Moyamoya disease (MMD) is a type of chronic cerebrovascular disease. Currently, revascularization surgery including direct/indirect procedure is recommended for symptomatic patients. However, some patients still respond poorly to the treatment or develop secondary symptoms.We report the first case of an MMD patient treated with deep brain stimulation (DBS) and thalamotomy. Symptoms of dystonia due to hemorrhage in the thalamus responded poorly to revascularization surgery, but were considerably alleviated by stereotactic neurosurgery.Our case report provides a potential strategy for management of refractory symptomatic MMD patients with dystonia and also supports the combined efficacy of DBS with thalamotomies.Approximately 30% of patients with Moyamoya disease (MMD) presenting movement symptoms do not respond well to revascularization surgery. We reported an MMD patient treated with deep brain stimulation (DBS) and thalamotomy with significant dystonia and dystonic tremor symptom amelioration. It indicates that DBS or stereotactic lesioning might be a potential treatment for the refractory movement symptoms of MMD. Moyamoya disease (MMD) is a cerebrovascular disease characterized by chronic progressive stenosis of bilateral supraclinoid internal carotid arteries and an abnormal vascular network at the base of the brain. Ischemic symptoms such as transient ischemic attacks are the most common clinical manifestations of MMD, while involuntary movements, usually observed among children, including chorea, dystonia, and dyskinesia are comparatively rare in MMD patients. The latter descriptive features are partially or minimally responsive to medications such as anticholinergic agents and baclofen . CurrentThe patient was a 15-year-old, right-handed girl who has experienced progressive involuntary movement of her left upper extremity for 5 years. At the age of 10 years, she suddenly developed jerky movements on her left upper limb accompanied by ipsilateral handshaking without triggers. The tremor worsened with activity and seriously affected her fine motor function. She was subsequently sent to a local hospital. Physical examinations showed increased muscular tension and reduced muscular strength (grade 4/5) of the patient’s left upper limbs. Computed tomography (CT) images suggested a recent hemorrhage in the right thalamus, and a typical manifestation of “puff of smoke” sign was revealed on CT angiography (CTA) Figure . DiseaseOur multidisciplinary DBS team discussed and agreed on a treatment strategy of unilateral VIM/VOA radiofrequency thalamotomy and GPi-DBS. Surgical implantation of the right posteroventral GPi electrode  and unilateral thalamotomy were performed during one operation, with informed consent provided by patient’s guardians for both surgery and video recordings. They acknowledged and fully understood the potential perioperative risks, specifically intracranial hemorrhage. Thalamotomy was performed before electrode implantation, leading to an immediate satisfactory outcome Videos . The dysFunctional stereotactic neurosurgery has revolutionized the treatment for dystonia following the success achieved in Parkinson’s disease (PD). In summary, our patient had dystonia over the left side of her body, which mainly affected her upper limb, with a presurgical BFMDRS score of 32 points. Her symptoms included rigidity, tremor, and involuntary movement of her left upper limb, causing significant disability. According to her diagnosis, GPi-DBS was a primary consideration because it is safe and effective for medically refractory dystonia, specifically the idiopathic group . However5Although DBS has been increasingly used for dystonia patients, ablative surgery still has not been fully replaced by DBS, considering that they both have advantages and limitations. DBS is a reversible and safe procedure that requires the replacement of pulse generators and batteries. However, this procedure is relatively expensive. Thus, considering that DBS is cost-prohibitive , the pat12133Unilateral GPi-DBS combined with VIM/VOA thalamotomy results in less financial burden and less risks of side effects. Additionally, previous cases have reported the remarkable efficacy of both DBS and thalamotomy for severe idiopathic dystonia  and combThe patient experienced a rapid amelioration after VIM/VOA ablation, which can be observed through the recorded video, and greater than 50% BFMDRS improvement was observed at the last follow-up visit. To confirm the separate and combined efficacy of VIM/VOA thalamotomy lesioning and GPi-DBS, we observed and evaluated the patient under DBS-off status (turn off the stimulator for 24 hours). At 1-month follow-up, the DBS-off BFMDRS motor score (16) was worse than the DBS-on status (14) (present on Video According to previously published reports , the effTo the best of our knowledge, this case reports the first application of stereotactic neurosurgery to movement disorders in MMD and is the first paper to report surgery of GPi-DBS with VIM/VOA thalamotomy for posthemorrhagic dystonia. Approximately 30% of the symptomatic MMD patients respond poorly to the established revascularization surgery . The suc


Despite several additive manufacturing techniques are commercially available in market, Fused Deposition Modeling (FDM) is increasingly used by researchers and engineers for new product development. FDM is an established process with a plethora of advantages, but the visible surface roughness (SR), being an intrinsic limitation, is major barrier against utilization of fabricated parts for practical applications. In the present study, the chemical finishing method, using vapour of acetone mixed with heated air, is being used. The combined impact of orientation angle, finishing temperature and finishing time has been studied using Taguchi and ANOVA, whereas multi-criteria optimization is performed using the Technique for Order of Preference by Similarity to Ideal Solution (TOPSIS). The surface finish was highly responsive to increase in temperature while orientation angle of 0° yielded maximum strength; increase in finishing time led to weight gain of FDM parts. As the temperature increases, the percentage change in surface roughness increases as higher temperature assists the melt down process. On the other hand, anisotropic behaviour plays a major role during tensile testing. The Signal-to-noise (S/N) ratio plots, and ANOVA results indicated that surface finish is directly proportionate to finishing time because a longer exposure results in complete layer reflowing and settlement. Additive manufacturing is a digital fabrication process used for the fabrication of tailor-made parts utilizing principle of layer-by-layer material addition . The proThe thermoplastic part material and support material in the form of thin wire is fed by rollers into heated nozzle which partially melts the wire. As the nozzle moves in a pre-decided path in