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Owing to the effect of temperature on the maximum power point of photovoltaic modules, the anticipated benefits of maximum power point tracking (MPPT) charge controller are also greatly affected under high-temperature environments. This paper presents experimental study results based on the investigations of the effect of high cell temperatures on the performance of MPPT charge controllers. The study was carried out over three months at Kenyatta University in Nairobi, Kenya under moderate and high temperature conditions. The experiments are carried out using two market-ready MPPT charge controllers and a normal pulse-width modulation (PWM) charge controller as the reference. The study establishes that below 50 °C, the MPPT charge controller exhibits significant advantages over its counterpart with about 24% - 29%, additional energy gain. However, at high cell temperatures, the MPPT charge controller loses its added advantage over the PWM charge controller and its performance benefits drop from an average of 28% to 0.2%. This demonstrates that an ordinary PWM charge controller would achieve the same yields under high-temperature conditions and at an even lower premium.

Utilization of loose waste biomass such as charcoal dust remains popular in low-to mid-income countries due to increased energy demands, poverty and heighten efforts to mitigate climate change. This study sought to investigate the effects of starch, paper and algae binders on the physical, mechanical and combustion characteristics and emission levels of manually-made acacia charcoal dust briquettes. Acacia charcoal dust is collected and mixed with different binders at binder proportions of 10 %, 15 %, 20 %, 25 % and 30 % of the total weight for individual samples. Briquette samples are produced using a manually-operated briquetting machine. Briquettes are dried, characterized, and analyzed. Response Surface Methodology is used to investigate the effect of binder type and binder proportions on calorific value, ignition time and burning rate of the briquettes. Results show that the density of the produced briquettes from Charcoal-Starch (CS), Charcoal-Paper (CP) and Charcoal-Algae (CA) samples ranged between 571.5 kg/m3- 678.1 kg/m3, 495.8 kg/m3-534.4 kg/m3, 535.6 kg/m3-578.2 kg/m3, respectively. A mean shatter resistance of 91.3 %, 97.9 % and 95.2 % is obtained for CS, CP and CA briquettes, respectively. Moisture content, volatile matter, ash content and fixed carbon for all the samples ranged between 4.7 % and 7.1 %, 30 % and 46 %, 14 % and 32 %, 23 % and 56 %, respectively. Moreover, optimization response results show that a 30 % starch binder proportion gives the best calorific value, ignition time and burning rate. The highest emissions for CO, NOx and SO2 are, respectively, 389 ppm, 2.86 ppm and 2.26 ppm for CS samples, 345.3 ppm, 5.46 ppm, and 2.66 ppm for CP, and 422.1 ppm, 5.46 ppm 3.06 ppm for CA samples. Those results show that it is practically feasible to manually-make quality acacia charcoal dust briquettes with starch, paper and algae as binders. Such briquettes hence provide a quality source of fuel curbing deforestation and waste dumping, reducing fuel shortages in cities and forming a source of livelihood especially in developing countries.

Effective and sustainable strategies are needed to address the burden of preventable deaths among children under-five in resource-constrained settings. The Tools for Integrated Management of Childhood Illness (TIMCI) project aims to support healthcare providers to identify and manage severe illness, whilst promoting resource stewardship, by introducing pulse oximetry and clinical decision support algorithms (CDSAs) to primary care facilities in India, Kenya, Senegal and Tanzania. Health impact is assessed through: a pragmatic parallel group, superiority cluster randomised controlled trial (RCT), with primary care facilities randomly allocated (1:1) in India to pulse oximetry or control, and (1:1:1) in Tanzania to pulse oximetry plus CDSA, pulse oximetry, or control; and through a quasi-experimental pre-post study in Kenya and Senegal. Devices are implemented with guidance and training, mentorship, and community engagement. Sociodemographic and clinical data are collected from caregivers and records of enrolled sick children aged 0–59 months at study facilities, with phone follow-up on Day 7 (and Day 28 in the RCT). The primary outcomes assessed for the RCT are severe complications (mortality and secondary hospitalisations) by Day 7 and primary hospitalisations (within 24 hours and with referral); and, for the pre-post study, referrals and antibiotic. Secondary outcomes on other aspects of health status, hypoxaemia, referral, follow-up and antimicrobial prescription are also evaluated. In all countries, embedded mixed-method studies further evaluate the effects of the intervention on care and care processes, implementation, cost and cost-effectiveness. Pilot and baseline studies started mid-2021, RCT and post-intervention mid-2022, with anticipated completion mid-2023 and first results late-2023. Study approval has been granted by all relevant institutional review boards, national and WHO ethical review committees. Findings will be shared with communities, healthcare providers, Ministries of Health and other local, national and international stakeholders to facilitate evidence-based decision-making on scale-up. Study registration: NCT04910750 and NCT05065320

