The intriguing multi-scale fractal patterns ubiquitously observed in nature similarly emerge as fascinating structures in two-phase fluid flows of bio-oil breakup and atomization processes. High-resolution microscopy of the two-phase flows under 15 flow conditions (cases of different flow rates of the liquid and co-flowing air streams as well as different degrees of liquid preheating) reveal that the geometrical complexities evolve under the competing/combined action of the instability mechanisms such as Kelvin–Helmholtz, Rayleigh–Taylor and Rayleigh–Plateau leading into the transition from break-up to atomization. A thorough analysis of the higher order moments of statistics evaluated based on the probability density functions from 15,000 fractal dimension samples suggest that a single-value analysis is not sufficient to describe the complex reshaping mechanisms in two-phase flows. Consistently positive skewness of the statistics reveal the role of abrupt two-phase mechanisms such as liquid column rupture, ligament disintegration, liquid sheet bursting and droplet distortions in a hierarchical geometrical entanglement. Further, large kurtosis values at increased flow inertia are found associated with turbulence-induced intermittent geometrical reshaping. Interestingly, the proposed power-law correlation reveals that the global droplet size obtained from laser-diffraction measurements declines as the two-phase geometrical complexity increases. Remarkably, fractal structures ubiquitously appear in nature and often characterize certain physical phenomena such as complex patterns in butterflies’ wings2, liquid phases undergoing diffusion in microgravity3, fractal universality of galaxies4, DNA-based spatio-temporal analysis of the species and their genetic similarity/diversity5, soil particle size distribution6, evolutionary sensitivity of the animals sensory systems towards geometrically complex environments7, molecular complexity8, structural complexity of the human brain9, organisation of landscapes and river networks10, generation of fractal laser beams11, chemotherapy effects on the micro-vascular fractal structures12, aerosol aggregate in Jupiter’s atmosphere13, thermodynamic phase transition of glass14, turbulence-induced fractal patches affecting the phytoplankton ecology15, Alzheimer’s disease assessment through the complexity analysis of human cerebral cortex16 and lung tumour spatial growth over time18. As a revolutionary modern art technique, Jackson Pollock used dripping paint to create geometrically complex patterns19. His work has been analysed in terms of fractal characteristics emerging due to the interaction of falling liquid paint on the canvas surface19. While fractals are commonly observed as regular or irregular shapes in natural, biological and physical structures, they are not always perfectly self-similar, but very few studies consider their statistical distribution20.In a pure mathematical point of view, fractals demonstrate aspatial self-similarity of geometrical patterns infinitely reproduced down to the smallest scales23, namely, Kelvin–Helmholtz (KH) instability in shear layers28 and viscous fingers30. Fractal complexities can be related to some important phenomena such as mixing and mass transport at the turbulent/non-turbulent interface of shear layers33 and boundary layers34 as well as turbulent particle agglomeration35. One of the most complex yet fascinating classes of fluid dynamics are the two-phase flows in which a competing effect from gravity36 and inertial forces against resistances arising from the differences in physical properties such as viscosity, surface tension and density produces a broad range of complex interfacial geometries. Such complexities arise from the onset of instability mechanisms such as Kelvin–Helmholtz (KH), Rayleigh–Taylor (RT) and Rayleigh–Plateau (RP) where initial linear growth of the perturbations transition into non-linear state and generate geometrically sophisticated structures in the form of liquid sheets, ligaments and distorted droplets of various length scales38. The KH instability sets in whenever a large velocity difference across an interface distorts the boundary between two layers of fluid and results in vortex roll-up25. As a result of the local acceleration at a section of a high-density fluid interface into the low-density medium, the RT instability is triggered39. The RP instability appears when nature tends to automatically minimize the interfacial energy through the act of surface tension39. In case of air-assisted atomization of liquid jets surrounded by high-momentum parallel air flow and subjected to shear layer instabilities40, atomization occurs in two successive steps. First, the gas/liquid interface experiences the KH type shear layer instability waves. In the second step, transient non-linear local accelerations on top of the primary KH waves trigger the RT instability. This results in the formation of ligaments that consequently break up into smaller droplets41. It should also be noted that the onset and growth of the instability mechanisms, which can be predicted by theory42, are highly affected by the mean velocity as well as turbulence levels introduced at the inlet. As a result of the combined effects from the mean flow as well as the turbulence intensity variations, determine the shape and length scale of the generated ligaments and droplets43. The morphology of primary and secondary gas/liquid interfacial waves, in terms of wavelength and amplitude, are the spatial manifestation of the instability mechanisms. Initiation and evolution of these surface instability waves are linked to the ligament and droplet formation45. In addition to experimental techniques, high-fidelity numerical methods such as direct numerical simulation (DNS) and large eddy simulation (LES) are able to resolve the smallest scales of turbulence affecting the atomization process. Detailed spatio-temporal analysis of the flow instabilities and their correlation with vortex dynamics reveal the complexities of break-up and atomization47.In the area of thermofluids, and possibly in general, fractals can be associated with some source of instability leading into organized/chaotic patterns50. In addition to fundamental significance, two-phase spray dynamics play important role in variety of applications. Particular attention is increasing towards the implementation of bio-oils as viable renewable alternative fuels to mitigate the existing global threats against the climate due to elevated greenhouse gas (GHG) emissions53. As conventional fossil fuels eventually run out, they can be replaced by the carbon neutral biofuels produced from the abundantly available biomaterials in some remote communities and contribute to reduced transportation cost and emissions56. Optimized performance of bio-oil sprays in novel power generation applications or upgraded existing systems such as alternative aviation fuels57, internal combustion engines58 and gas turbines61 are limited by our lack of understanding on their peculiar two-phase behaviour. Due to the complex physiochemical properties of pyrolysis oils, resulting in their resistance to break-up and atomization64, the current fundamental knowledge of their underlying two-phase physics need to be improved.Depending on the flow regime, ambient conditions and thermo-physical properties, the liquid break-up process may cascade down to the smallest possible length scales in fully atomized mode66, mainly due to the intensive computations involved in the analysis of high-resolution images. However, the remarkable random and irregular geometrical structures of the liquid break-up demand extensive fractal analysis. Therefore, the present study is motivated for a comprehensive statistical analysis of the fractal complexities for a broad range of bio-oil break-up atomization regimes. The rich and complex multi-scale two-phase dynamics are characterized in terms of the fractal dimensions evaluated for 15,000 high-resolution images obtained from long-distance microscopy. For each flow condition, the measured probability density functions (PDF) of the fractal dimensions are compared to the corresponding Gaussian normal distribution and interpreted based on the higher order moments of statistics (Skewness and Kurtosis). A power-law correlation is proposed between the image-based fractal dimensions and the droplet sizing obtained from laser-diffraction measurements . Total of 15 flow conditions are considered to generate a large database for the fractal analysis of the two-phase flow under a broad range of inertial (destabilizing) and viscous (stabilizing) effects.Very few studies have explored the fractal characteristics of the liquid break-up leading to the spray formationD) are evaluated to generate a comprehensive dataset from analysing 15,000 high-resolution images. This large dataset enables us for a detailed statistical analysis in terms of probability density functions (PDF), mean values (S) and kurtosis (K). The third category presents the results from laser diffraction droplet sizing for different flow conditions and a power-law correlation is developed between the fractal dimensions , liquid flow rate of L10 cases at 10 (lb/h)) and no air flow rate A00 case). The field of view (FOV) size of the snapshots is given in the figure caption based on the air discharge diameter A00L10 cases  instability69. The Young–Laplace relation suggests that the high-curvature contraction zones experience high pressures while the low-curvature expansion regions are associated with reduced local pressures70. This mechanism continuously induces internal liquid flows from the thinning high pressure zones to the thickening low pressure regions. Eventually, the thinnest liquid threads pinch-off leading into droplet formation . This mechanism is the nature’s ability to reduce the surface energy of a gas/liquid interface.The first row in Fig. mentclass2pt{minimA05L10 cases) results in the formation of non-symmetric deformations due to the sinusoidal mode of instability (downstream of arrow D) as demonstrated in the second row of Fig. A10L50 cases). In addition to the promoted short-wave (high frequency) sinusoidal jet meandering and the liquid column rupture, two new phenomena are observed. The first interesting phenomenon is the small local liquid ejections normal to the streamwise liquid column as pointed by arrow G. These local ejections are due to the Rayleigh–Taylor (RT) instability mechanism emerging as small patches of high-density liquid accelerating into the low-density gas phase. The local RT accelerations can also occur on top of the primary KH waves to form the ligaments further breaking up into smaller droplets41. With sufficient momentum, the ejecting patches of liquid detach from the main body of the liquid jet and reshape into droplets. Various levels of the gas/liquid inertia in Fig. 40. These stripped droplets could also be linked to the transition from initial linear KH instability to non-linear state and RT-type ligament formation on top of the primary KH vortex roll-ups at the gas/liquid interface41. As shown by arrow I, intense liquid column ruptures can also generate tiny ligaments and small droplets. Furthermore, droplet generation can also occur due to the leading tip vortex pinch-off (see arrow J) typically observed in transitioning/starting jets71.Introducing the atomizing air flow at mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimL04 cases at 4 (lb/h)) as shown in Fig. A05T24 cases). The thick falling liquid stripes  into a flow regime comprised of the spatio-temporal evolution of the wave groups exhibiting a broad spectrum of small-to-large wavelengths (frequencies). As visualized above, variable morphologies shaped under different flow conditions are basically the geometrical patterns arising from instability mechanisms as well as their corresponding vortex dynamics. Different observations of the local/global instabilities can be interpreted in terms of the wavelength distribution on the liquid surface, jet meandering, KH vortex roll-up or RT-shaped ligament distributions. In the following section, the dominant fractal character of the statistics can be correlated back to the above visualized shape of wavelength distribution on liquid jets, or the shape of ligament distributions.D) are evaluated using a box-counting73 method to create a comprehensive dataset