +/- 455.4 mL/day (p=0.8), respectively. CrCl declined in the furosemide and control groups at six months by an average of 5.55 +/- 20.4 mL/min and 4.52 +/- 29.0 mL (p=0.9), and at 12 months by 3.95 +/- 35.5 mL/min and 9.05 +/- 28.4 mL/min (p=0.5) respectively. Kt/V increased by 0.0850 +/- 0.890 in the furosemide group and declined by 0.0456 +/- 0.614 in the control group at six months (p=0.5), but after 12 months, Kt/V declined in both the furosemide and control groups by 0.00400 +/- 0.565 and 0.162 +/- 0.558 (p=0.5) respectively. Only one patient (five per cent) in the furosemide group developed anuria after one year on PD, whereas nine patients (22%) in the control group became anuric (p=0.1).ConclusionFurosemide did not have a statistically significant effect in either improving UV or preserving RRF in patients on PD for one year, but this study was not adequately powered to show an association. Although not statistically significant, fewer patients were anuric at one year in the furosemide group (five per cent versus 22%). Furosemide was not shown to be detrimental to either RRF or UV. [ABSTRACT FROM AUTHOR]

Acknowledging patients' spirituality is part of a nurse's holistic duty to a patient. A number of strategies can be employed to explore this, including the use of religious 'professionals' (priest, rabbi, imam, etc.) but this will exclude the 25% with no stated religion. Spirituality can exist without a theistic framework and can be more humanistic or existential. Nurses need to be aware of this important dimension in the patients' holistic needs. [ABSTRACT FROM AUTHOR]

Study Objectives: To evaluate the performance of a novel three-dimensional-printed customized nasal mask on patient satisfaction and adherence to continuous positive airway pressure (CPAP) therapy for obstructive sleep apnea., Methods: Patients prescribed CPAP therapy with suboptimal CPAP adherence using a conventional CPAP mask (< 70% of nights with ≥ 4 hours per night over 4 weeks) were recruited from the sleep investigation unit of a tertiary hospital. Patients underwent a three-dimensional facial mapping procedure to have a novel three-dimensional-printed customized nasal mask fabricated which was trialed for four weeks. CPAP adherence data download of the same period was conducted with their pre-existing conventional mask and customized mask. Questionnaires assessing symptoms of obstructive sleep apnea and mask-related side-effects were administered before and after the trial of the customized mask., Results: Thirty patients (22 males and 8 females, age 63.3 ± 12.5 years, body mass index 31.7 ± 5.2 kg/m 2 , apnea-hypopnea index 37.3 ± 21.9 events/h [mean ± standard deviation]) were studied. CPAP was used in a greater proportion of nights with the customized mask (85.7 [66.1, 98.2]% vs 63.2 [13.1, 96.8]%, P = .009) compared to the conventional mask. Hourly CPAP usage was higher with the customized mask (3.8 [2.7, 5.8] hours vs 2.4 [0.3, 5.0] hours, P = .016) compared to a conventional mask. Patients preferred the customized mask ( P = .008) and reported less mask-related side effects., Conclusions: The novel three-dimensional-printed customized mask improved CPAP usage in patients with suboptimal CPAP adherence. Customized CPAP masks may be a suitable option for patients experiencing poor CPAP adherence from mask-related side effects., Clinical Trial Registration: Registry: ANZCTR; Name: Conventional vs custom made nasal Continuous Positive Airway Pressure (CPAP) mask for treatment of Obstructive Sleep Apnoea: Pilot study A; URL: https://anzctr.org.au/Trial/Registration/TrialReview.aspx?id=382142; Identifier: ACTRN12621001301853., Citation: Tong BK, Cistulli PA, Ledger S, Chan ASL. A novel three-dimensional-printed customized nasal mask for improving CPAP adherence and satisfaction for the treatment of obstructive sleep apnea. J Clin Sleep Med . 2025;21(1):9-16., (© 2025 American Academy of Sleep Medicine.)

The use of Deaf role-models (DRMs) with Deaf children born into hearing families is a practice aimed at improving outcomes for Deaf children, yet there is little peer-reviewed research available to influence future direction of such. This scoping review directs attention to available research on DRMs as a socio-linguistic and cultural viewpoint for balancing a predominantly audiological approach for early intervention for Deaf children. Systematic database searches initially yielded 132 records, of which seven articles were included in this scoping review. Findings are presented as five themes: 'Deaf Gain' and associated cultural capital, effective communication, developmental influences, family (or caregiver) attitudes to Deafness, and administration of DRM programs. Few formalized DRM programs were identified within the literature. The review concludes with recommendations for further exploration of the DRM experiences of Deaf people and their families within Australia., (© The Author(s) 2024. Published by Oxford University Press.)