This study combines simulations and experiments to study the heat interactions between various types of roofs and the photovoltaic (PV) modules installed on them. Specifically, the performance of PV modules on a clay roof was compared with their performance on two types of metal roofs, a Box-profile metal roof and an Orientile metal roof, which differ in shape and geometry. Additionally, this study examined the cooling potential of three common metal roof pigments, iron (iii) oxide (Fe2O3), titanium dioxide (TiO2) and basalt, on roof-installed PV modules. An unpigmented roof was also studied for comparison purposes. Model development and simulation were implemented in COMSOL Multiphysics, and the simulation results were validated and compared with field experiments. The maximum open-circuit voltages of the PV installations were found to be 21.096 V for the clay roof, 20.945 V for the Box-profile metal roof and 20.718 V for the Orientile metal roof. This study revealed that the unpigmented roof had higher solar cell temperatures compared to the pigmented models, with temperature gains ranging from 2.2 °C to 2.71 °C. Moreover, the unpigmented model displayed significantly higher surface radiosity than the pigmented models. The performance output of the modules also varied depending on the metal roof sheet shape and geometry, with the Box-profile metal roof yielding better results than the Orientile metal roof sheet. These results indicate that a specific roof pigmentation may have a small impact on a single PV module, but it can become significant in a large array of modules, especially if cooling through natural convection is hindered.

Biodiesel is considered a suitable substitute for petroleum diesel because it is renewable, environment-friendly, and has a low carbon footprint. However, its high density, high viscosity and low heating value prevents it from replacing petroleum diesel completely. This study investigates the performance and emission characteristics of a compression ignition engine operating on oleander and croton biodiesel doped with graphene nanoparticles. Five fuel samples are used, including diesel (D100), diesel - 80% blended with oleander and croton biodiesel - 20% (OCB20) and OCB20 dosed with graphene nanoparticles at mass fractions of 50 ppm (mg/L), 75 ppm (mg/L) and 100 ppm (mg/L), respectively. The chemical composition of biodiesel and graphene nanoparticles is analyzed using Fourier Transform Infrared (FTIR) spectroscopy while the morphology of the nanoparticles is analyzed using Scanning Electron Microscope (SEM). Engine tests reveal a significant improvement in brake thermal efficiency, especially at 75 ppm concentration which is 2.76% and 18.93% higher than diesel and OCB20, respectively, and a reduction in brake specific fuel consumption by 2.44% and 16.67% compared to diesel and OCB20, respectively. Carbon monoxide (CO) and unburnt hydrocarbon emissions (UHC) decreases for the 50 ppm sample, recording 8.58% and 21.65% reduction in CO and 52.2% and 50% in UHC compared to the diesel and OCB20, respectively. However, Oxides of Nitrogen (NOx) emissions increase. The results indicate that graphene nanoparticle-enhanced biodiesel can adequately substitute petroleum diesel, albeit with NOx reduction techniques.

Manufacturing is an end-use sector that uses the most delivered energy, accounting for around 50% of all transported fuel globally and 40% of carbon dioxide emissions worldwide. Solar photovoltaic-thermal (PVT) energy can substitute the transported energy to meet thermal and electrical energy requirements, mitigating high energy costs and climatic problems. This research aimed to develop, simulate, and evaluate the capabilities of a solar photovoltaic-thermal system for potential use in Kenya's manufacturing sector. A multistage cluster sampling technique was used in the study to characterize the manufacturing industry. Additionally, a PVT system was simulated using MATLAB Simulink to ascertain the relationship of temperature and the PV electrical efficiency. The impact of incorporating a thermal collector into the PV system on electrical, thermal, and overall system efficiency, and also the system's potential for use in thermal processes in manufacturing, were assessed. From the characterization results, the agro-processing sector dominates with 35% representation, and the small-scale thermal energy category dominates at 80%. The simulation findings show that a small temperature increase leads to a small increment in short circuit current but a significant decline in open circuit voltage. As a consequence, the maximum power (Pmax) of the PV decreases, lowering its electrical efficiency. However, the integration of PV with thermal collector improved the electrical, thermal, and the entire system efficiencies by, 16.01%, 20%, and 36.13%, respectively. More than 75% of the electrical and thermal energy processes fall in the small energy category. Hence, the PVT system is suitable for small-scale low-to-medium heat thermal energy categories or as a substitute system for higher temperature processes to raise feed water temperatures and reduction of thermal energy cost. This study gives a new approach of the application of PVT system for thermal industrial applications.

Introduction Individuals with mental disorders are more susceptible to initiating and sustaining tobacco use; unfortunately, most do not get support to quit. Group behavioral counselling, an effective low-cost strategy for cessation has been shown to be effective, yet has not been studied among this population in Kenya. Methods Mentally ill tobacco users at Mathari Referral and Teaching Hospital’s Clinic for Substance Abuse Treatment in Nairobi, were recruited from September 2017 to March 2019. Participants were allocated into intervention and control groups (1:1). Intervention group participants met in groups of 10 over a 24-week period to participate in group behavioral counselling sessions using a structured curriculum to promote cessation. Control group participants received usual care. The primary outcome was tobacco cessation at 24 weeks measured through salivary cotinine strips. Secondary outcomes included self-reported number of cigarettes/sticks used daily and health-related quality of life (HRQOL), using the WHOQOL Brief Questionnaire at 24 weeks. Between-group event rates were compared using Cox proportional hazards models, while differences in HRQOL scores were analyzed using paired t-tests. Results Participants’ mean age was 35 (SD=9) years, 87% were male, and 42% had completed secondary education. Over half (65%) had substance use disorders (diagnosed) and 15% had major depressive disorders. Most participants (94%) used cigarettes at baseline and participants smoked for a mean of 13 (SD=11) years with an average of 14 (SD=7) sticks daily. Intervention group participants reported a higher cessation rate (15.2% vs 0%, p=0.02 at week 12, and 9.1% vs 0%, p=0.10 at week 24), with a lower number of sticks smoked (97% vs 58.6%, p