from analysing 15,000 high-resolution images (1000 images per flow condition). The large dataset produced is then used for a thorough statistical analysis. As also reported in earlier studies74, some of the visualized liquid fragments are highly three-dimensional and from a single viewing angle, presumably information is lost. However, this is accounted for due to the large number of statistics produced in the present study. As a result, the discrete probability density functions (PDF) are obtained from 1000 samples per case and compared to the corresponding normal Gaussian distribution. The higher order moments of statistics are also evaluated based on the data skewness (S) and kurtosis (K). In the following, mean fractal dimension values are denoted with an over-line as 76 to higher values associated with turbulent gas jets, shear layers and mixing layers 75. The geometrical complexity also varies instantaneously due to the chaotic/intermittent nature of the gas/liquid interface reshaping.In order to quantify the geometrical complexities observed in the 15 different two-phase flow conditions visualized above, the corresponding fractal dimensions  while the columns demonstrate gas phase inertial effects  and the liquid flow rate is maintained at L04 cases at 4 (lb/h)). One interesting general observation is that the skewness values are all positive, suggesting that sudden occurrence of certain physical mechanisms always result in increased geometrical complexity while the opposite is not true. Some examples of such mechanism are when consistently meandering liquid columns, oscillating thin ligaments or stretching liquid sheets suddenly burst into small-scale structures with more complex geometries. The A05T24 case is the only distribution in Fig. A15 cases in which the atomizing air flow inertia is the highest. This suggests higher probability of finding fractal dimension values away from the mean . Accordingly, the large kurtosis values imply intermittent variation of the geometrical complexity rather than exhibiting orderly reshaping in time.In addition to the inertial destabilizations, the stabilizing viscous effects on the distribution of the fractal complexities are explored. The rows in Fig. A05L04 cases with increased temperature, while the A10L04 and A15L04 cases exhibit a similar trend. Such a phenomenon is often observed in laminar to turbulent transition where flow properties demonstrate similarity beyond certain Reynolds numbers. Here, the geometrical complexity of the two-phase flow demonstrates a similar behaviour with elevated inertial effects. When preheated to A10L04 and A15L04 cases attain larger values of the mean fractal dimension compared to the A05L04 cases as a measure of their more complex structure.Results presented in the previous section suggest that a proper analysis of the geometrical complexity in two-phase flows is best achieved by analysing the statistical distribution of the fractal dimensions. However, the averaged value mm downstream of the injection orifice are considered. With increased interest in image classification techniques, fractal dimensions can be used to correlate a certain instantaneous image with a representative droplet size such as SMD. Therefore, a power-law correlation is developed between the mean fractal dimensions and SMD values for different flow conditions. As shown in Fig. Except for very few applications, the ultimate goal of the liquid injection is to produce fully atomized sprays where small scale droplets can enhance heat/mass transport. The global fineness of the droplets in two-phase flow systems can be defined based on the overall liquid volume to surface ratio characterised by the Suater mean diameter (SMD). As a representative, the centreline SMD values 10 Through careful analysis of instantaneous high-resolution snapshots, it is found that the geometrical complexity of the two-phase flow structures evolve in accordance with the onset of linear instability mechanisms and their non-linear growth. The competing effect between the destabilizing inertial forces, surface tension and the stabilizing (damping) fluid viscous forces govern the underlying two-phase physics. The competing instability mechanisms such as Kelvin–Helmholtz (KH), Rayleigh–Taylor (RT) and Rayleigh–Plateau (RP), which govern the two-phase dynamics, lead into the formation of very diversified geometrical shapes at different irregularity levels and length scales. While the 1000 instantaneous liquid structure for each of the 15 flow cases are very unlikely to be identical, each case represents a class of geometrical pattern. Under variable levels of gas/liquid inertial forces acting against different viscous resistance scenarios, geometrical complexities of liquid break-up are altered in accordance with the formation of meandering liquid columns, thick falling liquid stripes, stretched/distorted narrow liquid threads, oscillating ligaments, liquid sheets and deformed droplets which all appear within a broad range of length scales and shapes often dictated by the local vortex dynamics.A15 cases), it is found that the kurtosis values become significantly larger. This remarkable finding can be attributed to the intermittent temporal fluctuations of the geometrical complexity. This means that there is a higher probability of finding fractal dimension values either larger or smaller than mean values. Therefore, orderly temporal reshaping is replaced by chaotic geometrical deformations.Fractal dimensions are evaluated for all the 15,000 snapshots to characterize the visualized geometrical complexity of the liquid break-up. Even though this is a cumbersome box-counting task to evaluate the fractal dimensions for large number of high-resolution images, the output is a comprehensive set of data to conduct a thorough statistical analysis. The discrete probability density function (PDF) associated with the temporal variation of fractal dimensions reveal the complexity of reshaping two-phase structures. Compared to their corresponding Gaussian normal PDFs, the discrete distributions are found to behave differently for most of the flow cases. This is verified by calculating the higher-order moments of statistics, skewness and kurtosis. The skewness values suggest that the discrete PDFs are mostly non-symmetric with respect to the mean fractal dimensions. Interestingly, all the discrete PDFs are positively skewed (longer right tail) suggesting that during the regular/organized geometrical reshaping there may be some physical mechanisms abruptly taking place. Examples of such sudden distortions are liquid column rupture, ligament and sheet bursting and local liquid accelerations all of which result in increased geometrical complexity, hence larger fractal dimensions. At increased preheating temperatures (reduced viscosity) and/or inertial forces, where perturbations grow into chaotic sudden distortions, fractal dimension statistics tend to become more skewed and complexity increases. At the highest air flow inertia . Within the range of the present measurements an interesting decoupling appears between the large and small droplets where the SMD values are classified based on mean fractal dimensions. The smaller droplets class nicely follows a power-law correlation of the form Similar to that of various phenomena ubiquitously taking place in nature, fluid flows exhibit fascinatingly complex geometrical reshaping. Such geometrical intricacies are particularly remarkable in two-phase interfacial flows. In the present study, fractal analysis is conducted to characterize the random and irregular geometrical structures formed during the bio-oil break-up and atomization. High-resolution long-distance microscopy imaging is carried out to obtain 15,000 snapshots of the two-phase interactions for 15 different flow conditions during the air-assisted atomization of pyrolysis oils. For all the high-resolution images, fractal dimensions are evaluated to characterize the complex multi-scale two-phase dynamics. In addition, droplet sizing is carried out using laser-diffraction measurements . Results are discussed in three main categories:78 with liquid discharge area of 77 and kinematic viscosity are respectively denoted as Figure mentclass2pt{minimcm working range with clear aperture of 63 mm and magnification up to mm. In addition, laser diffraction droplet size measurements are carried out using Malvern’s Spraytec. The global fineness of the bio-oil break-up and atomization is represented by the Sauter Mean Diameter (SMD) which is a measure of liquid overall volume to surface ratio.Fifteen cases of pyrolysis bio-oil break-up regimes down to the fully atomized sprays are characterized using the high-resolution long distance microscopic imaging by generating a pulsed back-lighting illumination. Image acquisition is conducted using the 14-bit IMAGER PRO X 2M charge-coupled device (CCD) camera with CCD pixel size D) are evaluated by successively refining the grid size and extracting the cells representing the complexity of the two-phase flow structure, the so-called “box-counting” algorithm73. For each of the 15 flow cases, fractal dimension statistics are evaluated by analysing 1000 high-resolution snapshots. A systematic approach is taken to pre-process the images before they can be properly used for fractal analysis. All the RGB images are first converted into gray-scale and normalised to represent liquid fraction. Five images are taken from the background light source to obtain the average back light intensity. The averaged field is then subtracted form all the instantaneous images. In order to correct for the out of focus liquid structures images are slightly de-blurred and then binarized with zero pixels in the background. To start the box-counting algorithm, images are padded into a pixel dimension at a power of 2. The box size r is initialized equal to the image size. Then, number of boxes of size r containing a minimum of one liquid pixel are counted as N(r). This process is continuously repeated by setting A05T40 and A15T50. The slope of these lines gives the fractal dimension (D) of the image.Fractal dimensions (In order to verify the present box-counting approach, Fig. In order to ensure that the present measurements are repeatable and produce reasonably low errors, standard deviation mentclasspt{minima


Although a central role of the thalamus for sleep regulation is undisputed, the exact localization of the crucial structures within the thalamus remains controversial.Here we report a 35 year old woman with no prior comorbidities who developed severe and persistent hypersomnia with long sleep time after a small right-sided MRI-verified thalamic stroke affecting the dorsal part of the pulvinar and the dorsolateral boarders of the dorsomedial nuclei.The observed symptoms suggest a crucial role of posterior thalamus but not the midline parts of the thalamus in sleep-wake control.The online version contains supplementary material available at 10.1186/s12883-020-02018-2. Despite the importance of sleep for humans, the anatomical structures involved in sleep regulation and the transition from sleep to wakefulness are complex and not fully understood. Two models for the transition from sleep to wakefulness have been proposed. The hypothalamus-brainstem mutual inhibition model, known as the flip-flop model, is based upon the interaction between the wake-promoting nuclei, the ascending reticular activating system, and the sleep-promoting nuclei, the ventrolateral and median preoptic nuclei . The thaThe role of the thalamus during sleep and the anatomic structures involved remains therefore elusive. The classical view of thalamo-cortical connection during sleep focused on primary sensory nuclei with reciprocal projections to primary sensory cortical areas . HoweverThe patient gave written informed consent to this study, had no previous medical history (apart from migraine) and no history of substance abuse. In January 2016, she was acutely admitted to a local hospital with vertigo, nausea, headache and blurred vision. Vital signs and weight were normal (body mass index < 25). Neurological examination at admission revealed right sided hemihypesthesia. No ocular paresis or nystagmus was found. Acute 1.5 Tesla MRI showed an acute infarction in both cerebellar hemispheres and in the dorsal thalamus  and under (B) treatment with sodiumoxybat (4.5 g 2x at night).