X, Y and D directions, the thermoplastic layers are deposited on table. This layer by layer deposition of plastic material created an actual product in extremely small duration  depending upon size and shape. The overhanging parts are made of support material which can be simply cleaned after manufacturing . DespiteChohan et al.  exploredTakagishi and Umezu  studied To overcome such problems, an innovative experimental set-up has been developed for surface improvement of FDM components through hot air mixed with acetone vapours . Although experiments have been performed using in-house developed apparatus , the impA vapour finishing test-rig have designed and fabricated which has provision to alter temperature of chemical fumes. Moreover, the innovative apparatus has been designed to avoid wastage of chemical vapours during loading and unloading of FDM Parts, as shown in For present experimentation total twenty-seven parts are fabricated to carry out nine experiments taking three repetitions for respectively trials to evade random error. The specimens are fabricated at 0°, 45° and 90° orientation angle as previous literature revels that these settings have maximum impact on surface finish and tensile strength. After fabrication, the printed specimens are taken out of print bed carefully and treated in ultrasonic bath for cleaning and removal of support structure.The average surface roughness of FDM parts was calculated through Profile Measurement Equipment “SJ-210” . The average-roughness (Ra) is specifically examined for current investigation and observations were taken normal to deposition direction. The measurements were recorded using stylus tip angle of 60 °C tip and radius 2μm moving at 0.75 mN. The Gaussian filter was used for filtering the surface roughness profiles at cut off length 0.025 cm and 0.25 cm transverse length as per ISO 4287 . The surThe mass of samples has been calculated before and after finishing using Citizen digital weighing apparatus  having readability upto 0.0001 g and maximum limit of 220 g. During weight and roughness measurements, three readings were recorded, each pre- and post-finishing, and the average of three readings was taken as the final value. Moreover, surface-roughness was deliberately evaluated at the same location before, and after, the finishing process to avoid random errors. For Taguchi analysis, percentage-variation in weight and surface-roughness are used as response parameters and calculated as following Equation (1):After finishing process, the peak tensile strength of parts was measured using Universal Tensile Tester  with 500 mm/min cross head movement speed as per ASTM 638-14 standards  as shownFor percentage-variation in surface-roughness and tensile strength, the “larger is better” characteristic is required and thus, Signal to Noise Ratio (SN Ratio) is computed  using EqWhereas, the “smaller is better” characteristic is required for percentage change in weight and SN Ratio is calculated in Equation (3):In above equation, n represents number of replicated experiments while i is response which may vary from 1 to n.The micrographs were obtained to ascertain the manifestations occurring at micro scale on the surface and in cross-section of FDM parts. The micrographs are retrieved using Scanning Electron Microscope (SEM) apparatus . Tensile strength determines the maximum stress that a material can withstand without breaking. Hence, the images of failure point are taken at high vacuum mode, 10 KV accelerating voltage, 12 mm. working distance and double sided carbon tape. Initially, the site of the break point, five millimeters of the cross section was cut and subjected to gold coating to make it conductive. The coated parts were then placed in a vacuum chamber where the scanning of the parts at different magnification takes place.The Taguchi L9 orthogonal-array along with Signal to Noise (SN) Ratio of leading and trailing observations of surface-roughness, tensile strength and weight has been calculated. It can be clearly noticed that the percentage-variation in average surface roughness (%∆Ra) varies from 64.12% to 87.59% after vapor finishing. The maximum contribution (84.88%) of finishing temperature has been observed at 95% confidence level. The higher temperature ensures effective meltdown of upper layers of ABS polymer which intensified the surface enhancement phenomenon after parts are cooled. On the other hand, orientation angle does not affect the surface roughness as vapours could not to reach inside layers of printed polymer parts. Similarly, finishing time does not influence the surface finishing phenomenon as hot vapours instantly melted the upper layers after exposure. The measurements of surface-roughness also indicated that the percentage change in surface-roughness through vapor finishing is significantly more than other mechanical finishing techniques as well as pre-processing techniques . P-values and F-values. It can be deduced from F-values and P-values of ANOVA table that orientation angle significantly affects the tensile strength at 95% confidence level. The internal polymer bead deposition angle enhances the strength of parts in certain direction due to phenomenon of anisotropy in FDM parts.The tensile strength results were retrieved and percentage contribution of each parameter has been displayed in In case of weight measurements, the average weight of FDM samples has increased even after cooling down the parts for 24 h after chemical vapor exposure. In present experimentation, the percentage gain in weight ranges from 0.14% to 0.40%, for all the experiments. On other hand, 45° orientation have least strength due to weak bonding between layers fabricated at this angle. Although peak strength achieved at 90° orientation angle is greater than 45°, but due to change in deposition strategy, it could not yield maximum strength. Furthermore, the weight of FDM samples is directly proportional to finishing time, which is quite evident because with an increase in finishing time results in a higher exposure, which resulted in more absorption of vapors inside the FDM parts which resulted in weight gain. On the other hand, finishing temperature and orientation angle have least impact on the weight gain.Although, an optimization study has been conducted to analyze the impact of individual parameter on response, there is requirement for a multi-criteria optimization, which would yield the best results for every response. The present work objects to evaluate the optimum settings of FDM and finishing processes which would equally balance all the responses. Therefore, TOPSIS tool has been used to identify the optimized parameter settings was sub sequentially proposed . As suggInitially, the values of normalized matrix for experimentation abbreviated as rij are calculated using Equation (4) given below:In second step, the weights of response parameters are multiplied with respective columns