Among the emerging renewable energy technologies, solar photovoltaic (PV) power generation is growing steadily in the mainstream energy supply mix contributing about 2.58% of the global total power generation by 2018 from 2.1% in 2017. The negative high PV module temperature effects continue to pose significant hurdles though being addressed through active and passive cooling methods. Thermoelectric generator (TEG) technology, given its modularity, augments well in cooling PV modules’ and generating additional electricity. However, thermal coupling of the two technologies has remained an impediment to their good performance due to the microscopic roughness of the PV and TEG surfaces. Non-uniform temperature distribution from the PV cells hinders efficient heat transfer thus affecting the performance the two technologies. In this study, PV cell temperature distribution have been evaluated analytically and experimentally under outdoor setup environments. Further, cell temperatures distribution is investigated using three thermal interface materials (TIM) under air- and water-cooled environments with aluminium honeycomb cooling panels as the cooling contact medium. Results show that the three TIMs substantially reduced the temperature mismatch effects with the heat spreader (HS) presenting lower temperature and voltage mismatch compared with the other two TIMs under both air- and water-cooled test conditions exhibiting preference. Based on the best observed conditions, PV module power output increased by 1.8% and 2.5% under the two test conditions while the TEG generated an additional 19.7% and 24.85% of power, respectively. This translated to an improvement of 11.3% and 50.6%, respectively, compared to the bare cell TEG power generation. The use of TIMs hence has the potential to mitigate thermal coupling challenges associated with PV–TEG systems improving their overall power output.

Introduction Medical tourism is characterized by people seeking treatment abroad for various medical conditions due to varied reasons, many of whom benefit from specialized care for non-communicable diseases. Conversely, there are associated negative effects such as medical complications and weakened health systems. Currently, there is paucity of scientific evidence on patient-related factors influencing seeking treatment benefits abroad. This study sought to compare patient-related factors associated with choice of cancer treatment center locally or abroad, to understand reasons for seeking treatment outside Kenya. Materials and methods As a case-control study, 254 cancer patients were randomly sampled to compare responses from those who chose to receive initial treatment abroad or in Kenya. The cases were recruited from Ministry of Health while the controls from Kenyatta National Hospital and Texas Cancer Center. Data was analyzed using SPSS Software Version 21. Descriptive statistics, bivariate and multiple logistic regression analysis was carried out. Level of significance was set at 5%. Results Out of 254 respondents, 174 (69.5%) were treated for cancer in Kenya and 80 (31.5%) in India. We found that cost effectiveness was a significant factor for over 73% of all respondents. The study revealed independent predictors for seeking treatment in India were: monthly income higher than US$ 250; every additional month from when disclosure to patients was done increased likelihood by 1.16 times; physician advice (Odds Ratio(OR) 66; 95% Confidence Interval(CI) 7.9–552.9); friends and family (OR 42; 95% CI 7.07–248.6); and perception of better quality of care (OR 22.5; 95% CI 2.2–230.6). Conclusion Reasons patients with cancer sought treatment in India are multifactorial. Several of these can be addressed to reverse out-ward bound medical tourism and contribute to improving the in-country cancer healthcare. It will require strengthening the health system accordingly and sensitizing the medical fraternity and general public on the same.

Most of the tropical rainforests are subject to both anthropogenic and natural disturbances. Forest elephants (Loxodonta cyclotis) cause forest clearings within the tropics. This study was conducted at mid-elevations (1100–1700 m a.s.l.) in Mount Cameroon National Park. We assessed the difference in the structure of bird communities in the forest and areas located nearby affected by elephant activities. We used the point-count method; 22 points were established within each habitat. The vegetation was visually estimated within a 50 m radius surrounding each counting point. In total, 1603 birds from 85 species were recorded. The vegetation cover from 3 to 5 m at points with elephant activity was significantly lower compared to points without elephant activity. Bird species richness was significantly higher around points in pasture compared to points in intact forest. Habitat type and the percentage of vegetation layer from 3 to 5 m significantly impacted the bird community structure. The points in the pasture were especially characterized by the increased abundance of some open habitat species (e.g., Chubb’s Cisticola chubbi). Few studies have documented the effects of elephant activity on other species, although the effects are widely stated as important drivers of habitat diversity in tropical forests. In conclusion, disturbance caused by elephants leads to increased bird community diversity due to the increased heterogeneity of the environment, which documents the high importance of elephants as ecosystem engineers.