Live imaging of mRNA in cells and organisms is important for understanding the dynamic aspects underlying its function. in vivo make use of genetically encoded tags and reporters that significantly enhance the size of the mRNA of interest. Alternately, we utilize the 3′ poly(A) tail as a non-coding repetitive hallmark to covalently label mRNAs via bioorthogonal chemistry with different fluorophores from a wide range of spectra without significantly changing the size. We demonstrate that the labeled mRNAs can be visualized in cells and zebrafish embryos, and that they are efficiently translated. Importantly, the labeled mRNAs acquired the proper subcellular localization in developing zebrafish embryos and their dynamics could be tracked in vivo.Live imaging of mRNA in cells and organisms is important for understanding the dynamic aspects underlying its function. Ideally, labeling of mRNA should not alter its structure or function, nor affect the biological system. However, most methods applied  ASH1 in yeast,bicoid, oskar and nanos in Drosophila embryos,vg1 in Xenopus oocytes,beta actin in mammalian neurons.vasa and nanos (nos) mRNA in primordial germ cells, the progenitors of the germline lineage that gives rise to egg and sperm (PGCs).28The defined localization of different mRNAs to specific subcellular domains provides a mechanism for regulating gene expression temporally and spatially, in particular during dynamic processes such as embryonic development.Various approaches have been developed to visualize the subcellular localization of mRNAs in living systems and to gain a more comprehensive understanding of the life-cycle of an mRNA.in situ hybridization. Examples include single-molecule fluorescence in situ hybridization (smFISH),in situ hybridization (MERFISH).Visualization of mRNA can also be achieved by utilizing fluorescently labeled probes for To circumvent the introduction of exogenous probes like those mentioned above, several approaches for direct covalent labeling of mRNA with small fluorophores were devised. Body labeling by statistical incorporation of modified nucleotides during transcription and subsequent conjugation chemistry allows for multiple labeling.in vivo.62Alternatively, by adding a suitable genetically encoded tag in the UTR, RNAs can be labeled in cells using RNA-modifying enzymes recognizing this structural element.Experimental details are given in the ESI Section.in vitro transcription and then incorporated azido-modified adenosine nucleotides at their poly(A) tails using the yeast poly(A) polymerase (yPAP) and 2′-azido-2′-dATP (as co-substrate) as previously described.egfp and mcherry mRNAs in a strain-promoted alkyne–azide cycloaddition (SPAAC)  , namely  (SPAAC) .O-Me-m7G(5′)ppp(5′)G) resulting in correct cap orientation and translation was used in all cases, except for the negative control, where an ApppG-capped mRNA was used to rule out cap-independent translation. As an additional control, the non-labeled ARCA-capped mRNA with azido-modifications (N3pA) at the 3′ poly(A) tail was produced. Analysis of the labeled mRNAs showed that fluorescent signals at the respective wavelength settings could be observed in all cases for ∼1200 nt RNAs (3pA) did not show fluorescent signals. Together with SYBR Gold staining  these data demonstrate that the mRNAs remain intact and are efficiently labeled by various DBCO-fluorophore conjugates  was not translated, ruling out cap-independent translation. Together, these results show that bioorthogonal labeling of mRNA at the poly(A) tail via the SPAAC reaction can be used to incorporate different types of fluorescent dyes. Importantly, the differently labeled mRNAs can be visualized in cells and are actively translated by the cellular machinery.To confirm that the labeling of mRNAs with different fluorophores did not interfere with downstream processing, in vivo. We used zebrafish embryos as a model, since they can be injected with mRNAs at the 1-cell stage, with the RNAs distributed to all other cells of the embryo during subsequent cell divisions and development. Furthermore, the unique optical properties of the embryos allow imaging fluorescent signals resulting from injection of labeled mRNAs. We fused a reporter mRNA to the 3′ UTR of nanos (nos 3′ UTR), since this cis-acting element leads to protection and translation of the transcript in PGCs of zebrafish embryos,nos 3′ UTR-containing mRNAs are degraded and translationally suppressed by microRNAs.Next, we examined whether this labeling approach would be compatible with studying mRNAs nos 3′ UTR would be suitable for visualization in zebrafish embryos, we injected the in vitro-labeled mRNA into 1-cell stage embryos and assessed the dye signal in PGCs at 10 hours post fertilization (hpf) (mcherry sequence fused to the nos 3′ UTR (STOP mcherry-nos) was generated. This mRNA served for co-visualization of the injected labeled RNA with PGC-specific fluorescent markers  was constructed to assess whether PGC-specific translation of the labeled transcript can be achieved (To test whether poly(A) tail-labeling of mRNAs containing a on (hpf) . For thion (hpf) . First,  markers , and allng mRNAs . Second,achieved . We chosachieved  as it prmcherry-nos mRNA into 1-cell stage transgenic zebrafish embryos engineered to express EGFP on the membrane of PGCs and assessed the signal of the SRB-labeled RNA  tail-labeled mRNA remains functional with respect to translation in zebrafish embryos. To this end, t 10 hpf . We founnanos mRNA is known to be localized to germ granules (non-membrane-bound granules that contain RNAs and proteins important for germline development) within PGCs,mcherry-nos mRNA described above with the mRNA coding for the germ granule marker-protein Vasa-GFP and by assessing their subcellular localization within PGCs at 10 hpf  was localized to granules within which the endogenous nanos mRNA resides (nanos open reading frame targeted by an RNAscope probe). Furthermore, when the same experiment was performed with labeled STOP mcherry-nos mRNA, the SRB signals co-localized with the RNAscope signals for injected and endogenous nos mRNA (in vitro) resembles that of endogenous nanos mRNA, which is deposited in the egg during oogenesis in the mother and is associated with different mRNA-binding proteins that control its localization and function in PGCs—an important feature for application in biological studies. Importantly, the SRB-label does not significantly affect the behavior of the transcript in vivo.We found that the non-labeled STOP in vivo. This would enable studying the dynamics of subcellular mRNA localization in the living organism, which is important for understanding the extensive regulation of mRNA localization and fate from transcription to translation.gfp-nos mRNA into 1-cell stage zebrafish embryos. Time-lapse analysis of the SRB-signal in PGCs of 1 day-old embryos revealed dynamic changes in the structure and subcellular localization of the labeled mRNA (We next wanted to test whether poly(A) tail-labeling of mRNAs would allow us to track the dynamic positioning of transcripts led mRNA , that conos 3′ UTR-containing transcript by both systems in the same cell (see in vitro-labeled nos 3′ UTR-containing mRNA (STOP mcherry-nos) to visualize the transcript employing the click chemistry approach.nos 3′ UTR-containing mRNA that bears loop structures recognized by a YFP-tagged PP7 coat protein . In addition to the extended length of the RNA, unlike the click-based labeling, the PP7 system requires tethering 48 fluorescently-tagged proteins to the RNA, which could potentially further affect the dynamics of the labeled RNA.According to these data, the click chemistry labeling approach and the PP7-based system provide comparable information concerning the localization of the mRNA. Nevertheless, the two systems could show differences in certain cases concerning localization and dynamics of the RNA. Such differences could result from the fact that labeling RNAs by click chemistry does not involve alteration of the mRNA sequence, while the PP7-based detection involves large RNA sequence insertions (over 1400 bases). The added RNA sequence could potentially affect different characteristics of the tracked RNA, as it is relatively long  tail labeling technique we describe facilitates labeling mRNA with minimal effects or interference with its functions , and enables live-imaging. These methods are expected to aid in understanding the regulation of mRNA function in diverse processes in cells, embryonic development and disease contexts.In summary, we demonstrate a generally applicable bioorthogonal approach to label mRNAs in a way that maintains their translational activity in adherent cells and zebrafish embryos, and does not affect their subcellular localization in the latter. We show that an mRNA can be efficiently labeled at its 3′ poly(A) tail with different fluorophores, and that the label facilitates mRNA-visualization in cells and in developing embryos. Importantly, the labeled mRNA could be imaged and tracked in real-time in a living vertebrate, as demonstrated by experiments with mRNAs containing localization-specifying 3′ UTRs. Since mRNAs can be labeled with fluorophores of different spectra, this strategy is expected to pave the way for labeling and imaging more than one mRNA, enabling the correlation between localizations and functions of the mRNAs of interest. Furthermore, the procedure we present involves very small alterations of the mRNA, especially when compared to current methods such as the MS2 or PP7 system. Specifically, the stem-loops, coat-protein and fluorescent tag (GFP-MCP) in the MS2 system together are in the order of 25 000–50 000 g molThere are no conflicts to declare.Supplementary informationClick here for additional data file.Supplementary movieClick here for additional data file.