of normalized matrix. In present work, the equal importance has been given to each response parameter. The values of normalized matrix weightage normalized matrix have been shown in The positive-ideal  and negative-ideal  can be written as Equations (5), and (6), respectively, ℇThe evaluated values of positive-ideal (best) and negative-ideal (worst) results are displayed in Euclidean distance concept is utilized to evaluate the value of positive and negative separation measures. Hence, Equations (7) and (8) are used to compute the separation measures,The solutions calculated after implementing afore-mentioned equations have been shown in The ranking of alternates is done based on the relative closeness-value. The relative closeness of alternative Aj is written in Equation (9): Finally, Equation (9) has been used for calculation of relative closeness along with ranking for each experimental result has been shown in The optimization study has been exclusively conducted to ascertain the effect of combined operating parameters of FDM and vapour finishing process utilizing dedicated apparatus which creates controlled environment for vapour finishing. An apparatus has been prepared which utilizes air at variable temperature mixed with chemical vapours. TOPSIS have utilized for multi-objective optimization of process parameters which is highly effective tool used for industrial applications.The study has been conducted on dedicated vapor finishing apparatus which utilized influence of hot chemical vapors mixed with heated air for surface enhancement of ABS parts.The finishing temperature has a significant impact on surface finish, while orientation angle and finishing time were significant parameters for tensile strength and weight of FDM parts respectively.It was found that higher temperature resulted in better finish due to instant meltdown of upper plastic layers.Orientation angle of 0° led to highest value of tensile strength as layers are deposited in horizontal plane. Moreover, higher exposure duration induced permanent weight gain due to increase in absorption of vapours.Since each response parameter is impacted differently by input parameters, multi-criteria optimization tool TOPSIS has been utilized to identify optimal settings.The optimum parameter settings can be implemented to improve the surface-quality of FDM parts which can be utilized for end-use products and for rapid tooling applications.The study aims to investigate the impact of the orientation angle, finishing temperature and time on surface quality, tensile strength and weight gain of FDM parts during surface finishing process. A dedicated experimental set-up has been designed and made with provision of temperature control to investigate the impact of operating parameters on surface finish, tensile strength and weight of ABS parts. TOPSIS has been applied for multi-criteria optimization of FDM components to acquire single set of process parameters for mass production of finished FDM parts. Moreover, the optimal-set of process parameters for FDM and vapour finishing can be used for mass finishing of customized products. The study can be further broadened to evaluate the impact of process-parameters on dimensional accuracy, compressive strength and wear behaviour. Following conclusions are derived:


Highly cross-linked polyethylene (HXLPE) enhances the anti-wear characteristics of the conventional polyethylene (PE). Early failure for wear after ceramic-on-highly cross-linked polyethylene (CoHXLPE) total hip arthroplasty (THA) is extremely rare.We described the case of a 60-year-old man who underwent right CoHXLPE THA because of the developmental dysplasia hip (DDH) complained pain 32 months after this procedure. Plain radiographs showed that eccentric wear existed at the polyethylene insert. However, the patient refused surgery at that time and did not stop weight-bearing. The right hip pain continued for 7 months. Plain radiographs of the pelvis showed that the HXLPE liner was penetrated and partial inner wall of acetabular shell was worn. Acetabular cup revision was performed, and the ceramic head and HXLPE were exchanged.Difficult reduction during primary THA, especially for DDH, can result in higher abductor tension, which may lead to early eccentric wear of the prosthesis. Whenever eccentric wear of HXLPE liner was found, weight-bearing must be stopped to avoid the accelerated wear and adverse reactions to metal debris (ARMD). THA is an effective treatment for various end-stage hip diseases, such as osteonecrosis, rheumatoid arthritis, degenerative joint diseases and degenerative diseases caused by developmental dysplasia . NowdaysWear debris production from bearing surfaces is regarded as the main factor limiting THA survival. In order to improve the anti-wear characteristics of the conventional PE, HXLPE has been developed to reduce the amount of wear particle production. These improved PEs have shown better wear characteristics in vitro and in vivo . AlthougA 60-year-old man diagnosed with developmental dysplasia of the right hip Fig. a underweInfection was excluded before revision, as the white blood cell count, C- reactive protein level and erythrocyte sedimentation rate were negative. Metal ion concentrations in the blood were not measured because this was not covered by his health insurance. 3D computed tomography reconstruction revealed numerous metal debris around hip joint, and the worn between ceramic head and the acetabular shell Fig. a. SuperoThe patient was permitted partial weight-bearing 3 days after surgery, and fully weight-bearing after 4 weeks. The muscle strength recovered 4 weeks after operation. The anteversion angle, abduction angle were all within normal range. Plain radiographs of the pelvis showed ideal size and position of the prosthesis Fig. c. Pain wHXLPE was developed to reduce wear in conventional PE liners. Meanwhile, the incidence and severity of osteolysis secondary to wear would also be decreased . MutimerThe wear of HXLPE after THA is multifactorial, such as body weight, high activity level, large abduction angle of the cup and decrease of femoral offset –10. WhatWith the image test, the early wear of the prosthesis can be found. Wear debris production may lead to ARMD, which will worsen the fibrosis and necrosis of soft tissue, the erosion of metal and the loosening of prosthesis . Thus, oRevision THA is the definitive treatment for the wear of PE . HoweverDifficult reduction during primary THA, especially for DDH, can result in higher abductor tension, which may lead to early eccentric wear of the prosthesis. Whenever eccentric wear of HXLPE liner was found, weight-bearing must be stopped to avoid the accelerated wear and ARMD. Partial or total revision THA is the primary treatment for such wear of HXLPE. A careful assessment of preoperative radiographs and intraoperative testing are imperative to avoid subsequent dislocation and loosening.