Human T cell leukaemia virus type-1 (HTLV-1) is an oncogenic retrovirus that causes lifelong infection in ~5-10 million individuals globally. It is endemic to certain First Nations populations of Northern and Central Australia, Japan, South and Central America, Africa, and the Caribbean region. HTLV-1 preferentially infects CD4 + T cells and remains in a state of reduced transcription, often being asymptomatic in the beginning of infection, with symptoms developing later in life. HTLV-1 infection is implicated in the development of adult T cell leukaemia/lymphoma (ATL) and HTLV-1-associated myelopathies (HAM), amongst other immune-related disorders. With no preventive or curative interventions, infected individuals have limited treatment options, most of which manage symptoms. The clinical burden and lack of treatment options directs the need for alternative treatment strategies for HTLV-1 infection. Recent advances have been made in the development of RNA-based antiviral therapeutics for Human Immunodeficiency Virus Type-1 (HIV-1), an analogous retrovirus that shares modes of transmission with HTLV-1. This review highlights past and ongoing efforts in the development of HTLV-1 therapeutics and vaccines, with a focus on the potential for gene therapy as a new treatment modality in light of its successes in HIV-1, as well as animal models that may help the advancement of novel antiviral and anticancer interventions., Competing Interests: The authors declare no conflicts of interest.

Early gene therapy studies held great promise for the cure of heritable diseases, but the occurrence of various genotoxic events led to a pause in clinical trials and a more guarded approach to progress. Recent advances in genetic engineering technologies have reignited interest, leading to the approval of the first gene therapy product targeting genetic mutations in 2017. Gene therapy (GT) can be delivered either in vivo or ex vivo. An ex vivo approach to gene therapy is advantageous, as it allows for the characterization of the gene-modified cells and the selection of desired properties before patient administration. Autologous cells can also be used during this process which eliminates the possibility of immune rejection. This review highlights the various stages of ex vivo gene therapy, current research developments that have increased the efficiency and safety of this process, and a comprehensive summary of Human Immunodeficiency Virus (HIV) gene therapy studies, the majority of which have employed the ex vivo approach.