Brain complexity estimated using sample entropy and multiscale entropy (MSE) has recently gained much attention to compare brain function between diseased or neurologically impaired groups and healthy control groups. Using resting-state functional magnetic resonance imaging (rfMRI) blood oxygen-level dependent (BOLD) signals in a large cohort (n = 967) of healthy young adults, the present study maps neuronal and functional complexities estimated by using MSE of BOLD signals and BOLD phase coherence connectivity, respectively, at various levels of the brain’s organization. The functional complexity explores patterns in a higher dimension than neuronal complexity and may better discern changes in brain functioning. The leave-one-subject-out cross-validation method is used to predict fluid intelligence using neuronal and functional complexity MSE values as features. While a wide range of scales was selected with neuronal complexity, only the first three scales were selected with functional complexity. Fewer scales are advantageous as they preclude the need for long BOLD signals to calculate good estimates of MSE. The presented results corroborate with previous findings and provide a baseline for other studies exploring the use of MSE to examine changes in brain function related to aging, diseases, and clinical disorders. Fluctuations in the resting-state functional magnetic resonance imaging (rfMRI) blood oxygen-level dependent (BOLD) signals have recently received considerable attention for their use in studies of functional brain networks and discovering “neuromarkers” for brain function in diseases and clinical disorders . FunctioIn the past, many studies made the simplifying assumption that the correlation or functional connection between different regions of the brain is static . HoweverThe variability and complexity of the brain’s neural signals are two quantitative measures that can be used to assess the health of a brain . These mm being the pattern length) and displays relative consistency compared to approximate entropy. SampEn is the natural logarithm of the conditional probability that a pattern length of m points will repeat itself, without including self matches, for r in a time series of length N  and z-score normalized [MATLAB-zscore] before being concatenated to yield a total of 4800 data points. The time series were not filtered because studies have shown that useful neuronal related signals are present at higher frequencies up to 0.5 Hz [Brain parcellation was performed using an atlas with 90 functional brain regions of interest, also known as nodes, defined across 14 major RSNs . Three parameters must be defined: (i) pattern length m—the number of data points for pattern matching; (ii) tolerance (similarity factor) r—the fraction of standard deviation of the time series; and (iii) scale factor l—the scale factor of coarse-graining.Brain complexity was estimated in two steps using MSE. First, the multiple coarse-grained time series of length dFC matrix for each subject resulted in a column vector with dimension 90 l. In the higher dimensional eMSE case, the (4005 ×l) matrix for each subject resulted in a column vector with dimension 4005 l. Second, the correlation between the features and fluid intelligence was calculated. Third, the features were sorted from the lowest to the highest p-value of correlation, and the first n features were selected [selected ,58 for uselected . Finallyp-value then represents the probability of observing the reported accuracy by chance.The statistical significance of the leave-one-subject-out method was assessed using permutation testing ,58,59, w


Background: Right ventricular (RV) infarction is as an extremely rare cause of isolated anterior ST-segment elevation. Occlusion of the RV branch in a recessive right coronary artery (RCA) causing isolated RV infarction and only anterior ST-elevation is extremely rare. To date, the handful of such cases reports do not describe any arrhythmia associated with this presentation. Although ventricular fibrillation (VF) has been well-documented with interruption of flow in the conus branch of the RCA, here we describe VF occurring in a patient with occlusion of the RV branch of a recessive RCA presenting with isolated anterior ST-segment elevation.Case: A 51-year-old man presented with acute chest pain and isolated anterior ST-segment elevation on electrocardiogram (ECG). The patient developed ventricular fibrillation prior to coronary angiography requiring cardiopulmonary resuscitation. Coronary angiography revealed an unobstructed left coronary system and a recessive right coronary artery with ostial occlusion of the RV branch which was treated with a drug eluting balloon, resulting in resolution of the chest pain and ECG changes.Conclusion: Isolated RV infarction due to RV branch occlusion can cause ECG changes mimic anterior left ventricular infarction. This presentation may be complicated by VF, even in the setting of a recessive RCA. The ST-segment of an electrocardiogram (ECG) represents the time between ventricular depolarization and repolarization. In the majority of healthy individuals, the ST-segment is isoelectric. In the context of chest pain, ST-segment elevation in the left anterior precordial leads typically reflects anteroseptal infarction due to occlusion of the left anterior descending (LAD) artery. However, there are a broad range of differentials to consider .Right ventricular (RV) infarction is as an extremely rare cause of isolated anterior ST-segment elevation –4. RV inA 51-year-old man with a history of hypertension and cigarette smoking developed acute onset chest pain radiating to his jaw and back whilst at work. After the pain worsened over 2 h, the patient called for an ambulance and an electrocardiogram (ECG) revealed significant anterior ST elevation see .The patient was treated as a suspected acute ST-elevation myocardial infarction (STEMI) and taken immediately to the cardiac catheterization laboratory. Before the procedure could commence, the patient developed ventricular fibrillation (VF) requiring 20 s of cardiopulmonary resuscitation and one direct current shock before return of spontaneous circulation. Coronary angiography via the right radial artery revealed a normal left main stem (LMS) and a dominant and unobstructed left circumflex artery (LCX). The left anterior descending artery (LAD) had good antegrade flow with only mild disease in the mid-vessel . OpticalRight coronary angiography revealed a recessive vessel with occlusion of the right ventricular (RV) branch at the ostium , 3A,C. FFollowing the procedure, the patient was transferred to the coronary care unit and was found to have a high-sensitivity troponin I of 22,880 ng/L. The following day, transthoracic echocardiography revealed normal left and right ventricular size and systolic function with no significant valvular disease. The patient experienced no further chest pain or arrhythmias and was subsequently discharged 2 days after admission.RV infarction most commonly occurs due to occlusive coronary lesions in the RCA. It is associated with inferior ST-segment elevation due to concomitant infarction of the inferior left ventricular (LV) wall. Rarely, isolated RV infarction can occur, presenting with anterior ST-segment elevation. The ECG in such patients typically demonstrates significant ST-elevation in V1–2 and smaller ST-elevation in V5–6 , causes isolated RV infarction and the right ventricular electrical potentials are recorded in the left precordial leads producing anterior ST-segment elevation. The reason for this lies in the anatomical position of the RV which overlies the LV and thus infarction of the RV is recorded particularly in the first two precordial leads of the ECG  branch of the RCA is interrupted . The conus branch supplies the RVOT and ischaemia in this myocardial region can generate electrical disturbances and even VF. Such ventricular arrhythmias occurring due to occlusion of the RV (2nd) branch of a recessive RCA is extremely rare. Clinicians should be mindful that a patient presenting with isolated RV branch occlusion in a recessive RCA can present with only anterior ST-segment elevation and can develop life-threatening ventricular arrhythmias.All datasets generated for this study are included in the article/Written informed consent was obtained from the individual (who is over the age of 16) for the publication of any potentially identifiable images or data included in this article.Both authors contributed to the writing and proof-reading of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Image-guided thermal ablations are increasingly applied in the treatment of renal cancers, under the guidance of ultrasound (US) or computed tomography (CT). Fusion imaging allows exploitation of the strengths of all imaging modalities simultaneously, eliminating or minimising the weaknesses of every single modality. We present a case of a 68-year-old patient treated using US/CT fusion imaging to guide radiofrequency ablation for local recurrence of renal cell carcinoma undetectable by ultrasound. Renal cell carcinoma (RCC) is the most common primary malignancy of the kidney, accounting for about 2% of all cancer diagnoses in humans . Image-gA 68-year-old-male patient with a history of right nephrectomy was treated 3 months before by microwave ablation for RCC of the left kidney . He preset al [et al [RCC accounts for 2%–3% of all adult malignancies . The detet al  fusion iet al , 24. Howl [et al  have alrEspecially when treating residual or recurrence of renal lesions, either after surgery or percutaneous thermal ablation, tumour could not be sufficiently defined with grey-scale US alone. The combination of real-time US scans with CT images is an essential tool to place applicators for ablation, thus offering to the patients a chance to be treated in a minimally invasive way.The authors declare that they have no conflicts of interest.The authors received no specific funding for this work.