Genomic structural variations, previously considered rare events, are widely recognized as a major source of inter-individual variability and hence, a major hurdle in optimum patient stratification and disease management. Herein, we focus on large complex germline structural variations and present challenges towards target treatment via the synergy of state-of-the-art approaches and information technology tools. A complex structural variation detection remains challenging, as there is no gold standard for identifying such genomic variations with long reads, especially when the chromosomal rearrangement in question is a few Mb in length. A clinical case with a large complex chromosomal rearrangement serves as a paradigm. We feel that functional validation and data interpretation are of outmost importance for information growth to be translated into knowledge growth and hence, new working practices are highlighted. Multi-allelic copy number variations (CNV) and duplications (median length larger than 10 kb) are prominent In the clinic, such hurdles need to be overcome. Indeed, quality of diagnosis for rare complex structural rearrangements would be remarkably improved, if exact breakpoints could be detected with base-pair-resolution. Further, accurate breakpoint mapping, gene identification with high accuracy, precision, and robustness for those being rearranged may empower clinical diagnosis. A clear insight into the pathogenesis of the genomic landscape sheds light into the molecular mechanisms of the genetic rearrangement in question.A clinical phenotype of severe developmental delay (DD), possibly indicating a nested or large SV, may serve as a paradigm. For someone to explore the molecular mechanisms that generated such a SV, a multi-step approach is presented that consists of cytogenetic pre-screening, next generation sequencing (NGS) of a region of interest, followed by clinical phenotype interpretation and conformational SV analysis. Cytogenetic approaches  allow for low resolution genome screening. Notwithstanding, insertions and deletions can also be detected by CNV analysis (short-read sequencing). Next, NGS enables the in-depth characterization for the genome regions of interest. Due to a high number of false positive variant calls, emphasis may be put on the SVs that are validated by cytogenetics. Using NGS, breakpoints can be detected along with the genes or their parts involved in the rearrangement of interest. The latter may be validated further by Sanger sequencing and/or long-range PCR coupled by NGS. Functional studies, although at their infancy, may validate datasets and hypotheses and enable clinical insights.Herein, we build on the principles and strategies of clinical cytogenetics and present encountered challenges in the identification of large germline structural variants. Long read sequencing technologies hold promise as a theranostics roadmap and for this, a specific technical aspect of a clinical case with known complex structural rearrangement was selected for the demonstration. State-of-the-art methodologies were employed and integrated to allow for high diagnostic accuracy. To this end, the added value of multi-omics and 3D cell co-cultures is a potenital path towards better-informed decision-making in the clinic and clinically relevant biomarkers.2in situ hybridisation (FISH) and comparative genomic hybridisation (CGH) methods to interrogate the structural phenomena of the human genome In the last 62 years, since the identification of the exact chromosome number in a diploid human cell by Tjio and Levan in 1956 per se, except for the aforementioned methodological aspects.Chromosome G-banding, historically, has been the most widely adopted chromosome banding and staining technique, based on the partial trypsin digestion of the chromosomal protein scaffold followed by Giemsa staining of fixed metaphases In 1985, Landegent and colleagues mapped the first single-copy human gene to a specific genomic location using FISH In brief, when performing a FISH experiment, multiple specific chromophore labeled oligonucleotide probes, complementary to the region of interest (ROI), are applied to the fixed metaphase slides. During the hybridisation process, which involves partial DNA denaturation and renaturation, the probes attach to their specific location along the ROI. After the removal of excessive non-bound and poorly bound probes and addition of a counter-stain to visualise chromosomes and/or nuclei, the ROI are usually visualized as coloured dots on the counter-stained chromosomes or interphase nuclei by a fluorescent microscope system. Upon analysis, depending on the number of ROI copies present in the chromosomes studied, there may be single, double or multiple signals detected in the metaphase (or interphase) nuclei. Overall, FISH is a relatively straightforward method, when interrogating relatively simple structural rearrangements using up to three different probes. Challenges arise when mapping complex chromosomal rearrangements utilising multiple probes is desired, accompanied by technical and financial burdens, as expensive equipment  and technical skills become indispensable. It should be also noted that there may be a profound crosstalk among multiple probes, as their emission spectra may be too close to each other and hence, available filters cannot eliminate such non-specific signals in-situ on a glass slide For larger chromosomal CNV (deletions and insertions) screening, CGH (comparative genomic hybridization), which is also known as metaphase CGH, was the first method employed. CGH is based on the comparison of a fluorescence-labeled control vs. the metaphase chromosomes of a sample, hybridized on glass slides and analyzed by fluorescence microscopy 3Oxford Nanopore Technologies (ONT) has introduced nanopore DNA sequencing Long read sequencing has the potential to capture clinically important large genomic structural rearrangements as well as repetitive sequences and single nucleotide variants, overcoming the limitations of NGS short reads, which produce reads spanning 50–600 bp, as the detection of SVs from short read data often suffers from low sensitivity (30–70%) and high false discovery rate (up to 85%) de novo assembly followed by reference-based assembly alignment  mapper or the Minimap2 aligner, followed by Sniffles or SVIM variant callers NGMLR was designed to quickly and correctly align the reads of interest, including those spanning (complex) SVs. NGMLR uses the convex gap-cost scoring model to accurately align long reads across small indels that commonly occur as sequencing errors. Moreover, larger and complex SVs are captured through spot-read alignments Within PacBio reads, large SVs can be identified by PBHoney or SMRT-SV, too. PBHoney comprises two variant identifications approaches; a) PBHoney-Spots considers intra-read discordance by a subsequent increase or decrease in error along the reference sequence and b) PBHoney-Tails identifies structural variants by realigning soft-clipped tails of long reads  to the reference genome in silico by merging two haploids, have missed more than a half (∼59%) of the heterozygous SVs Even though read aligners as Minimap2 and NGMLR explicitly take large SVs > 50 bp into account, it is still not clear how these aligners and variant callers are detecting complex variants spanning few Mbp lengths with multiple structural rearrangement events. SV detection and identification is challenging using current analytical approaches, especially when SVs are longer than the average read length. For SVs that were identified using Sniffles after NGLMR alignment, the SV validation status (per length of SVs) failed to detect any true variants spanning over 7.5 kb Simple large chromosomal rearrangements, like multi-locus deletions, are easy to be determined. 