Denosumab can improve bone health in advanced kidney disease (CKD) but is associated with hypocalcemia. We created a clinical care pathway focused on the safe provision of denosumab in advanced CKD that reduced the risk of hypocalcemia by 37% at our hospital. Similar pathways could be adopted and tested in other centers., Purpose: There is an increased risk of hypocalcemia with denosumab in advanced chronic kidney disease (CKD). We aimed to reduce the proportion of patients with advanced CKD who experienced denosumab-induced hypocalcemia at our center., Methods: We conducted a quality improvement (QI) project of patients with CKD stage 3b or less (i.e., estimated glomerular filtration rate <45 mL/min/1.73m 2 including dialysis) who were part of the Osteoporosis and Bone Disease Program at St. Joseph's Health Care London (Canada) between December 2020 and January 2023. Our intervention was a clinical care pathway which optimized CKD mineral and bone disorder (CKD-MBD) and 25-hydroxyvitamin levels; provided calcium and vitamin D prophylaxis; promoted multidisciplinary communication between bone and kidney specialists; and carefully monitored calcium post-denosumab injection. Our primary outcome measure was the proportion of patients with hypocalcemia (defined by albumin-corrected serum calcium <1.9mmol/L) at 60 days. Process measures included the appropriate provision of calcium and vitamin D prophylaxis. Balance measures included the development of hypercalcemia and hyperphosphatemia following prophylaxis. We used plan-do-see-act cycles to study four tests of change and presented results using descriptive statistics and run charts., Results: There were 6 patients with advanced CKD treated with denosumab prior to the implementation of our care pathway (March 2015-October 2020; 83% receiving dialysis). At the time of their denosumab injection, 83% were using 500-1000 mg of calcium, and 83% used 1000-2000 IU of vitamin D 3 . Fifty percent developed denosumab-induced hypocalcemia. Following the implementation of our care pathway, 15 patients (40% receiving dialysis) were treated with denosumab. Ninety-three percent received calcium at a daily dose of 350 to 2250 mg and 87% received 1000-2000 IU of vitamin D 3 . Thirteen percent developed denosumab-induced hypocalcemia. There was no hypercalcemia or hyperphosphatemia., Conclusions: A clinical care pathway focused on the safe provision of denosumab in advanced CKD reduced the risk of hypocalcemia in patients treated in our hospital. Similar pathways could be adopted and tested in other centers., (© 2023. International Osteoporosis Foundation and Bone Health and Osteoporosis Foundation.)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a respiratory virus that causes COVID-19 disease, with an estimated global mortality of approximately 2%. While global response strategies, which are predominantly reliant on regular vaccinations, have shifted from zero COVID to living with COVID, there is a distinct lack of broad-spectrum direct acting antiviral therapies that maintain efficacy across evolving SARS-CoV-2 variants of concern. This is of most concern for immunocompromised and immunosuppressed individuals who lack robust immune responses following vaccination, and others at risk for severe COVID and long-COVID. RNA interference (RNAi) therapeutics induced by short interfering RNAs (siRNAs) offer a promising antiviral treatment option, with broad-spectrum antiviral capabilities unparalleled by current antiviral therapeutics and a high genetic barrier to antiviral escape. Here we describe novel siRNAs, targeting highly conserved regions of the SARS-CoV-1 and 2 genome of both human and animal species, with multi-variant antiviral potency against eight SARS-CoV-2 lineages - Ancestral VIC01, Alpha, Beta, Gamma, Delta, Zeta, Kappa and Omicron. Treatment with our siRNA resulted in significant protection against virus-mediated cell death in vitro, with >97% cell survival (P < 0.0001), and corresponding reductions of viral nucleocapsid RNA of up to 99.9% (P < 0.0001). When compared to antivirals; Sotrovimab and Remdesivir, the siRNAs demonstrated a more potent antiviral effect and similarly, when multiplexing siRNAs to target different viral regions simultaneously, an increased antiviral effect was observed compared to individual siRNA treatments (P < 0.0001). These results demonstrate the potential for a highly effective broad-spectrum direct acting antiviral against multiple SARS-CoV-2 variants, including variants resistant to antivirals and vaccine generated neutralizing antibodies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: S.L., E.B.R, A.D.K. and C.A. hold a patent for antiviral siRNA targeting SARS-CoV-2 and have no personal relationships which may be considered as potential competing interests. All remaining authors declare they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Providing adequate writing instruction and practice in schools is an essential cornerstone of writing development and it affords a diagnostic approach for teachers. But what writing instruction is being practiced in Australian primary schools? The aim of this study was to survey a sample of teachers (n = 310) about their instructional practices for writing and their preparation and self-efficacy to teach writing. The majority of the teachers surveyed indicated they allocated on average less than three hours per week for writing practice in their classrooms, with findings further showing a large variability in the frequency of writing practice ranging from 15 min to 7.5 h per week. Findings suggested an emphasis placed on teaching foundational skills, such as spelling, over the teaching of process skills, such as planning and revising. Results further indicated that less emphasis is placed on teaching handwriting and typing. The majority of participating teachers reported implementing only six of the 20 different instructional practices included in the survey on a weekly basis, with school-home strategies being the least frequently reported strategies to foster students' writing development. Most teachers expressed positive beliefs about their preparation and self-efficacy for teaching writing. Results from multiple regression analysis showed that preparation and self-efficacy for teaching writing significantly and statistically accounted for variability in using evidence-based practices, teaching foundational skills, and teaching process skills. However, only self-efficacy made a statically significant contribution to predicting strategies to extend writing to the home environment. Implications for teaching and recommendations for research are provided., (© The Author(s) 2022.)

Our understanding of HIV infection has greatly advanced since the discovery of the virus in 1983. Treatment options have improved the quality of life of people living with HIV/AIDS, turning it from a fatal disease into a chronic, manageable infection. Despite all this progress, a cure remains elusive. A major barrier to attaining an HIV cure is the presence of the latent viral reservoir, which is established early in infection and persists for the lifetime of the host, even during prolonged anti-viral therapy. Different cure strategies are currently being explored to eliminate or suppress this reservoir. Several studies have shown that a functional cure may be achieved by preventing infection and also inhibiting reactivation of the virus from the latent reservoir. Here, we briefly describe the main HIV cure strategies, focussing on the use of RNA therapeutics, including small interfering RNA (siRNA) to maintain HIV permanently in a state of super latency, and CRISPR gRNA to excise the latent reservoir. A challenge with progressing RNA therapeutics to the clinic is achieving effective delivery into the host cell. This review covers recent nanotechnological strategies for siRNA delivery using liposomes, N-acetylgalactosamine conjugation, inorganic nanoparticles and polymer-based nanocapsules. We further discuss the opportunities and challenges of those strategies for HIV treatment.