A high burden of severe disease and death from the coronavirus disease 2019 (COVID-19) has been consistently observed in older patients, especially those with pre-existing medical co-morbidities. The global pandemic lockdown has isolated many patients with chronic illnesses from their routine medical care. This narrative review article analyses the multitude of issues faced by individuals with underlying medical conditions during the COVID-19 pandemic.Sources for this publication were identified through searches of PubMed for articles published between 31st December 2019 and 4th June 2020, using combinations of search terms. Guidelines and updates from reputable agencies were also consulted. Only articles published in the English language were included.The volume of literature on COVID-19 continues to expand, with 17,845 articles indexed on PubMed by 4th June 2020, 130 of which were deemed particularly relevant to the subject matter of this review. Older patients are more likely to progress to severe COVID-19 disease requiring intensive care unit (ICU) admission. Patients with pre-existing cardiovascular disease, especially hypertension and coronary heart disease, are at greatly increased risk of developing severe and fatal COVID-19 disease. A controversial aspect of the management of COVID-19 disease has been the use of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers. Obese COVID-19 patients are more likely to require complex ICU management. Putative mechanisms of increased COVID-19 disease severity in diabetes include hyperglycaemia, altered immune function, sub-optimal glycaemic control during hospitalisation, a pro-thrombotic and pro-inflammatory state. Patients with mental health disorders are particularly vulnerable to social isolation, and this has been compounded by the suspension of non-emergency care in hospitals around the world, making it difficult for patients with chronic mental illness to attend outpatient appointments.The global pandemic of COVID-19 disease has had a disproportionately negative impact on patients living with chronic medical illness. Future research should be directed at efforts to protect vulnerable patients from possible further waves of COVID-19 and minimising the negative impact of pandemic mitigation strategies on these individuals. From its presumptive origins in late 2019 at an indoor seafood and live wild animal market in the major transportation hub of Wuhan, China . UltimatThere is growing interest among the travel medicine community in assessment and mitigation of the non-communicable disease risks faced by international travellers . There wThe widespread global lockdown and reconfiguration of health services to cope with the first wave of COVID-19 cases have isolated many patients with chronic illnesses from their routine medical care, leading to concerns about an anticipated surge in non-COVID medical cases as lockdown measures are gradually lifted. The rapid adoption of telehealth care has been a hallmark of the health system’s response to this pandemic , and theGiven the rapid growth in medical literature relating to this global pandemic, this narrative review article will focus on the issues facing adults with predisposing medical conditions, especially cardiorespiratory and metabolic, in an effort to provide greater clarity to the travelling public and guidance to their healthcare providers.Two of the authors (GTF and CHL) identified the references for this review through weekly searches of PubMed between 1st May 2020 and 4th June 2020 for articles published between 31st December 2019 and 4th June 2020, using combinations of the terms “coronavirus”, “novel coronavirus”, “nCoV”, “COVID-19”, “SARS-CoV-2”, “diabetes”, “obesity”, “cardiovascular disease”, “respiratory disease”, “gastrointestinal disease”, “cancer”, “kidney disease”, “immunosuppression”, and “co-morbidities”. All articles retrieved using the general search term “COVID-19” or variations thereof were further screened by title and/or abstract for additional sources not yielded by the focused search. Articles which did not relate to at least one chronic medical comorbidity were excluded, unless they contained important general information which provided context to our narrative discussion. We reviewed guidelines for the management of COVID-19 published by the World Health Organization, the European Centre for Disease Prevention and Control, and the US Centers for Disease Control and Prevention. We also examined relevant references cited in retrieved articles. Only articles published in the English language were retrieved but the English language abstract of one Chinese language article by Peng et al. was also consulted. The final reference list was agreed by all authors on the basis of its relevance to the topics covered in this review, with the aim of exploring the clinical presentation and complications of COVID-19 in patients with co-morbid conditions, and in presenting authoritative guidance to practitioners who manage patients with these chronic illnesses.The volume of literature on COVID-19 continues to expand at a rapid rate, with 17,845 associated articles indexed on the PubMed database alone as of 4th June 2020. After screening titles, abstracts and, in some cases, the full text version of indexed articles, 130 articles were deemed relevant to the subject matter of this review.The rapid global spread of the SARS-CoV-2 infection has challenged traditional efforts to communicate medical information in a timely manner to older patients living with chronic illness. The majority of older travellers attending a specialist travel clinic in Ireland were found to have a pre-existing medical condition . In the Older patients have borne a disproportionate burden of severe disease and death from COVID-19. Elderly patients with COVID-19 are more likely to progress to severe disease requiring intensive care unit (ICU) admission . In a caThe devastating effect of COVID-19 clusters in residential care settings has been a tragic feature of this pandemic. Older people living in residential care facilities have been over-represented among the victims of this infection, and there has been much discussion about the adequacy of the public health response in safeguarding older residents by ensuring that carers have access to personal protective equipment. People living with dementia have been particularly marginalised, given the difficulties they face in registering and recalling safety advice, such as the wearing of barrier face masks, or in processing the public health messages relayed by the media . This plDespite earlier suggestions that cigarette smoking, by up-regulating the expression of angiotensin-converting enzyme 2 (ACE-2) receptors in distal airways, may be responsible for causing more severe COVID-19 disease , a subsePatients with cystic fibrosis (CF) who develop COVID-19 lung disease must contend with the risk of suffering a negative effect on lung function , and thep = 0.007) [Patients with pre-existing cardiovascular disease (CVD), especially hypertension and coronary heart disease, are at greatly increased risk of developing severe and fatal COVID-19 disease. A meta-analysis of six studies with 1527 patients identified that hypertension and cardio-cerebrovascular disease were present as co-morbidities in 17.1 and 16.4% of the patients, respectively . In thei= 0.007) .Potential mechanisms of myocardial injury in COVID-19 include viral entry via the ACE-2 receptor, hypoxia-induced myocyte damage, and an immune-mediated cytokine storm . Recent While the positive environmental effects of the societal lockdown, in terms of reduced particulate air pollution and diminished traffic noise, are likely to have an appreciable population-level cardioprotective effect , these bA controversial aspect of the management of COVID-19 has been the use, in some patients with hypertension, of ACE inhibitors (ACEI) and angiotensin receptor blockers (ARB). ACE-2 receptor upregulation in infected patients taking these antihypertensive agents is believed to generate higher viral loads . This is2) and under the age of 60 years predicted an approximately two-fold increased risk of requiring acute and critical care [The challenges faced by obese international travellers have attracted recent attention in the medical literature . Obese Ccal care . This rical care , chroniccal care , and an cal care . There iThe effects of prolonged societal lockdown measures on obese individuals’ attempts at weight management also deserve attention. It has been suggested that obese subjects may require a longer period of quarantine than normal weight individuals, owing to their prolonged viral shedding . StrategDiabetes mellitus has rapidly become established as a major co-morbidity for severe COVID-19 disease . PatientThis group of patients benefits from the renoprotective effect of RAAS blocking antihypertensive agents. As discussed earlier, it is currently recommended that ACEI and ARB be continued in COVID-19 patients. Continuous blood glucose monitoring is recommended in hospitalised COVID-19 patients with diabetes, with use of sliding scale insulin therapy . The posIn common with other chronic illness populations, diabetic patients should have access to telemedicine services in order to benefit from the continuity of routine primary or outpatient care during the pandemic lockdown period. This may be supplemented by the transmission in real-time of capillary blood glucose readings to the diabetes health care team using remote sensor technology. Lifestyle advice which encompasses physical activity should be provided by members of the multidisciplinary diabetes health care team. A summary of practical clinical guidance for diabetes management during the current pandemic is presented in Table Meta-analysis has demonstrated a significant independent association of chronic kidney disease (CKD) with severe COVID-19 . The useThe volume of calls to a leading UK-based cancer charity during the COVID-19 pandemic lockdown reflects the understandable anxiety among patients undergoing treatment for, or recovering from, cancer . It is nDeferral of adjuvant chemotherapy or elective surgery for stable cancer patients has been advocated during the pandemic . It has Jindal et al. concluded in their recent guidance that, notwithstanding the potential risk of infection at chemotherapy day wards, chemotherapy should be continued with stringent measures taken to prevent transmission of the virus . This inPatients with a compromised immune system are anticipated to be at risk of more severe COVID-19 disease. A study by Sawalha et al. suggested that epigenetic dysregulation of the ACE-2 gene may confer an increased susceptibility and severity of COVID-19 disease in patients with systemic lupus erythematosus . There iQualitative analysis of participants in a US-wide longitudinal observational registry revealed that 42% of patients with RA experienced a disruption to their normal treatment in the preceding 2 weeks . The folPatients living with HIV infection suffer from more respiratory illnesses than HIV-negative individuals . Case seThe finding of prolonged shedding of SARS-CoV-2 viral ribonucleic acid in stool  has raisA cross-sectional study found a much higher prevalence of depression , anxiety , and anxiety-depression  during the COVID-19 outbreak in Wuhan, China . The COVPandemics tend to engender anxiety and depression, and lockdown is recognised as a cause of insomnia . The psyPatients with mental health disorders are particularly vulnerable to social isolation. This has been compounded by the effective suspension of non-emergency care in hospitals around the world, making it difficult for patients with chronic mental illness to attend outpatient appointments, for example. Some patients with psychosis face healthcare inequalities relating to a lack of a sense of self-protection and difficulty adhering to self-isolation guidelines .The importance of safeguarding access to clozapine monitoring clinics and depot antipsychotic injections for patients with chronic schizophrenia has been highlighted by the psychiatric profession . While tA particularly distressing aspect of this pandemic has been the increase in racist attitudes towards people of presumed Chinese ethnicity, given the probable origin of the novel coronavirus in Wuhan, China . As the Increasing levels of alcohol misuse during lockdown are also of concern and this has contributed to an increase in levels of reported domestic abuse. The potential public health effects of long-term isolation on rates of alcohol misuse are largely unknown , but theMale pattern baldness, though not generally regarded as a serious chronic medical condition, does predict a higher risk of developing coronary heart disease, hypertension and prostate cancer. An intriguing recent observation that patients being treated with hormonal therapy for prostate cancer showed a degree of protection against severe COVID-19 disease has led to suggestions that androgenetic alopecia may be a risk factor for severe infection with SARS-CoV-2. This has been invoked as a plausible explanation for why adult males develop more severe disease than females , why preTo the best of our knowledge, this is the first review article addressing the impact of the COVID-19 pandemic on patients with chronic medical conditions. We have attempted to critically discuss the issues relating to a wide range of common and important medical illnesses but our coverage cannot be considered exhaustive and relevant disorders which have been omitted from this work may be the subject of future reviews on the subject. A summary of the major issues relating to COVID-19 in patients with long-term illnesses is presented in Table The volume of new publications relating to COVID-19 continues to increase at a rapid rate. As such, this narrative literature review may not retain its currency for long. At the time of writing, there was still a paucity of original studies specifically addressing the issues facing patients with chronic medical illness, and some of the sources represented published editorial and expert consensus material. Future reviews should build on our current knowledge and provide updates during this dynamic period based on a larger proportion of research articles and meta-analyses, which provide a higher level of evidence. Our review was largely restricted to articles published in the English language, and potentially relevant studies published in Mandarin Chinese and other languages may have been omitted for consideration.This review has raised important questions, which should be addressed in future research grant calls. The effects of prolonged societal lockdown on cardiovascular and metabolic health parameters will help to guide future public health strategy and avoid an increased burden of preventable non-communicable disease. Whether certain vulnerable socio-economic groups were particularly disadvantaged, as suggested by recent reports of increased COVID-19 burden in ethnic minority groups in the UK and USA , is wortAs countries take tentative steps towards the relaxation of stringent border control measures, the future landscape of international travel is being hotly debated . InevitaThe global pandemic of COVID-19 disease has had a disproportionate impact on patients living with chronic medical illness. Underlying hypertension, cardiovascular disease, diabetes, obesity and respiratory disease have emerged as significant risk factors for development of severe COVID-19 pneumonia and systemic inflammation. Patients who are immunosuppressed by virtue of their chronic disease or its therapy have also been identified as vulnerable groups. The realignment of health services towards providing emergency and critical care for large numbers of COVID-19 patients has compromised the delivery of routine care to patients with chronic diseases. Lockdown measures have also negatively impacted the metabolic health of many patients by limiting opportunities for physical activity. Travel restrictions and media saturation with COVID-19-related information have also challenged the mental health of some individuals, especially those with underlying mental illness or its risk factors. Future research should be directed at efforts to protect vulnerable patients from possible further waves of COVID-19 and to minimise the negative impact of pandemic mitigation strategies on these individuals.