3.2de novo assembly can also identify the structure of nested SVs. In such case, Canu, wtbg2, and FALCON are frequently used tools for the de novo assembly of long reads. Canu and wtdbg2 can assemble long nosy reads produced by ONT and PacBio sequencing, while FALCON can assemble PacBio reads only. Wtbg2 is using the fuzzy Bruijn graph approach when assembling the human genome, which has a great advantage of being tens of times faster than Canu and FALCON, while producing contigs of comparable base accuracy To complement reference-based alignment with variant calling, de novo genome assembly aligned to a reference genome. Assemblytics can identify all the insertions and deletions from 1b up to a maximum 10 kb in size. The maximum limit is defined by the minimum amount of the unique contig sequence anchor, contained in no other alignments of that contig. In that way, it prevents translocations and complex variants from being interpreted as indels After having a consensus sequence, the next step is to align it to the reference genome and investigate, if the structure of the rearrangement(s) in question can be assessed. Genome sequence aligner nucmer (part of the MUMmer system) has been widely applied to align whole genome sequences, compare different assemblies of the same genome and align reads to the reference, even though it is less sensitive and accurate than the dedicated read aligners 4When thinking of important technological advances for the discovery and identification of SVs, BioNano optical mapping, 10x Genomics or chromatin conformation capture (Hi-C) crosslinking protocols should not be overlooked .Table 2Lde novo genome assemblies BioNano Genomics combines long-read technology with low resolution sequencing. Enzymes nick and fluorescently label specific sequences within DNA fragments that are up to ∼1 Mb long. Then, fragments are assembled and/or aligned to the reference genome to map the locations of the probes in question. This approach can identify SVs that span up to tens of kb, however it does not provide a nucleotide-level resolution. For detecting the precise structure of genomic rearrangements, BioNano optical mapping can serve as a good companion to NGS technologies by providing a long-range scaffold to A multi-platform comparison between BioNano optical mappings, Illumina short read sequencing and PacBio long read sequencing revealed that insertions and deletions between 10 kb and 1 Mb are most accurately detected by BioNano optical mapping. Insertions between 1 kb and 5 kb can be detected by BioNano, PacBio as well as their synergy, whereas deletions can be identified either with short-reads or long-reads as well as by BioNano optical mapping. Additionally, median size insertions (between 50 b and 1 kb) are mostly detected by PacBio, while some deletions can be detected only with Illumina short reads. Large inversions (>50 kb) were detected only by single-cell/single-strand genome sequencing de novo assembly and hence, SV structures, as they are both coupled with short read sequencing to provide base-pair-resolution. The chromium technology from 10x Genomics enables identification determination of a diploid genome sequence at high resolution. It does so, by partitioning large DNA fragments into micelles, which typically contain < 0.3x copies of the genome and one unique barcode. In each micelle, smaller fragments are amplified and barcoded, afterwards the pooled DNA undergoes a standard library preparation and sequencing. The reads are aligned and linked together to form a series of anchored fragments, which can span up to 100 kb in length 10x Genomics or Hi-C crosslinking protocols can also determine a 5Experiencing the era of big data and technological advances, a series of wet- and dry-lab approaches hold the promise of translating information growth into knowledge growth. In this context, synergies play a pivotal role, in particular if clinical relevance and cost-effectiveness are considered; multi-omics may map inter-individual variability via holistic profiling, 3D cell (co)cultures may dissect molecular mechanisms and provide mechanistic insight, and information technologies may inform decision-making.Upon interpretation of complex SV, prominent key questions go beyond inferring their architecture, questioning their role (if any). To this end, reconstructing and visualizing such complex variant structures is not trivial, while functional predictions remain a bottleneck. Looking for sustainable and cost-effective strategies given the scale of current and forthcoming genome sequencing endeavours, one might consider the synergy of artificial and human intelligence Mechanistically minded studies aim to reconstruct the mutational events that resulted in the SV of interest, as already experienced in ancestral genome reconstruction using breakpoint graphs Karyotyping with or without FISH are considered effective ways of identifying large scale structural variation, despite their relatively low resolution .Multi-omics could be of great benefit in resolving the enigma of the functional role of SVs. A multi-omics design was employed to explore the presence of SVs in heart failure patients due to dilated cardiomyopathy, in which genomic aberrations were linked to myocardial gene expression by performing heart-specific SV-eQTL and SV-load correlations [48]3D cell co-cultures may address the challenge of heterogeneous cell mixtures with possibly different numbers of mutations. Cancer serves as a paradigm, as admixture between normal and tumor cells is present or cell subpopulations that may contain a range of SVs, including driver or drug resistance mutations. Despite single cell technologies 6Our pipeline was employed to resolve a clinical case where a large structural rearrangement was observed by G-banded karyotype , A, follTaking into account that the analysis of large complex rearrangements and high-resolution breakpoints profiling remain difficult, cytogenetic approaches do not suffice and hence, multi-step synergies of state-of-the-art genomic sequencing and mapping technologies are emerging to shed light on clinical phenotypes.Nanopore MinION technology was applied to determine the precise variant configuration of the large complex SV previously observed by G-banded karyotype, aCGH and FISH. Median read quality was 12.44, representing a 13.2x theoretical coverage of the human genome, with an average N50 read length of 10.2 kb. Currently, there is no gold standard bioinformatics approach for detecting and identifying SVs with long reads, especially when the chromosomal rearrangement in question is few Mbp in length. To identify the structural variant(s) and break points that could explain the underlying chromosomal rearrangement in the clinical case in question several computational approaches have been explored .Fig. 3ThA read depth analysis was performed , A to deIn addition to true SVs, we have also observed many large false positives SVs detected by any combination of aligner and variant caller. To our knowledge, CNV detection for long read whole genome sequencing is not yet available, thus pointing towards the need to combine long read sequencing with cytogenetic or optical mapping approaches to better define the structural rearrangement(s) and region of interest. Assembly approaches did not give us any additional valuable information, most probably because of insufficient coverage. In diagnostics, reaching a high coverage as 50x or more with Nanopore technology is still costly and requires a relatively higher amount (up to 10^3) of high molecular weight DNA samples in comparison to short read sequencing. As shown in our case study, it remains difficult to ensure a sufficient amount of DNA to acquire optimal coverage. No doubt, continuous optimization of the library preparation protocols as well as sequencing pipelines are in place, with the aim to lower required DNA input for the same data quality.7The success in the identification of genomic structural rearrangement(s) in routine clinical