Background: History starts from where we are now - it is not just things that happened a long time ago. The global pandemic began in 2019. It has changed the lives of people with learning disabilities. We began our project during the first lockdown in April 2020. We came together to set up a website to collect stories and support and learn from each other about how to survive and keep strong. Storytelling is very important because it helps us understand what is going on. It is also a way to capture the history of people with learning disabilities at a very difficult time. We know that thousands of people with learning disabilities became ill and died in the flu epidemic of 1918. But nobody recorded their stories in their own words. We want to make sure this does not happen again, so we created an archive to help us remember., Methods: The project was managed with an advisory group of people with and without learning disabilities who met monthly to monitor the collection and analysis of stories on the site. A site audit was performed regularly to determine the themes in the stories and who had submitted. The article describes the progress of the project, the stories we have shared, and the challenges we have faced., Conclusions: We discuss how people with learning disabilities have been presented in the media and our views about the way we are not heard, or always shown as vulnerable victims. We have found many sad stories, but also positive ones about people being creative and supportive. We look forward to the future and share our ideas about how society could be different and more inclusive. Being part of this project has given us confidence to know we are not alone, and shown us how we can help with the recovery., Competing Interests: Some of the authors have received payment for talks and training associated with their work for Surviving through Story., (© 2022 The Authors. British Journal of Learning Disabilities published by John Wiley & Sons Ltd.)

Since December 2019, a pandemic of COVID-19 disease, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread across the globe. At present, the Food and Drug Administration (FDA) has issued emergency approval for the use of some antiviral drugs. However, these drugs still have limitations in the specific treatment of COVID-19, and as such, new treatment strategies urgently need to be developed. RNA-interference-based gene therapy provides a tractable target for antiviral treatment. Ensuring cell-specific targeted delivery is important to the success of gene therapy. The use of nanoparticles (NPs) as carriers for the delivery of small interfering RNA (siRNAs) to specific tissues or organs of the human body could play a crucial role in the specific therapy of severe respiratory infections, such as COVID-19. In this review, we describe a variety of novel nanocarriers, such as lipid NPs, star polymer NPs, and glycogen NPs, and summarize the pre-clinical/clinical progress of these nanoparticle platforms in siRNA delivery. We also discuss the application of various NP-capsulated siRNA as therapeutics for SARS-CoV-2 infection, the challenges with targeting these therapeutics to local delivery in the lung, and various inhalation devices used for therapeutic administration. We also discuss currently available animal models that are used for preclinical assessment of RNA-interference-based gene therapy. Advances in this field have the potential for antiviral treatments of COVID-19 disease and could be adapted to treat a range of respiratory diseases.

Jetlag and travel fatigue can impair functioning, but it is unknown what strategies are used by travelers to minimize these consequences. Passengers on Qantas Airways flights were invited to take part in online surveys. Long-haul flights of ≥8 h into and out of Australia were targeted, which involved time differences of 1 to 18 h between the origin and destination. Passengers were queried about the use of travel booking choices before the flight, and the use of behavioral strategies before, during, and after flight for reducing jetlag and travel fatigue. Surveys were completed by N = 460 passengers aged 18 to 78 (43% male; mean age 50 y). Selecting a seat location (59%) and choosing a direct flight (52%) were the most common booking strategies. Almost all (99%) employed specific behavioral strategies during flight, with fewer implementing strategies before flight (73%) and after flight (89%). During the journey, 81% consumed or avoided caffeine and alcohol, 68% altered food intake, 68% used comfort/relaxation strategies, 53% light exposure, 35% physical activity, 31% compression stockings, 15% pharmaceutical sleep aids, and 8% melatonin. Surprisingly, only 1 of 460 passengers reported using a jetlag app. Younger travelers were more likely to adopt any strategy before the flight than older travelers (χ 2 2  = 14.90, p =.01), while female travelers appeared more likely than male travelers to use strategies before (77% vs. 68%) and after flight (91% vs. 85%). Reason for travel, flight cabin, leg of journey, and country of residence were not significantly associated with the use of behavioral strategies. Nearly all passengers took measures to improve the experience and consequences of long-haul flying. The results suggest that interventions around food/drink and physical activity may be highly acceptable to passengers for mitigating travel fatigue and that greater public education on evidence-based strategies may be helpful for reducing travel fatigue and jetlag.