CT and its comprehensive usage have become one of the most indispensable components in medical field especially in the diagnosis of several diseases. SECT and DECT have developed CT diagnostic potentials in several means. In this review article we have discussed the basic principles of single-energy and dual-energy computed tomography and their important physical differences which can cause better diagnostic evaluation. Moreover, different organs diagnostic evaluations through single-energy and dual-energy computed tomography have been discussed. Conventional or single-energy CT (SECT) uses a single polychromatic X-ray beam (ranging from 70 to 140 kVp with a standard of 120 kVp) emitted from a single source and received by a single detector. The concept of dual-energy computed tomography (DECT) is almost as old as the CT technology itself; DECT initially required substantially higher radiation doses (nearly two times higher than those employed in single-energy CT) and presented problems associated with spatial misregistration of the two different kV image datasets between the two separate acquisitions. The basic principles of single-energy and dual-energy computed tomography and their important physical differences can cause better diagnostic evaluation. Moreover, different organs diagnostic evaluations through single-energy and dual-energy computed tomography have been discussed. According to diverse data and statistics it is controversial to definitely indicate the accurate comparison of image quality and dose amount. Computerized tomography (CT) has been hailed as a revolutionary diagnostic imaging tool in medicine ever since its introduction in the early 1970s , 3. TherThis review attempts to discuss the basic and fundamental factors of image formation in single- and dual-energy CT and to compare the image quality and dose in various clinical organs.Z (eff) values comparable to those of water) remain almost constant when varying the X-ray beam energy. DECT is based on the principle that the attenuation of tissues (reflected by their CT attenuation number in Hounsfield units [HUs]) depends not only on their density but also on their atomic number Z, as well as on the energy of the photon beam. To get the best material characterization, DECT should be performed in these conditions ..33].Renal stone composition:Renal calculi are composed of different substances such as uric acid, calcium phosphate, calcium oxalate, cystine, and brushite. Clinical administration diverges by stone type. Dual-energy CT uses progressive postprocessing techniques to determine the composition of the calculi accurately –39. For Pseudoenhancement of renal cysts:Virtual monoenergetic images ranging from 80 to 90 keV created from dual-energy datasets can be fashioned to overcome beam hardening and partial volume of indeterminate renal masses. This can be done retrospectively and act as a convenient utensil when calculating for pseudoenhancement of renal cysts , 41.Dual-energy CT has a number of clinical applications in the assessment of the urinary system particularly in the realm of artifact reduction and material composition.Virtual noncontrast imaging is an image postprocessing technique used to create “noncontrast” images of contrast-enhanced scans via the subtraction of iodine. It is an imaging technique unique to dual-energy CT. Clinical applications of this method have been bolded in differentiation of hemorrhage from iodinated contrast. Contrast staining of the brain parenchyma after iodinated contrast can lead to interpretation issues, especially when the density of contrast is similar to that of blood. Virtual noncontrast imaging is a precise  method iOne of the most alluring uses of dual-energy CT is direct CT angiography. In this method, the dual-energy algorithm classifies and eradicates bone, permitting direct imagining of iodinated vessels . On the Also the DLP factor in surgical group who are undergoing intracranial aneurysm repair in SECT is 925 mGy and in DECT is 534 mGy which shows remarkable DLP in SECT.Coronary computed tomographic (CT) angiography provides angiographic images of coronary stenosis but is a poor predictor of the physiologic importance of any given stenosis. The addition of functional imaging with other modalities such as echo, magnetic resonance imaging which has been compared with DECT in CT and its comprehensive usage have become one of the most indispensable components in medical field especially in the diagnosis of several diseases. SECT and DECT have developed CT diagnostic potentials in several means. In this review article we have discussed the basic principles of single-energy and dual-energy computed tomography and their important physical differences which can cause better diagnostic evaluation. Moreover, different organs diagnostic evaluations through single-energy and dual-energy computed tomography have discussed. According to diverse data and statistics it is controversial to definitely indicate the accurate comparison of image quality and dose amount. In more items single-energy computed tomography has shown better image quality rather than to dual-energy computed tomography also in more items and organs single-energy computer tomography has shown greater CT dose index volume in comparison to dual-energy computed tomography.


LDLR) resulting in the non-clearance of LDL from the blood by hepatocytes and consequently the formation of plaques. LDLR is synthesized and glycosylated in the endoplasmic reticulum (ER) and then transported to the plasma membrane via Golgi. It is estimated that more than 50% of reported FH-causing mutations in LDLR result in misfolded proteins that are transport-defective and hence retained in ER. ER accumulation of misfolded proteins causes ER-stress and activates unfolded protein response (UPR). UPR aids protein folding, blocks further protein synthesis, and eliminates misfolded proteins via ER-associated degradation (ERAD) to alleviate ER stress. Various studies demonstrated that ER-retained LDLR mutants are subjected to ERAD. Interestingly, chemical chaperones and genetic or pharmacological inhibition of ERAD have been reported to rescue the transport defective mutant LDLR alleles from ERAD and restore their ER-Golgi transport resulting in the expression of functional plasma membrane LDLR. This suggests the possibility of pharmacological modulation of proteostasis in the ER as a therapeutic strategy for FH. In this review, we picture a detailed analysis of UPR and the ERAD processes activated by ER-retained LDLR mutants associated with FH. In addition, we discuss and critically evaluate the potential role of chemical chaperones and ERAD modulators in the therapeutic management of FH.Familial hypercholesterolemia (FH) is an autosomal genetic disease characterized by high serum low-density lipoprotein (LDL) content leading to premature coronary artery disease. The main genetic and molecular causes of FH are mutations in low-density lipoprotein receptor gene ( LDLR) account for more than 80% of monogenic FH     is a genetic disorder that results in altered lipid metabolism and consequently leading to elevated levels of plasma low-density lipoprotein cholesterol (LDL-C) . Clinicagenic FH   , PCSK9 (#603776) , and LDL 603813) . FH can  603813) . The cli 603813)  though f 603813) . On the  603813) . If left 603813) . In rece 603813) .de novo intracellular synthesis or by uptake of dietary cholesterol  is the prototype receptor of a group of structurally and functionally similar cell surface receptors. LDLR is encoded by the o the ER   each composed of 40 amino acid residues . Six cysO-linked sugars domain plays a major role in the post-translational modification of LDLR. This domain is encoded by exon 15 and is composed of 48 amino acids consisting of 18 threonine and cysteine residues that act as attachment sites for O-linked sugar chains. The membrane-spanning domain is responsible for LDLR integration and attachment to the cell membrane  , rangingAround 50% of reported LDLR mutations are Class II mutants which are implicated to be transport-defective . At presIn eukaryotes, an estimated one-third of all newly synthesized proteins enter the ER to undergo post-translational modifications and achieve their three-dimensional native conformation, before reaching their proper cellular destination . HoweverN-linked glycosylation of Asn-X-Ser/Thr motif is an important post-translational modification that help nascent proteins to remain soluble and prevent aggregation by masking the hydrophobic stretches in the protein , to the Asn residue  . Though  protein . N-glyco residue .N-glycan to Glc1Man9GlcNAc2 by glucosidases I (GI) and 2 (GII), makes it a high-affinity ligand for lectin chaperones such as calnexin (CNX) and calreticulin (CRT). Binding of CNX/CRT to glycoproteins facilitates their retention in ER, prevention of aggregation and recruitment of PDIs such as ERp57 (PDIA3) (Enzymatic deglucosylation of the  (PDIA3) . Removal (PDIA3)  and termN-glycans with deglucosylated, demannosylated forms (Man5–Man7) that are incompatible with UGGT-mediated reglucosylation (Demannosylation by the ER-resident mannosidases such as ER mannosidase 1 (ERMan1) and ER-degradation enhancing mannosidase-like proteins  results in sylation . The degsylation  , transmembrane (ERAD-M), and cytosolic domains (ERAD-C) . An emerSince the proteins targeted for degradation have diverse structures and topologies, distinct combinations of adaptors that recognize these features are recruited by the E3-ubiquitin ligases. In mammals, the transmembrane (TM) protein SEL1L works in conjunction with the HRD1 E3 ligase and is necessary to deliver the ERAD substrates from ER lectins  to HRD1 . DepletiDerlin family of proteins DER1, 2, and 3 have been proposed to play a role in substrate dislocation through association with HRD1/SEL1L . During The quality control of TM proteins containing defects in their membrane-spanning domain appear to be distinct from that of ERAD-L and ERAD-C substrates, since luminal or cytosolic factors do not have access to the domain location. An intramembrane rhomboid protease, RHBDL4 has been shown to cleave TM-anchors of unstable single-membrane spanning or polytopic membrane proteins in an ubiquitin-dependent manner and divert them to the UPS coupled to VCP/p97 ATPase  Fleig e. Recent While ERAD is highly efficient in handling a variety of misfolded proteins, some membrane and soluble proteins form aggregates and place constraints on ER retrotranslocation machinery. These aggregates are diverted to the lysosome for degradation via alternative pathways collectively termed as ER-to-lysosome–associated degradation (ERLAD) . ERLAD ide novo biosynthesis of cholesterol and LDLR for the uptake of cholesterol , a rate-limiting enzyme in the mevalonate pathway which produces cholesterol and other isoprenoids . The accOther than transcriptional regulation by SREBP, LDLR is post-translationally regulated by ubiquitin-dependent degradation mediated by the E3 ligase- inducible degrader of the LDLR (IDOL) . IDOL is2-to-COOH terminus and the folding is co-translational, the nascent LDLR polypeptide is demonstrated to fold rapidly into compact structures by forming non-native disulfide bonds linking distant domains of the receptor  