protocols mainly depends on the complexity and size of SVs. Short and/or simple SV are being successfully identified by cytogenetic techniques or short read sequencing, while large nested and complex rearrangements demand case-specific investigation via the application of novel emerging technologies as those presented in our clinical example. A clinical phenotype of unexplained severe DD or DD with multiple embedded or associated gain/loss genomic events identified by aCGH may be indicative for long-read sequencing application, accompanied by the presented bioinformatics approaches. The identification of the exact composition of the underlying structural rearrangement may improve treatment and prognosis counselling as well as potential future family planning. Such novel technologies will be of great benefit when standardized and validated analytical protocols become widely available. There is still a missing gap in guidelines and standards for identifying the detailed composition of large structural rearrangements. When facing a rare few Mbp in size nested SV it is difficult to decide which approach to use to provide the most suitable diagnosis to the patient. Long read sequencing carries a huge potential to become the routinely used technology for identifying large structural rearrangements in clinical diagnostics, yet several challenges need to be resolved, among others, increasing the average length of the reads ideally encompassing the whole region of the rearrangements of interest. Of note, the technology should be cost-effective, to be of benefit to a health care. To set a benchmark system, herein, we performed cytogenetic screening with low resolution, first, to select cases where long read sequencing would be of benefit. Notwithstanding, relatively high error rates are still a bottleneck in genetic testing by long reads.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


A.PCa belongs to the cancer types with the highest incidence worldwide. Corresponding economic burden is enormous. Moreover, the costs of treating PCa are currently increasing more quickly than those of any other cancer. Implementing individualised patient profiles and adapted treatment algorithms would make currently too heterogeneous landscape of PCa treatment costs more transparent providing clear “road map” for the cost saving.B.PCa is a systemic multi-factorial disease. Consequently, predictive diagnostics by liquid biopsy analysis is instrumental for the disease prediction, targeted prevention and curative treatments at early stages.C.The incidence of metastasising PCa is rapidly increasing particularly in younger populations. Exemplified by trends observed in the USA, prognosis is that the annual burden will increase by over 40% in 2025. To this end, one of the evident deficits is the reactive character of medical services currently provided to populations. Innovative screening programmes might be useful to identify persons in suboptimal health conditions before the clinical onset of metastasising PCa. Strong predisposition to systemic hypoxic conditions and ischemic lesions  and low-grade inflammation might be indicative for specific phenotyping and genotyping in metastasising PCa screening and disease management. Predictive liquid biopsy tests for CTC enumeration and their molecular characterisation are considered to be useful for secondary prevention of metastatic disease in PCa patients.D.Particular rapidly increasing PCa incidence rates are characteristic for adolescents and young adults aged 15–40 years. Patients with early onset prostate cancer pose unique challenges; multi-factorial risks for these trends are proposed. Consequently, multi-level diagnostics including phenotyping and multi-omics are considered to be the most appropriate tool for the risk assessment, prediction and prognosis. Accumulating evidence suggests that early onset prostate cancer is a distinct phenotype from both aetiological and clinical perspectives deserving particular attention from view point of 3P medical approaches.In the early twenty-first century, societies around the world are facing the paradoxal epidemic development of PCa as a non-communicable disease. PCa is the most frequently diagnosed cancer for men in several countries such as the USA. Permanently improving diagnostics and treatments in the PCa management causes an impressive divergence between, on one hand, permanently increasing numbers of diagnosed PCa cases and, on the other hand, stable or even slightly decreasing mortality rates. Still, aspects listed below are waiting for innovate solutions in the context of predictive approaches, targeted prevention and personalisation of medical care (PPPM / 3PM). The prostate cancer (PCa) management was for a long-time preoccupied by quite conservative beliefs which can be shortly summarised as follows: PCa is a life non-threatened disease of organ-specific aetiology (rather than systemic) being characteristic for elderly and, therefore, could be easily detected by PSA screening applied to ageing male population followed by radical prostatectomy as an optimal approach to treat PCa.Consequently, in the early twenty-first century, societies around the world are facing the paradoxal epidemic development of PCa as a non-communicable disease Fig.  1]. PCa. PCa1]. PCa is the second most common cause of cancer death in men after lung cancer  and the Similarly to already recognised and currently extensively discussed epidemic developments of other non-communicable diseases such as type 2 diabetes  and breaBody Mass Index (BMI) and PCa-related mortality. A population-based cohort study of 3.6 million adults has been recently performed in the UK analysing association of BMI with overall and cause-specific mortality [PCa is a multi-factorial disease resulting from imbalanced interplay between exogenous and endogenous risks against protective factors. The overall picture is extremely comprehensive that perhaps can be illustrated by the complex association between the ortality . NoteworThe incidence of PCa is linked to the human development index (HDI). Its age-standardised incidence rate is the highest for high-HDI countries , while the age-standardised mortality rate is the highest in low-HDI countries (Middle Africa) . FurtherA family history of PCa is a well-established risk factor of PCa . The finUnderstanding determinants of ethnic and racial disparities in treatment, mortality, health resource utilisation and associated costs is crucial for developing effective healthcare policies and improving quality of care for PCa patients . Recent Progressing age is a well acknowledged non-modifiable risk factor of increasing PCa incidence at global scale. This trend is well documented by long-term statistics also in the Czech Republic as demonstrated in Fig. Consequently, factors contributing to accelerated ageing may further increase PCa risks. Amongst them are both non-modifiable (genetic) factors such as ageing-relevant DNA mutations  as well Immunosenescence plays a central role in many tumours including PCa . Chronicversus inhibiting effects.Unhealthy lifestyle such as smoking, excessive alcohol consumption, sleep deprivation etc., further, in combination with inappropriate dietary habits demonstrate adverse health effects leading to cancer development and progression. In contrast, some specific dietary patterns may have protective function. Below summarised facts provide clear indication for both PCa promoting The intrauterine microenvironment plays a pivotal role in physiologic embryonal development including prostate development, growth and potential predisposition to the PCa later on in life . Using aIt is generally known that a environmental pollution has a negative impact on human health. Chlordecone (CLD) is one of environmental pollutants linked to the PCa development . AnotherFurther environmental pollutants impacting the prostate health are pesticides. An