Rationale, Aims, and Objectives: Maternity training is a critical global issue. In the United Kingdom (UK), the need for safer care and patient safety is emphasized through NHS policy. Health Education England (HEE) recommends that training should support a culture of continuous learning and improvement, particularly in the area of reducing the rates of stillbirths, neonatal and maternity deaths, and other adverse outcomes, such as intrapartum brain injuries. Training has been shown to play a crucial role in improving quality of care and reducing maternal and perinatal mortality and morbidity. This evaluation was undertaken to determine both the immediate and sustained impact of multiprofessional training in cardiotocograph (CTG) interpretation and community-based simulation training in obstetric emergencies: childbirth emergencies in the community (CEC). The impact was measured in terms of practitioner knowledge, confidence, and empowerment immediately pretraining and posttraining and at 12 weeks following training., Methods: A longitudinal mixed methods design was used. Attendees to maternity training sessions on cardiotocograph interpretation and management of childbirth emergencies in the community provided the sample. Quantitative data were collected using questionnaires to assess knowledge, confidence, and empowerment. Qualitative data were collected using open-ended questions embedded in the questionnaires. Quantitative data were analysed using within-subject t test to compare differences in the dependent variable measures. Qualitative data analysis was guided by Braun and Clarke (2013) method thematic analysis., Results: The combined qualitative and quantitative results lucidly highlight that training positively impacts upon knowledge, confidence, and empowerment, an impact which is observed across three time points., Conclusions: Training in CTG and CEC is effective in improving knowledge, confidence, and empowerment across all groups. Furthermore, the provision of training packages in these subject areas facilitates improvements in the longer term., (© 2019 John Wiley & Sons, Ltd.)

Objective measures of circadian disruption are difficult to capture in a free-living environment hence the importance of validating subjective measures of jetlag. We aimed to assess the internal consistency of the 15-item Liverpool Jetlag Scale and its convergent and divergent validity with indicators of fatigue and anxiety in a large sample of air passengers. Online survey of passengers was conducted after travel on a range of long-haul flights. Jetlag was captured using the Liverpool scale, fatigue was measured using the Vitality subscale of the Short-Form Health Survey (SF-36), and the presence of anxiety or worry before, during, and after flight was self-reported. Inter-item correlations and Cronbach's alpha were calculated to assess the internal consistency of the scale. Exploratory factor analysis was used to examine whether the scale was consistent with one underlying construct of circadian disruption. Correlations between fatigue and anxiety (flying, situational, symptoms) with jetlag were used to assess convergent and divergent validity. Linear regression was used to determine the most important symptoms contributing to subjective jetlag rating. N = 460 passengers (57% female, mean age 50, SD 16 years) were surveyed. Cronbach's alpha indicated high internal reliability (alpha = 0.85). Jetlag was more strongly correlated with fatigue (rho = 0.47) than any type of anxiety (rho = 0.10-0.22). Exploratory factor analysis indicated responses were consistent with four factors: (i) fatigue/daytime impairment, (ii) sleep disturbance, (iii) changes in appetite and (iv) changes in bowel function. Regression analysis indicated that only changes in concentration, sleep time, fatigue, sleep quality and frequency of bowel motions were independent correlates of subjective jetlag (R 2 = 27%). The Liverpool Jetlag Scale is internally consistent and demonstrates the expected relationships with fatigue and anxiety. Patterns of response are not consistent with all items being derived from one underlying factor, i.e. circadian disruption. Further, not all items contributed to the jetlag rating, suggesting the single-item rating may be useful for capturing the subjective experience of jetlag, whilst a total jetlag score is useful for also capturing circadian symptoms considered by passengers to be unrelated to jetlag. Validation of subjective jetlag against objective measures of circadian disruption is required.

Traditional anatomical analyses captured only a fraction of real phenomic information. Here, we apply deep learning to quantify total phenotypic similarity across 2468 butterfly photographs, covering 38 subspecies from the polymorphic mimicry complex of Heliconius erato and Heliconius melpomene . Euclidean phenotypic distances, calculated using a deep convolutional triplet network, demonstrate significant convergence between interspecies co-mimics. This quantitatively validates a key prediction of Müllerian mimicry theory, evolutionary biology's oldest mathematical model. Phenotypic neighbor-joining trees are significantly correlated with wing pattern gene phylogenies, demonstrating objective, phylogenetically informative phenome capture. Comparative analyses indicate frequency-dependent mutual convergence with coevolutionary exchange of wing pattern features. Therefore, phenotypic analysis supports reciprocal coevolution, predicted by classical mimicry theory but since disputed, and reveals mutual convergence as an intrinsic generator for the unexpected diversity of Müllerian mimicry. This demonstrates that deep learning can generate phenomic spatial embeddings, which enable quantitative tests of evolutionary hypotheses previously only testable subjectively.