is an ER-localized oxidoreductase containing J domain and thioredoxin domains important for its disulfide exchange activity . ERDJ5 iIn addition to the aforementioned chaperones, several private chaperones are involved in LDLR folding. The LBD of LDLR family members require the assistance of the receptor associated protein (RAP) for maturation, which prevents premature interaction of the domain with its ligands in the same compartment . The BOCIt was demonstrated that different Class II mutants of LDLR affecting the LBD  and EGF domain (C646Y) were retained in the ER and degraded by a proteasome-dependent pathway in cell lines stably expressing the mutants . One of Very little information is available on the ERAD components involved in the substrate recognition, retrotranslocation and degradation of LDLR mutants. Recent results from our lab indicate that the LDLR mutants interact with HRD1 and its partners SEL1L and OS9 . Our resThe role of non-canonical ERAD pathways in the degradation of LDLR class II mutants have not been explored to our knowledge. We have reported previously that a small fraction of ER-retained VLDLR missense mutants are aggregation-prone and may undergo an autophagy-related process for degradation . It is pThe mechanisms by which the mutations affecting the TM domain of LDLR cause FH are only emerging. It was reported that a mutation affecting the TM domain of the LDLR (G805R), undergoes ectodomain cleavage by a metalloproteinase in the ER and results in lower LDLR levels at the cell surface . The ER-Accumulation of unfolded proteins in ER activates a battery of cellular stress responses, altogether called as UPR. The UPR aims to restore the normal ER-homeostasis, however, if the stress is severe and irreversible, then UPR switches to apoptosis. Endoplasmic reticulum serves as a site for protein synthesis, folding as well as the internal cellular calcium reservoir and hence plays a critical role in cell physiology. Disruption of ER homeostasis due to derailed calcium physiology; redox imbalance; accumulation of misfolded proteins causes ER-stress . Cells rActivated PERK phosphorylates the eukaryotic initiation factor 2 alpha  . PhosphoActivation of UPR is aimed to resolve the stress in ER and bring back the normal ER homeostasis. Notably, major targets of all three ER stress sensors are directed to mitigate any harmful consequences of ER stress. However, when stress is overwhelming and beyond the capacity of ER adaptive stress machinery then the initial adaptive UPR switches to ER-stress activated apoptosis. Persistent activation of three major ER sensors results from unresolvable ER-stress which activates ER-apoptotic signaling principally mediated by pro-apoptotic proteins involving JNK, CHOP and BCL-2 family proteins . ER-streSurprisingly, only few studies pertaining to the association of ER-stress with the molecular pathology of FH have been reported  in contrOur lab has recently reported that two missense LDLR mutants D482H and C667F associated with FH were misfolded and retained in ER . FurtherAnother important aspect to consider is the role of ER-synthesized sterol regulators such as proprotein convertase subtilisin/kexin type 9 (PCSK9) in the ER physiology of class II LDLR mutant expressing cells. Both PCSK9 and LDLR are transcriptionally upregulated by SREBP2, an ER-resident transcriptional factor that binds to sterol regulatory elements in the promoter regions of sterol inducible genes including LDLR and PCSK9 . PCSK9 iInterestingly, ER stress induced by pharmacological ER stress inducers appear to inhibit the secretion of PCSK9 due to their retention in the ER by GRP94 . This isAlso, it is quite important to note that in protein conformation diseases such as alpha-1 antitrypsin deficiency, ER-stress induced apoptosis plays a critical role in the molecular pathology associated with liver failure . Adding Induced Pluripotent Stem Cell (iPSCs) have been developed by reprogramming fibroblasts from a FH patient carrying a homozygous three-base pair deletion in LDLR exon 4. The mutation results in ER-retention of LDLR and hence comes under class II mutation . Apart fResearch underpinning the molecular pathology of FH from various labs including our group have demonstrated that class II mutations in LDLR cause misfolding, ER retention and consequent protein degradation via ERAD . As abunXenopus oocyte grown at room temperature , a low molecular weight bipolar fatty acid derivative appears to rescue ER-retained transport defective class II mutant LDLR G544V in cell line overexpressing model. Although, 4-PBA mediates the rescue of only 30% of mutant LDLR expressed, the rescued LDLR mutant is expressed on the cell surface and capable of LDL binding and internalization compared to wild type . InteresOur lab has been studying proteostasis regulation of various missense mutants implicated in various genetic diseases including FH. We have demonstrated that the genetic ablation of ERAD components leads to the stabilization of ER-retained VLDLR missense mutants . HoweverThe UPS is the end component of ERAD where the retrotranslocated misfolded proteins are finally processed. We and others have shown that proteasome inhibitors such as MG132 aid protein transport and significantly improve the protein function of missense mutants which otherwise retained in ER and subsequently subjected to ERAD . It is qEven though, the cell line-based studies are encouraging, targeting normal cellular process such as ERAD and proteasome has deleterious effects. One has to be cautious of the fact that majority of the information on the molecular pathology of class II LDLR mutants come from cell line-based overexpression studies. The cell line-based data has to be validated with studies involving tissues from FH patients. Low efficiency also poses a problem while considering PCs/PRs as therapeutic agents. Cell line-based study shows only 30% functional recovery of rescued LDLR by 4-PBA, an extensively studied chemical chaperone for various mutants associated with protein conformational diseases. Pre-clinical studies show that application of PCs result in variable increase in the activity of mutant enzymes associated with lysosomal storage diseases . Some muIt is quite interesting to note that 4-PBA mediated rescue of LDLR mutants is mutation specific. The authors diligently prove that the other mutations in the same domain of the protein which are also class II mutations are not rescued by 4-PBA . One canLDLR gene. In homozygous FH patients where a functional LDLR is lacking, the current LDL-C lowering drugs have minimal effect (In this review, we have outlined the impact of class II LDLR mutants on ER-proteostasis and how it can be modulated for the therapeutic management of FH. Conventional lipid-lowering drugs are effective in maintaining LDL-C levels in heterozygous FH patients since these patients have residual LDLR function due to the presence of a normal copy of the l effect . A growil effect . AdditioDO, PK, and AJ drafted different sections of the manuscript. PK and BA compiled and edited the manuscript. PK and DV created the figures. DO, PK, DV, AJ, and BA critically reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The concerns, published on the Medicine Correspondence Blog, detail a misrepresentation of data as post-treatment efficacy data and the inappropriate use of selected studies in the meta-analysis. Authors were unresponsive in providing corrections to address the raised concerns.The article “A comparison of tiotropium/olodaterol vs tiotropium alone in terms of treatment effect for chronic obstructive pulmonary disease: A meta-analysis”,


Tobacco use is prevalent amongst individuals infected with human immunodeficiency virus (HIV). In resource-constrained settings, pharmacological smoking cessation interventions are unfeasible because of their high cost. There is a need to develop and evaluate behavioural interventions to address the unique challenges of tobacco use in the HIV-infected populations in these settings.The authors aimed to assess the feasibility and acceptability of the Behavioural Activation/Problem Solving for Smoking Cessation (BAPS-SC) intervention programme to determine whether it should be tested in an adequately powered randomised controlled trial.The authors merged behavioural activation therapy (BAT) with the principles of problem-solving therapy to create a novel five-session counselling model to address the unique challenges of tobacco cessation amongst those infected with HIV. Feasibility measures included the rate of enrolment amongst those eligible and the retention rate and descriptive analysis of intervention acceptability. The authors’ secondary outcome was 7-day point smoking prevalence abstinence, confirmed with breath carbon monoxide.A total of 128 individuals were screened over 8 weeks with 50 deemed eligible and 40 enrolled (80%). Retention at week 12 was 53% (21/40). The 7-day point prevalence abstinence, co-confirmed, at week 12 was 37.5% (15/40). All respondents indicated that they would recommend BAPS-SC to other smokers who want to quit, and would be willing to participate in the programme again up to the point of exit if they did not stop smoking.A full-scale randomised control trial comparing BAPS-SC with usual practice is warranted to evaluate the efficacy of this novel intervention in these settings. Additionally, problem-solving approaches have been used with PLWHA to improve medication adherence and decrease depressive symptoms.17 Behavioural activation therapy and problem-solving approach may help smokers select and implement activities that will replace smoking, thereby reducing smoking rates.Depressive symptoms are common in HIV-infected populations, often comorbid with smoking, and associated with poor smoking cessation rates.The authors developed a novel counselling model incorporating elements of behavioural activation and problem solving to address the unique challenges of tobacco cessation amongst those with HIV infection. They aimed to assess the feasibility and appeal of Behavioural Activation/Problem Solving for Smoking Cessation (BAPS-SC) intervention to determine whether it should be tested in an adequately powered clinical trial.18 The authors excluded participants if they reported current untreated and unstable alcohol dependence; current use or discontinuation within last 14 days of smoking cessation medications; current diagnosis of unstable and untreated major depression or current or past diagnosis of psychotic disorder; use of chewing tobacco, snuff or snus; current participation in a smoking cessation programme; or plans to use nicotine substitutes or smoking cessation treatments in the next 7 months. The restriction to 7 months was based on the anticipated period to conduct the trial so as to avoid contamination of the intervention with other smoking cessation modalities not under investigation.The authors conducted a single-arm pilot trial of the BAPS-SC intervention in Botswana amongst HIV-infected individuals aged 18–65 years who smoked ≥ 5 cigarettes/day, on average, at four outpatient HIV clinics. The target accrual goal was 40 participants. This was based on the assumption that at least five participants would be enrolled per week, thus ensuring that the