Agricultural Health Study (AHS) in the USA revealed strong association between the utilisation of insecticides and/or herbicides and aggressive type of PCa incidence (GS ≥ 7), when the period of exposure was longer than 4 years .Further, heavy metals have been demonstrated to increase PCa risks. Detailed investigations have been performed for cadmium (Cd) which is considered as one of the main pollutants in economically developed countries contributing to the development of many types of cancer, including PCa . ConsideFinally, specific professional occupation linked to the application of toxic chemicals as well as abnormal physical and emotional strain, such as in the case of firefighting professionals, both have been demonstrated to predispose the exposed individuals most frequently to the gastric carcinoma and PCa .Excessive release of reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been identified as initiators, mediators and regulators of cellular oxidative stress (OxiS). OxiS can damage almost all biomolecules including chromosomal and mitochondrial DNA . Recent Nitric oxide (NO) is a signalling molecule that plays an important role in both - physiologic processes and cancer promotion. Regarding the latter, it has been suggested that low levels of NO are cancer promoting, while high levels of NO are protective against cancer. Both ROS and RNS can be carcinogenic by modifying the inflammatory status as well as influencing cellular lipid structures, angiogenesis and anti-apoptotic pathways, amongst others. For example, low concentrations of NO can result in the redox imbalance, increased inflammation and damage to sub-cellular components accelerating neoplastic process. NO production modifies the sensitivity of AR; in turn this receptor becomes sensitised to lower levels of androgens in the microenvironment of the prostate. Those effects are currently considered for targeted therapies in PCa management .Adverse health effects of smoking should be considered in the context of both metabolic changes and detoxification processes . ConsequPatients with sleep disorders (SDs) have increased primary PCa risk: the adjusted hazard ratio corresponds to 1.42-times higher risk and specifically in the group aged ≥ 65 years to 1.35-times higher risk . FurtherSexual activity is a factor which may play a role in the risk of prostate cancer; however, the exact mechanism is unknown . FurtherHormonal regulatory axis affects nuclear receptors and transcriptional regulators, thereby changing the gene expression important for tumour development, progression and metastasising . AndrogeInsulin-like growth factor 1 (IGF-1) plays a crucial role in PCa pathophysiology . An elevVasectomy is an intervention commonly practised in the USA and Europe. Although some studies have associated vasectomy with potential PCa risks , clear pUrinary tract infections (UTI) such as cystitis and urethritis are associated with a primary PCa risk. The primary risk of PCa increases with recurrent low urinary tract infection (LUTI) linked to chronic inflammation , 74.Understanding the relationship between the urinary microbiome set-up and prostatic chronic inflammation is crucial in developing PCa preventive strategies. Chronic inflammation initiated by microbial species persisting in the urinary tract promotes prostate inflammatory atrophy which may result in the PCa development . In currFurther, low-grade inflammation could be associated with the presence of more aggressive forms of PCa . Non-bacMetabolic syndrome (MetS) is associated with increased primary PCa risk but not with GS value. Higher circulating CRP levels were positively correlated with higher GS and secondary PCa risk . FurtherPatients with chronic obstructive pulmonary disease (COPD) have 1.62-times increased primary risk of PCa development compared with non-COPD patients. Both the systemic hypoxic condition and systemic inflammation associated with COPD have been proposed as promoting PCa progression and secondary risks. Treatments by short-acting muscarinic antagonists (SAMAs) and short-acting beta-agonists (SABAs) increase the risk of primary PCa. In contrast, primary risk of PCa decreases, when statins are implemented to COPD patients . IncreasAbnormal BMI affects overall risks and mortality rates in a large spectrum of pathologies including several cancer types . InformaDoubtless obesity negatively impacts microcirculation and sex steroid hormones, therefore, influencing the aggressiveness of PCa ; data fr2) as well as in the group with low BMI (<22.5 kg/m2) compared with the reference group (BMI 22.5–25 kg/m2) [2 was associated with better overall survival amongst PCa patients compared to those with BMI < 25 kg/m2 [On the other hand, a study focused on PCa-specific mortality and overall mortality in relation to BMI demonstrated statistically significant risk of PCa-specific mortality and overall mortality in the group with high BMI (≥27.5 kg/m5 kg/m2) . However25 kg/m2 . ObviousExtensive alcohol consumption may increase risks of PCa development . ResearcAnimal fat consumption is positively associated with PCa incidence and mortality . High-enThere is a controversial presentation of potential association between eggs consumption and PCa risks in the literature. On the one hand, several studies have associated high choline intake with an increased risk of the disease development , aggressThe World Health Organisation (WHO) classified processed meat as “carcinogenic to humans” and red meat as “potentially carcinogenic to humans” referring to the evidence-based association between an extensive red meat consumption and increased risk of advanced PCa . While sInborn genetic predisposition is characteristic for minor portion of cancer cases. In contrast, modifiable risk factors contribute to the development of the majority of cancers. Contextually, general and targeted prevention is in focus of the cost-effective approach by 3P medicine.Vigorous physical activity is defined as activity that causes sweating, increased heart and respiratory rates. Typically, vigorous activities like jogging, biking, swimming, and bicycling are considered . A comprHigh fish intake, especially containing high levels of omega-3 fatty acids, is associated with decreased incidence , progresEpidemiological studies report plant-based foods including cruciferous vegetables, tomatoes, garlic, pomegranate and green tea as associated with a significant reduction in the progression of PCa , 122. ReTraditional garlic consumption is highly protective against the primary PCa risk. Accordingly to currently provided recommendations, the minimal daily dose of garlic intake is 10 g .Grape powder extract (GPE) contains resveratrol or its natural analogue—pterostilbene. Resveratrol and pterostilbene are stilbenes involved in numerous biochemical and molecular pathways preventing PCa progression and metastatic disease . In vitrPomegranate peel extract, black berries, wild strawberries and raspberries are rich on EA which is effectively protecting against PCa . EA showBroccoli and its active compound SFN demonstrate highly protective properties useful for targeted prevention against primary PCa risks and possibly for secondary risks mitigation . Other cRed onions, white onions, green hot peppers, elderberries cranberries are rich on QCT contents. Similarly to SFN, QCT demonstrates highly protective properties useful for targeted prevention against primary PCa risks and possibly for secondary risks mitigation . QCT is The incidence of PCa recorded in East-Asian countries is significantly lower compared with Western countries that has been associated with a traditionally abundant consumption of green tea there. Green tea is rich on catechin  demonstrating chemopreventive effects against PCa development and metastatic disease in experimental models –139.A series of studies reported a strong association between regular coffee consumption and