Abstract Background Children who experience sexual abuse undergo various negative psychosocial outcomes such as depressive symptoms. Unfortunately, not many studies have been conducted on the incidence of depressive symptoms among sexually abused children in Kenya. This study sought to ascertain the incidence of depressive symptoms among children who have experienced sexual abuse in Kenya. Methods This was a longitudinal study design. It was conducted at Kenyatta National Teaching and Referral Hospital and Nairobi Women’s Hospitals in Kenya. One hundred and ninety-one children who had experienced sexual abuse and their parents/legal guardians were invited to participate in the study. The study administered the Becks Depression Inventory and the Child Depression Inventory to the children. Results The incidence of depressive symptoms after 1 month of sexual abuse revealed that amongst children who were below 16 years old, 14.6% had minimal-mild depressive symptoms while 85.4% had moderate-severe depressive symptoms. In comparison, children who were 16 years or older, 6.4% had minimal-mild depressive symptoms while 93.6% had moderate-severe depressive symptoms. Children below 16 years old whose parents were separated were found to have depressive symptoms (p

The incidence and correlates of breast milk HIV-1 RNA detection were determined in intensively sampled women receiving highly active antiretroviral therapy (HAART) for the prevention of mother-to-child HIV-1 transmission.Women initiated HAART at 34 weeks of pregnancy. Breast milk was collected every 2-5 days during 1 month postpartum for measurements of cell-associated HIV DNA and cell-free HIV RNA. Plasma and breast milk were also collected at 2 weeks, 1, 3 and 6 months for concurrent HIV-1 RNA and DNA measurements. Regression was used to identify cofactors for breast milk HIV-1 RNA detection.Of 259 breast milk specimens from 25 women receiving HAART, 34 had detectable HIV-1 RNA (13%, incidence 1.4 episodes/100 person-days 95% CI = 0.97-1.9). Fourteen of 25 (56%) women had detectable breast milk HIV-1 RNA [mean 2.5 log(10) copies/ml (range 2.0-3.9)] at least once. HIV-1 DNA was consistently detected in breast milk cells despite HAART, and increased slowly over time, at a rate of approximately 1 copy/10(6) cells per day (p = 0.02). Baseline CD4, plasma viral load, HAART duration, and frequency of breast problems were similar in women with and without detectable breast milk HIV-1 RNA. Women with detectable breast milk HIV-1 RNA were more likely to be primiparous than women without (36% vs 0%, p = 0.05). Plasma HIV-1 RNA detection (OR = 9.0, 95%CI = 1.8-44) and plasma HIV-1 RNA levels (OR = 12, 95% CI = 2.5-56) were strongly associated with concurrent detection of breast milk HIV-1 RNA. However, no association was found between breast milk HIV-1 DNA level and concurrent breast milk HIV-1 RNA detection (OR = 0.96, 95%CI = 0.54-1.7).The majority of women on HAART had episodic detection of breast milk HIV-1 RNA. Breast milk HIV-1 RNA detection was associated with systemic viral burden rather than breast milk HIV-1 DNA.

In in vitro model of short-term therapeutic inhalation of Xe/O 2 mixture, xenon in millimolar concentrations led to a pronounced decrease in induced platelet aggregation in the platelet-enriched blood plasma. The maximum and statistically significant decrease occurred in response to induction by collagen (by ≈30%, p≤0.01) and ADP (by ≈25%, p≤0.01). A slightly weaker but statistically significant reduction in aggregation appeared in response to ristocetin (by ≈12%, p≤0.01) and epinephrine (by ≈9%, p≤0.01). It should be noted that the spontaneous aggregation exceeded the reference values in the control group. Nevertheless, even at minimal absolute values, spontaneous platelet aggregation decreased by 2 times in response to xenon (p≤0.01). The reasons for the decrease of spontaneous and induced aggregation are xenon accumulation in the lipid bilayer of the membrane with subsequent nonspecific (mechanical) disassociation of membrane platelet structures and specific block of its distinct from neuronal NMDA receptor., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