recruitment of human subjects into the trial is timely as this is vital to the success of the trial.A trained recruiter approached each patient in clinic to ascertain smoking status. Those acknowledging smoking were asked to participate in a questionnaire related to their smoking and offered participation in the pilot trial. Those who agreed were referred to the research assistant who arranged to meet with them to determine eligibility and administer informed consent.The authors merged BAT with the principles of problem-solving therapy to create a novel five-session counselling model to address the unique challenges of tobacco cessation amongst PLWHA (BAPS-SC). Members of the team created a formal treatment manual, which was evaluated for cultural appropriateness by Botswana co-investigators, including assessment of translation and back translation and pre-pilot testing using videoconferencing to role play. Key components of BAPS-SC include activity monitoring and rewarding activity scheduling, assessment of personal goals and values, assessment and altering of avoidance behaviour and other maladaptive coping strategies, and contingency management. Behavioural Activation/Problem Solving for Smoking Cessation focuses on reducing stress pile-up and loss of pleasure that accompanies the cessation process and on identifying and establishing environmental/social changes to promote abstinence. Behavioural Activation/Problem Solving for Smoking Cessation addresses smoking as a behaviour that prevents and restricts opportunities for contact with healthy rewarding behaviours. These changes are achieved through altering daily routines previously associated with smoking in ways that increase pleasure and mastery across life domains, reducing rumination and increasing behavioural skills to prevent return to smoking as a means of avoiding stressors.A pre-quit session (session 1) introduces participants to: (1) self-monitoring of mood and behaviour; (2) assessment of personal values to refine the treatment plan; and (3) scheduling of substitute rewarding activities that align with their abstinence goal. At the target quit date (TQD) session (session 2), participants’ experiences with abstinence are reviewed and functional analysis of behaviour is introduced, especially as it relates to smoking and avoidance patterns. Information obtained is used to help generate a tailored behavioural activation plan by using the problem-solving framework to increase rewarding activities and relationships, reduce avoidant responses to distressing experiences and facilitate successful implementation of smoking trigger management strategies. Sessions 3–5 incorporate strategies to address avoidance patterns, especially those involving smoking, and replace them with adaptive coping strategies, again by using problem solving. Sessions were conducted by telephone over a 12-week period (with the first session lasting 1 h and subsequent sessions lasting 30–45 min on average) and involved weekly homework assignments.19), anhedonia [using the Snaith–Hamilton Pleasure Scale (SHAPS)20] and feasibility . Participant accrual rate was defined as the number of participants enrolled over 8 weeks. Retention was calculated as a proportion of participants enrolled who were available at week 12 contact session. Study staff not acting as interventionists interviewed participants at baseline, week 6 and week 12 to determine if they were still smoking and whether they implemented the suggested intervention strategies. The timeline follow-back procedure assessed daily smoking between measurement time points.21 Additionally, the amount of carbon monoxide (CO) in participants’ breath was tested to confirm self-reported abstinence from tobacco. Data were collected on paper and transferred to an electronic REDCap database for analysis.Collected data included information related to smoking behaviour . The primary efficacy outcome was 7-day point prevalence abstinence at week 12 (12 weeks post-TQD), defined as self-reported abstinence for 7 days prior to the assessment and breath CO <8 ppm.Ethical approval was obtained from the Botswana Ministry of Health .Characteristics of the sample are described in The 7-day point prevalence abstinence at week 12 was 37.5% (15/40). Notably, all respondents indicated that they would recommend BAPS-SC to other smokers who want to quit and would be willing to participate in the programme again up to the point of exit if they did not stop smoking. When a more stringent threshold of exhaled CO ≤ 4 ppm was used as the criterion for success, the quit rate in this pilot was 9/40 (22.5%).24 The results of the CO monitoring and exit interviews suggest that BAPS-SC is likely to be efficacious.The authors conducted a single-arm pilot trial of the BAPS-SC intervention programme, a novel behavioural smoking cessation intervention for PLWHA in a high-HIV-burden sub-Saharan country wherein all 40 participants received the intervention. This design is ideal for studies aiming to determine if a novel intervention is appealing and feasible in the setting and warrants a full-scale clinical trial.25 There is no comparable trial that tested the intervention developed by the team of the authors with an HIV-negative cohort of smokers. Two comparisons are worth noting, and both indicate a substantial benefit from authors’ intervention. Firstly, meta-analyses of behavioural interventions (without medications) for smoking cessation rooted in classic cognitive-behavioural theory show that they yield quit rates of generally less than 15% at the end of treatment.26 Secondly, MacPherson et al. (2010), in their pilot test of BAT for smoking cessation, reported an end-of-treatment quit rate of 17%.27 The authors used a higher threshold of CO to determine smoking cessation as opposed to a more stringent cut-off in the range of 3 ppm – 4 ppm recently proposed by other researchers.28 However, 8 ppm is the cut-off used in most smoking cessation clinical trials for PLWHA, so the authors chose this cut-off to compare their results to the current literature. With regard to past behavioural smoking cessation treatments for PLWHA, a cell phone-based intervention was found to be associated with significantly higher initial quit rates compared with usual care.29 Other studies including a group-based tailored intervention, motivational interventions and a web-based intervention (vs in-person or self-help) have not yielded significant increases in quit rates.36 Two pilot studies of behavioural treatments that address negative affect (depression and anxiety) show promise for PLWHA.38This study’s results of a high degree of smoking cessation success with BAPS-SC suggest a potentially highly impactful smoking cessation programme given that behavioural smoking cessation programmes rarely yield quit rates higher than 10% – 15%.39 There were proportionately fewer female candidates enrolled in the present study consistent with data from demographic and health surveys in sub-Saharan Africa.40 Although these smoking rates appear to be comparatively lower than reports from North America and Europe, these rates are projected to increase in the African continent whilst they are falling in other parts of the world.41 Given the limited behavioural health infrastructure in low middle income countries (LMICs) such as Botswana, the authors leveraged HIV clinical care sites and telephone-delivered counselling to extend the reach of skilled practitioners. The authors found this strategy to be effective and acceptable by the participants in the pilot trial.The BAPS-SC trial proved feasible. Firstly, the target sample size of 40 participants was reached within 8 weeks of recruitment, a rate of enrolment that would make large trials feasible. The authors’ findings indicated that a large-scale clinical trial would be feasible to determine the efficacy of the BAPS-SC programme in this setting where HIV prevalence is relatively high. Furthermore, smoking prevalence in the setting of their pilot study was estimated to be as high as 51% for male candidates and 6% for female candidates with HIV infection.Smoking is a major cause of morbidity and mortality, yet smokers find it very difficult to quit. If individuals quit because of this intervention, they will reduce their risk of cardiovascular disease, chronic obstructive pulmonary disease and cancers, particularly lung cancer. This will be of direct benefit to the individual participant. Further, if the authors are able to mount a full-scale trial based on the results of this pilot trial, high HIV burden LMICs such as Botswana may benefit because the intervention may become standard of care in the country, reducing disease burden caused by tobacco use. Being a behavioural intervention without pharmacological intervention, the cost is expected to be substantially less with the BAPS-SC compared with a pharmacological intervention. A formal cost-effective analysis will be needed to be undertaken following the roll out of the programme.42 This may be improved by asking participants to provide contact numbers of associates such as friends and family members who can be contacted if the participant is unreachable after a number of attempts and increasing incentives to complete follow-ups. From an evidence-based medicine perspective, if efficacy of the intervention is determined to be high, implementation science methods would need to be employed in the clinical care setting to incorporate the intervention into HIV programmes. The intervention leverages the existing HIV care infrastructure and will likely facilitate scale-up in sub-Saharan African settings where HIV is common and smoking continues to emerge as a threat to HIV-positive individuals’ health and survival.Results of the exit questionnaires to evaluate the acceptability of the BAPS-SC trial indicate that HIV-infected smokers in Botswana find the programme to be appealing and acceptable. The results indicate a lower-than-expected retention rate with slightly over half of the participants remaining in the study after a 12-week follow-up period. The retention rates across most smoking cessation trials generally exceed 75%.This pilot study evaluating the feasibility of BAPS-SC amongst HIV-infected smokers in Botswana has notable limitations. This was a single-arm trial, and therefore the results indicate only preliminary evidence of efficiency of the intervention and do not confirm efficacy. However, the authors’ main aim was to evaluate feasibility of implementing the intervention. The planned full clinical trial informed by these results will be powered to address efficacy. Additionally, the follow-up period was of only 12 weeks as the aim was to evaluate feasibility of the novel intervention. Thus, the follow-up period was not long enough to assess long-term abstinence. However, this was a pilot study to inform a future clinical trial.The results of this single-arm pilot trial demonstrate the feasibility of leveraging HIV clinical infrastructure for implementing BAPS-SC as a smoking cessation intervention programme amongst HIV-infected smokers in a resource-limited setting with high HIV burden. A full-scale clinical trial comparing BAPS-SC with standard counselling is thus warranted to evaluate the efficacy of this novel intervention in these settings.