Background: Current antiretroviral therapy is effective in controlling HIV-1 infection. However, cessation of therapy is associated with rapid return of viremia from the viral reservoir. Eradicating the HIV-1 reservoir has proven difficult with the limited success of latency reactivation strategies and reflects the complexity of HIV-1 latency. Consequently, there is a growing need for alternate strategies. Here we explore a "block and lock" approach for enforcing latency to render the provirus unable to restart transcription despite exposure to reactivation stimuli. Reactivation of transcription from latent HIV-1 proviruses can be epigenetically blocked using promoter-targeted shRNAs to prevent productive infection. We aimed to determine if independent and combined expression of shRNAs, PromA and 143, induce a repressive epigenetic profile that is sufficiently stable to protect latently infected cells from HIV-1 reactivation when treated with a range of latency reversing agents (LRAs)., Results: J-Lat 9.2 cells, a model of HIV-1 latency, expressing shRNAs PromA, 143, PromA/143 or controls were treated with LRAs to evaluate protection from HIV-1 reactivation as determined by levels of GFP expression. Cells expressing shRNA PromA, 143, or both, showed robust resistance to viral reactivation by: TNF, SAHA, SAHA/TNF, Bryostatin/TNF, DZNep, and Chaetocin. Given the physiological importance of TNF, HIV-1 reactivation was induced by TNF (5 ng/mL) and ChIP assays were performed to detect changes in expression of epigenetic markers within chromatin in both sorted GFP - and GFP + cell populations, harboring latent or reactivated proviruses, respectively. Ordinary two-way ANOVA analysis used to identify interactions between shRNAs and chromatin marks associated with repressive or active chromatin in the integrated provirus revealed significant changes in the levels of H3K27me3, AGO1 and HDAC1 in the LTR, which correlated with the extent of reduced proviral reactivation. The cell line co-expressing shPromA and sh143 consistently showed the least reactivation and greatest enrichment of chromatin compaction indicators., Conclusion: The active maintenance of epigenetic silencing by shRNAs acting on the HIV-1 LTR impedes HIV-1 reactivation from latency. Our "block and lock" approach constitutes a novel way of enforcing HIV-1 "super latency" through a closed chromatin architecture that renders the virus resistant to a range of latency reversing agents.

Background: The gene therapeutic Cal-1 comprises the anti-HIV agents: (i) sh5, a short hairpin RNA to CCR5 that down-regulates CCR5 expression and (ii) maC46 (C46), a peptide that inhibits viral fusion with the cell membrane. These constructs were assessed for inhibition of viral replication and selective cell expansion in a number of settings., Methods: HIV replication, selective outgrowth and cell surface viral binding were analysed with a single cycle infection assay of six pseudotyped HIV strains and a static and longitudinal passaging of MOLT4/CCR5 cells with HIV. Pronase digestion of surface virus and fluorescence microscopy assessed interactions between HIV virions and transduced cells., Results: Cal-1 reduced CCR5 expression in peripheral blood mononuclear cells to CCR5Δ32 heterozygote levels. Even low level transduction resulted in significant preferential expansion in MOLT4/CCR5 gene-containing cells over a 3-week HIV challenge regardless of viral suppression [12.5% to 47.0% (C46), 46.7% (sh5), 62.2% (Dual), respectively]. The sh5 and Dual constructs at > 95% transduction also significantly suppressed virus to day 12 in the passage assay and all constructs, at varying percentage transduction inhibited virus in static culture. No escape mutations were present through 9 weeks of challenge. The Dual construct significantly suppressed infection by a panel of CCR5-using viruses, with its efficacy being independently determined from the single constructs. Dual and sh5 inhibited virion internalisation, as determined via pronase digestion of surface bound virus, by 70% compared to 13% for C46., Conclusions: The use of two anti-HIV genes allows optimal preferential survival and inhibition of HIV replication, with the impact on viral load being dependent on the percentage of gene marked cells., (Copyright © 2018 John Wiley & Sons, Ltd.)