This study evaluated the use of interactive, branched videos compared with traditional passive linear delivery for enhancing student engagement and learning in online courses. Undergraduate biology students were provided with either branched decision-based or linear videos on cell biology and protein purification as self-guided or consolidation activities. While the interactive branched videos did not improve learning gains, thematic analysis revealed that students found them more enjoyable and preferable for revision. However, most students felt linear passive videos were more logically structured for core content delivery. In a revised format, with clearer scaffolding, the interactive branched videos were perceived as significantly more engaging and useful when utilised for a problem-solving activity. Students welcomed the autonomy of directing their learning path but desired support to avoid missing critical information. Overall, thoughtfully designed branched videos can increase student motivation, but their utility depends on context. Our findings indicate the importance of balancing interactivity, clear organisation and purpose when incorporating these innovative formats into online learning. Branched videos show promise for increasing engagement but require intentional instructional design tailored to learning objectives., (© 2024 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The Drosophila PRC1 complex regulates gene expression by modifying histone proteins and chromatin architecture. Two PRC1 subunits, PSC and Ph, are most implicated in chromatin architecture. In vitro, PRC1 compacts chromatin and inhibits transcription and nucleosome remodeling. The long disordered C-terminal region of PSC (PSC-CTR) is important for these activities, while Ph has little effect. In cells, Ph is important for condensate formation, long-range chromatin interactions, and gene regulation, and its polymerizing sterile alpha motif (SAM) is implicated in these activities. In vitro, truncated Ph containing the SAM and two other conserved domains (mini-Ph) undergoes phase separation with chromatin, suggesting a mechanism for SAM-dependent condensate formation in vivo. How the distinct activities of PSC and Ph on chromatin function together in PRC1 is not known. To address this question, we analyzed structures formed with large chromatin templates and PRC1 in vitro. PRC1 bridges chromatin into extensive fibrillar networks. Ph, its SAM, and SAM polymerization activity have little effect on these structures. Instead, the PSC-CTR controls their growth, and is sufficient for their formation. To understand how phase separation driven by Ph SAM intersects with the chromatin bridging activity of the PSC-CTR, we used mini-Ph to form condensates with chromatin and then challenged them with PRC1 lacking Ph (PRC1ΔPh). PRC1ΔPh converts mini-Ph chromatin condensates into clusters of small non-fusing condensates and bridged fibers. These condensates retain a high level of chromatin compaction and do not intermix. Thus, phase separation of chromatin by mini-Ph, followed by the action of the PSC-CTR, creates a unique chromatin organization with regions of high nucleosome density and extraordinary stability. We discuss how this coordinated sequential activity of two proteins found in the same complex may occur and the possible implications of stable chromatin architectures in maintaining transcription states.

The Polycomb group (PcG) complex PRC1 localizes in the nucleus in condensed structures called Polycomb bodies. The PRC1 subunit Polyhomeotic (Ph) contains an oligomerizing sterile alpha motif (SAM) that is implicated in both PcG body formation and chromatin organization in Drosophila and mammalian cells. A truncated version of Ph containing the SAM (mini-Ph) forms phase-separated condensates with DNA or chromatin in vitro, suggesting that PcG bodies may form through SAM-driven phase separation. In cells, Ph forms multiple small condensates, while mini-Ph typically forms a single large nuclear condensate. We therefore hypothesized that sequences outside of mini-Ph, which are predicted to be intrinsically disordered, are required for proper condensate formation. We identified three distinct low-complexity regions in Ph based on sequence composition. We systematically tested the role of each of these sequences in Ph condensates using live imaging of transfected Drosophila S2 cells. Each sequence uniquely affected Ph SAM-dependent condensate size, number, and morphology, but the most dramatic effects occurred when the central, glutamine-rich intrinsically disordered region (IDR) was removed, which resulted in large Ph condensates. Like mini-Ph condensates, condensates lacking the glutamine-rich IDR excluded chromatin. Chromatin fractionation experiments indicated that the removal of the glutamine-rich IDR reduced chromatin binding and that the removal of either of the other IDRs increased chromatin binding. Our data suggest that all three IDRs, and functional interactions among them, regulate Ph condensate size and number. Our results can be explained by a model in which tight chromatin binding by Ph IDRs antagonizes Ph SAM-driven phase separation. Our observations highlight the complexity of regulation of biological condensates housed in single proteins.

Blended learning is becoming the expected norm for core content delivery in many institutions. Pre-recorded videos in the form of screencasts are the primary delivery method, with students being asked to engage with the content in this medium. Usage is only likely to increase into the future as delivery moves away from traditional lectures and seminars. In this perspective, we look at the use of video material as a means of content delivery and how to help students engage with it. Theoretical literature around cognitive loading and active learning, alongside personal experience of delivery, is drawn on to give a framework for creating engaging recordings and learning activities., (© 2022 The Author(s).)

Surgical practice during the coronavirus disease 2019 (COVID-19) pandemic has changed significantly, without supporting data. With increasing experience, a dichotomy of practice is emerging, challenging existing consensus guidelines. One such practice is elective tracheostomy. Here, we share our initial experience of head and neck cancer surgery in a COVID-19 tertiary care centre, emphasizing the evolved protocol of perioperative care when compared to pre-COVID-19 times. This was a prospective study of 21 patients with head and neck cancers undergoing surgery during the COVID-19 pandemic, compared to 193 historical controls. Changes in anaesthesia, surgery, and operating room practices were evaluated. A strict protocol was followed. One patient tested positive for COVID-19 preoperatively. There was a significant increase in pre-induction tracheostomies (28.6% vs 6.7%, P=0.005), median hospital stay (10 vs 7 days, P=0.001), and postponements of surgery (57.1% vs 27.5%, P=0.01), along with a significant decrease in flap reconstructions (33.3% vs 59.6%, P=0.03). There was no mortality and no difference in postoperative morbidity. No healthcare personnel became symptomatic for COVID-19 during this period. Tracheostomy is safe during the COVID-19 pandemic and rates have increased. Despite increased rescheduling of surgeries and longer hospital stays, definitive cancer care surgery has not been deferred and maximum patient and healthcare worker safety has been ensured., (Copyright © 2020 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

The pandemic has brought challenges to teaching lab and research skills. Here Nigel Francis and colleagues explore the diverse approaches taken to replace lab-based immunology teaching, explain how networks of educators have driven this innovation and discuss the importance of retaining best practice into the future., (© 2021 The Authors. Immunology published by John Wiley & Sons Ltd.)

R-loops are non-canonical nucleic structures composed of an RNA-DNA hybrid and a displaced ssDNA. Originally identified as a source of genomic instability, R-loops have been shown over the last decade to be involved in the targeting of proteins and to be associated with different histone modifications, suggesting a regulatory function. In addition, R-loops have been demonstrated to form differentially during the development of different tissues in plants and to be associated with diseases in mammals. Here, we provide a single-strand DRIP-seq protocol to identify R-loop-forming sequences in Drosophila melanogaster embryos and tissue culture cells. This protocol differs from earlier DRIP protocols in the fragmentation step. Sonication, unlike restriction enzymes, generates a homogeneous and highly reproducible nucleic acid fragment pool. In addition, it allows the use of this protocol in any organism with minimal optimization. This protocol integrates several steps from published protocols to identify R-loop-forming sequences with high stringency, suitable for de novo characterization. Graphic abstract: Figure 1.Overview of the strand-specific DRIP-seq protocol., Competing Interests: Competing interestsThe authors declare no competing interests., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)

Histones carry information in the form of post-translational modifications (PTMs). For this information to be propagated through cell cycles, parental histones and their PTMs need to be maintained at the same genomic locations. Yet, during DNA replication, every nucleosome in the genome is disrupted to allow passage of the replisome. Recent data have identified histone chaperone activities that are intrinsic components of the replisome and implicate them in maintaining parental histones during DNA replication. We propose that structural and kinetic coordination between DNA replication and replisome-associated histone chaperone activities ensures positional inheritance of histones and their PTMs. When this coordination is perturbed, histones may instead be recycled to random genomic locations by alternative histone chaperones., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Polycomb Group (PcG) proteins organize chromatin at multiple scales to regulate gene expression. A conserved Sterile Alpha Motif (SAM) in the Polycomb Repressive Complex 1 (PRC1) subunit Polyhomeotic (Ph) has been shown to play an important role in chromatin compaction and large-scale chromatin organization. Ph SAM forms helical head to tail polymers, and SAM-SAM interactions between chromatin-bound Ph/PRC1 are believed to compact chromatin and mediate long-range interactions. To understand the underlying mechanism, here we analyze the effects of Ph SAM on chromatin in vitro. We find that incubation of chromatin or DNA with a truncated Ph protein containing the SAM results in formation of concentrated, phase-separated condensates. Ph SAM-dependent condensates can recruit PRC1 from extracts and enhance PRC1 ubiquitin ligase activity towards histone H2A. We show that overexpression of Ph with an intact SAM increases ubiquitylated H2A in cells. Thus, SAM-induced phase separation, in the context of Ph, can mediate large-scale compaction of chromatin into biochemical compartments that facilitate histone modification.

Polycomb Group proteins regulate gene expression by modifying chromatin. Polycomb Repressive Complex 1 (PRC1) has two activities: a ubiquitin ligase activity for histone H2A and a chromatin compacting activity. In Drosophila, the Posterior Sex Combs (PSC) subunit of PRC1 is central to both activities. The N-terminal of PSC assembles into PRC1, including partnering with dRING to form the ubiquitin ligase. The intrinsically disordered C-terminal region of PSC compacts chromatin and inhibits chromatin remodeling and transcription in vitro. Both regions of PSC are essential in vivo. To understand how these two activities may be coordinated in PRC1, we used crosslinking mass spectrometry to analyze the conformations of the C-terminal region of PSC in PRC1 and how they change on binding DNA. Crosslinking identifies interactions between the C-terminal region of PSC and the core of PRC1, including between N and C-terminal regions of PSC. New contacts and overall more compacted PSC C-terminal region conformations are induced by DNA binding. Protein footprinting of accessible lysine residues reveals an extended, bipartite candidate DNA/chromatin binding surface in the C-terminal region of PSC. Our data suggest a model in which DNA (or chromatin) follows a long path on the flexible disordered region of PSC. Intramolecular interactions of PSC detected by crosslinking can bring the high-affinity DNA/chromatin binding region close to the core of PRC1 without disrupting the interface between the ubiquitin ligase and the nucleosome. Our approach may be applicable to understanding the global organization of other large intrinsically disordered regions that bind nucleic acids., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Polycomb Group (PcG) proteins form memory of transient transcriptional repression that is necessary for development. In Drosophila, DNA elements termed Polycomb Response Elements (PREs) recruit PcG proteins. How PcG activities are targeted to PREs to maintain repressed states only in appropriate developmental contexts has been difficult to elucidate. PcG complexes modify chromatin, but also interact with both RNA and DNA, and RNA is implicated in PcG targeting and function. Here we show that R-loops form at many PREs in Drosophila embryos, and correlate with repressive states. In vitro, both PRC1 and PRC2 can recognize R-loops and open DNA bubbles. Unexpectedly, we find that PRC2 drives formation of RNA-DNA hybrids, the key component of R-loops, from RNA and dsDNA. Our results identify R-loop formation as a feature of Drosophila PREs that can be recognized by PcG complexes, and RNA-DNA strand exchange as a PRC2 activity that could contribute to R-loop formation.

Background: The survival benefit of neoadjuvant therapy in resectable carcinoma esophagus has been elucidated. We performed a meta-analysis in light of new studies and long-term results of past trials. The search strategy was refined to include only "neoadjuvant" so that any bias by adjuvant treatment is eliminated., Methods: A detailed search of MEDLINE, Embase, and Cochrane Library was done. Only published randomized English language trials were included. Data were categorized as neoadjuvant concurrent chemoradiation (NACRT), neoadjuvant chemotherapy (NACT), neoadjuvant radiotherapy (NART), and neoadjuvant sequential chemoradiotherapy (SCRT). Meta-analysis was done using odds ratio (OR) and 95% CI using fixed/random effects model. Heterogeneity was tested by chi-square and I 2 test. Z probability calculated significant difference across subgroups. Outcomes assessed were overall survival (OS) and disease-free survival (DFS) at 3 and 5 years, respectively, mortality (30/90 day) and failures (local/systemic)., Results: Twenty-five randomized trials involving 5272 patients were included for quantitative analysis. NACRT was evaluated in 12 studies (2676 patients). Superior 3-year OS (OR = 0.68 CI 0.52-0.90, p = 0.007), 3-year DFS (OR = 0.55 CI 0.45-0.68, p = 0.00001), and 5-year DFS (OR = 0.59 CI 0.47-0.74, p = 0.00001), with lower failures (OR = 0.52 CI 0.37-0.73, p = 0.0001), were seen in favor of NACRT at the cost of increased perioperative mortality (OR = 1.79 CI 1.15-2.80, p = .01). However, 5-year OS (OR = 0.78 CI 0.60-0.1.01, p = 0.06) was not found to be significantly superior. NACT, NART, and SCRT were not found to have any benefit over surgery alone., Conclusion: This meta-analysis presents strong evidence favoring NACRT over upfront surgery. It also shows no survival advantage of neoadjuvant chemotherapy.

Metabolic pathways that regulate T-cell function show promise as therapeutic targets in diverse diseases. Here, we show that at rest cultured human effector memory and central memory CD4+ T-cells have elevated levels of glycolysis and oxidative phosphorylation (OXPHOS), in comparison to naïve T-cells. Despite having low resting metabolic rates, naive T-cells respond to TCR stimulation with robust and rapid increases in glycolysis and OXPHOS. This early metabolic switch requires Akt activity to support increased rates of glycolysis and STAT5 activity for amino acid biosynthesis and TCA cycle anaplerosis. Importantly, both STAT5 inhibition and disruption of TCA cycle anaplerosis are associated with reduced IL-2 production, demonstrating the functional importance of this early metabolic program. Our results define STAT5 as a key node in modulating the early metabolic program following activation in naive CD4+ T-cells and in turn provide greater understanding of how cellular metabolism shapes T-cell responses.

This study aims to evaluate the antibiotic prescribing rate for acute bacterial rhinosinusitis in community pharmacies and to study the corresponding attitude and behavior of participants. A cross-sectional, nationwide study was conducted using a patient-simulated case of bacterial rhinosinusitis. Descriptive data were reported for the medications prescribed, questions asked, and recommendations made. Bivariate analysis was conducted to identify factors affecting the aforementioned. Out of the 250 community pharmacies visited, 77 (30.8%) prescribed antibiotics, 15 (6%) referred the patient to a physician, and 79 (32%) made the right diagnosis. Amoxicillin/clavulanic acid (69.7%) was the most prescribed antibiotic. The majority of the participants prescribed antibiotics according to guidelines. Overall, 108 (43.2%) participants questioned about symptoms and few questioned about patient age, pregnancy, and history of rhinosinusitis. None counseled about interactions or in case a dose is missed. We concluded that antibiotics are easily prescribed in Lebanese community pharmacies. This misuse should be tackled by legislative authorities to restrict such practices., Competing Interests: The authors declare that they have no conflicts of interest., (© Atlantis Press International B.V.)

Linking immunometabolic adaptation to T-cell function provides insight for the development of new therapeutic approaches in multiple disease settings. T-cell activation and downstream effector functions of CD4 + and CD8 + T-cells are controlled by the strength of interaction between the T-cell receptor (TCR) and peptides presented by human leukocyte antigens (pHLA). The role of TCR-pHLA interactions in modulating T-cell metabolism is unknown. Here, for the first time, we explore the relative contributions of the main metabolic pathways to functional responses in human CD4 + and CD8 + T-cells. Increased expression of hexokinase II accompanied by higher basal glycolysis is demonstrated in CD4 + T-cells; cytokine production in CD8 + T-cells is more reliant on oxidative phosphorylation. Using antigen-specific CD4 + and CD8 + T-cell clones and altered peptide ligands, we demonstrate that binding affinity tunes the underlying metabolic shift. Overall, this study provides important new insight into how metabolic pathways are controlled during antigen-specific activation of human T-cells.

Nucleosomes are believed to carry epigenetic information through the cell cycle, including through DNA replication. It has been known for decades that parental histones are reassembled on newly replicated chromatin, but the mechanisms underlying histone inheritance and dispersal during DNA replication are not fully understood. We monitored the fate of histones H3 or H4 from a single nucleosome through DNA replication in two in vitro systems. In the SV40 system, histones assembled on a single nucleosome positioning sequence can be inherited by their own daughter DNA but are dispersed from their original location. In Xenopus laevis extracts, histones are dynamic, and nucleosomes are repositioned independent of and prior to DNA replication. Nevertheless, a high fraction of histones H3 and H4 that are inherited through DNA replication remains near its starting location. Thus, inheritance of histone proteins and their dispersal can be mechanistically uncoupled., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Polycomb Group (PcG) proteins assemble a chromatin state that maintains developmental gene repression. A new study combining structure and in vivo analysis details a molecular network from DNA recognition to PcG recruitment, highlighting the essential role of Sterile Alpha Motifs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The genomes of metazoa are organized at multiple scales. Many proteins that regulate genome architecture, including Polycomb group (PcG) proteins, form subnuclear structures. Deciphering mechanistic links between protein organization and chromatin architecture requires precise description and mechanistic perturbations of both. Using super-resolution microscopy, here we show that PcG proteins are organized into hundreds of nanoscale protein clusters. We manipulated PcG clusters by disrupting the polymerization activity of the sterile alpha motif (SAM) of the PcG protein Polyhomeotic (Ph) or by increasing Ph levels. Ph with mutant SAM disrupts clustering of endogenous PcG complexes and chromatin interactions while elevating Ph level increases cluster number and chromatin interactions. These effects can be captured by molecular simulations based on a previously described chromatin polymer model. Both perturbations also alter gene expression. Organization of PcG proteins into small, abundant clusters on chromatin through Ph SAM polymerization activity may shape genome architecture through chromatin interactions.

Leucocytes respond rapidly to pathogenic and other insults, with responses ranging from cytokine production to migration and phagocytosis. These are bioenergetically expensive, and increased glycolytic flux provides adenosine triphosphate (ATP) rapidly to support these essential functions. However, much of this work is from animal studies. To understand more clearly the relative role of glycolysis and oxidative phosphorylation in human leucocytes, especially their utility in a translational research setting, we undertook a study of human peripheral blood mononuclear cells (MNCs) bioenergetics. Glycolysis was essential during lipopolysaccharide (LPS)-mediated interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α production, as 2-deoxy-D-glucose decreased significantly the output of all three cytokines. After optimizing cell numbers and the concentrations of all activators and inhibitors, oxidative phosphorylation and glycolysis profiles of fresh and cryopreserved/resuscitated MNCs were determined to explore the utility of MNCs for determining the bioenergetics health profile in multiple clinical settings. While the LPS-induced cytokine response did not differ significantly between fresh and resuscitated cells from the same donors, cryopreservation/resuscitation significantly affected mainly some measures of oxidative phosphorylation, but also glycolysis. Bioenergetics analysis of human MNCs provides a quick, effective means to measure the bioenergetics health index of many individuals, but cryopreserved cells are not suitable for such an analysis. The translational utility of this approach was tested by comparing MNCs of pregnant and non-pregnant women to reveal increased bioenergetics health index with pregnancy but significantly reduced basal glycolysis and glycolytic capacity. More detailed analysis of discrete leucocyte populations would be required to understand the relative roles of glycolysis and oxidative phosphorylation during inflammation and other immune responses., (© 2015 British Society for Immunology.)

Glioblastoma, the most common and aggressive adult brain tumor, is characterized by extreme phenotypic diversity and treatment failure. Through fluorescence-guided resection, we identified fluorescent tissue in the sub-ependymal zone (SEZ) of patients with glioblastoma. Histologic analysis and genomic characterization revealed that the SEZ harbors malignant cells with tumor-initiating capacity, analogous to cells isolated from the fluorescent tumor mass (T). We observed resistance to supramaximal chemotherapy doses along with differential patterns of drug response between T and SEZ in the same tumor. Our results reveal novel insights into glioblastoma growth dynamics, with implications for understanding and limiting treatment resistance., (©2014 American Association for Cancer Research.)

Aberrant expression of chromatin-modifying enzymes (CMEs) is associated with a range of human diseases, including cancer. CMEs are now an important target area in drug discovery. Although the role that histone and protein (lysine) methyltransferases (PMTs) play in the regulation of transcription and cell growth is increasingly recognized, few small-molecule inhibitors of this class of enzyme have been reported. Here we describe an assay suitable for primary compound screening for the identification of PMT inhibitors. The assay followed the methylation of histones in the presence of the PMT SET7/9 and the radioactive cofactor S-adenosyl-methionine using scintillating microplates (FlashPlate) and was used to screen approximately 65 000 compounds (% coefficient of variation = 10%; Z' = 0.6). The hits identified from a library of more than 63 000 diverse small molecules included a series of rhodanine compounds with micromolar activity. A screen of the National Cancer Institute Diversity Set (2000 compounds) identified an orsein derivative that inhibited SET7/9 (~20 µM) and showed modest growth inhibition associated with the expected cellular phenotype of reduced histone methylation in a human tumor cell line. The assay represents a useful tool for the identification of inhibitors of PMT activity.