significantly reduced risks of the PCa development, progression, recurrence and related mortality –142. NotCurcumin slows down proliferation and induces apoptosis in the PCa cells. Further, curcumin demonstrates strong anti-angiogenic properties and downregulates AR expression. Its regular consumption as a dietary supplement is highly recommended .Gut microbiota have been demonstrated as a strong contributor to tumorigenesis and may, further, influence the tumour environment . SpecifiVitamins A, C, D, E, K and folate have been demonstrated as potentially affecting PCa pathogenesis and progression , 147–152In the early twenty-first century, societies around the world are facing the paradoxal epidemic development of PCa as a non-communicable disease: PCa is the most frequently diagnosed cancer for men in several countries such as the USA . PermanePCa patients are benefiting a lot from personalisation of medical services: the general approach by the radical castration has been revised for several subtypes of PCa, since keeping urinary and sexual functions intact allows for significantly higher quality of life for many PCa patients without diminishing the survival rates .Three pillars are currently involved in the standard PCa diagnostics: prostate biomarker panel, medical imaging and tissue biopsy . Current18F-methylcholine and 68Ga-PSMA-11 positron emission tomography (PET)/MRI [Comprehensive medical imaging is utilised in the overall PCa management: transrectal ultrasound (TRUS), multi-parametric magnetic resonance imaging (mpMRI) to evaluate local staging and hybrid imaging methods: PET)/MRI , 168.Main indication for initiating PCa treatment is the histological analysis of the biopsy. The TRUS-navigated biopsy is used as a basic procedure. The second variant is the cognitive biopsy in which the result of the imaging technique, most often mpMRI, is known. Currently, the preferred procedure is the fusion biopsy, where mpMRI and TRUS images are merged by software. MRI information is used to guide biopsy cores to suspicious areas within the prostate. PET/TRUS and PET/MRI/TRUS fusions have also been tested . Based oA.PCa belongs to the cancer types with highest incidenceStill, several aspects are waiting for innovate solutions, which obviously can be provided only if the paradigm shift from reactive to predictive, preventive and personalised medicine will get implemented to the overall PCa management as detailed below.The PCa incidence is annually increasing. Corresponding economic burden is enormous . Being tB.PCa is a systemic multi-factorial diseasePCa is a systemic multi-factorial disease. Consequently, predictive diagnostics by liquid biopsy analysis is instrumental for the disease prediction, prevention and curative treatments at early stages. Further, the absolute majority of PCa patients suffer from comorbid conditions as demonstrated for oncologic patients in general . To thisC.The incidence of metastasising PCa is rapidly increasingExemplified by trends observed in the USA, metastatic PCa began to increase by 0.58% per year after 2008 and accelerated to 2.74% annually following the 2012. The pattern was magnified amongst men aged below 69 years. Prognosis in 2025 is that men aged 45–54 years might demonstrate more rapid annual increase by 2.29% versus men aged 55–69 years by 1.53% per year reaching the annual burden increase of 42% by 2025 . To thisD.Rapidly increasing incidence rates are characteristic for adolescents and young adults aged 15–40 years Multi-factorial risks for these trends have been proposed. Consequently, multi-level diagnostics including phenotyping and multi-omics is considered to be the most appropriate tool for risk assessment, prediction, targeted prevention and prognosis . To this


The direct delivery of central nervous system (CNS) drugs into the brain after administration is an ideal concept due to its effectiveness and non-toxicity. However, the blood–brain barrier (BBB) prevents drugs from penetrating the capillary endothelial cells, blocking their entry into the brain. Thus, alternative approaches must be developed. The nasal cavity directly leads from the olfactory epithelium to the brain through the cribriform plate of the skull bone. Nose-to-brain drug delivery could solve the BBB-related repulsion problem. Recently, it has been revealed that insulin improved Alzheimer’s disease (AD)-related dementia. Several ongoing AD clinical trials investigate the use of intranasal insulin delivery. Related to the real trajectory, intranasal labeled-insulins demonstrated distribution into the brain not only along the olfactory nerve but also the trigeminal nerve. Nonetheless, intranasally administered insulin was delivered into the brain. Therefore, insulin conjugates with covalent or non-covalent cargos, such as AD or other CNS drugs, could potentially contribute to a promising strategy to cure CNS-related diseases. In this review, I will introduce the CNS drug delivery approach into the brain using nanodelivery strategies for insulin through transcellular routes based on receptor-mediated transcytosis or through paracellular routes based on escaping the tight junction at the olfactory epithelium. The blood–brain barrier (BBB) pharmacokinetically blocks the entry of therapeutic agents into the brain. This impermeability represents a serious problem in drug discovery or the development of efficient treatments for central nervous system (CNS)-related diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) . TherefoDrug delivery across the cell membrane attracts significant attention in various biomedical fields, including medicinal chemistry. Furthermore, nanoparticles based on nanodelivery strategies have been extraordinarily used for membrane permeation. I have already described carrier-mediated transport methods based on tissue-specifically expressed transporters using arbitrary substrate–cargo conjugates , molecul2) could be divided into three regions: the vestibular (approximately 0.6 cm2), olfactory (approximately 10 cm2), and respiratory (approximately 130 cm2) regions. In the olfactory region, the olfactory epithelium, covered with mucosa, is composed of olfactory sensory neurons, supporting cells, basal cells, and Bowman’s glands that secrete mucin, including sialic acid and sulfate. The lamina propria between the olfactory epithelium and cribriform plate contains blood vessels and lymphatic vessels . These bindings trigger neuronal activation, and the resulting odor signal impulses are electrically transmitted to the glomeruli along the olfactory sensory neuronal axons that penetrate through the cribriform plate of the skull bone into the brain. Within the glomeruli in the olfactory bulb, the information is transmitted through synaptic connectivity, using glutamates as major neurotransmitters , to mitr vessels  6,10]. ,10. 2) cIntriguingly, it is known that viruses, such as influenza, enter into the brain transcellularly through or extracellularly along the olfactory nerve that is exposed to the outer environment and is directly connected to the CNS, although the transport mechanisms are not precisely understood. Influenza virus penetration into the brain slightly differed after intranasal inoculation in an in vivo assay using ferrets based on isolation detection or immunohistochemistry, depending on the virus species or strains . MoreoveIntranasal administration-mediated drug delivery into the brain attracts considerable attention. This CNS drug administration strategy is an alternative method to cross the BBB and shows good bioavailability due to the considerable transport processes through the olfactory epithelium, rapid distribution to the CNS, and avoidance of first-pass metabolism in the liver ,6. SeverWhen intranasally administered drugs are not absorbed through the olfactory epithelium, they move to the respiratory epithelium that forms the nasal cavity, subsequently to the pharynx, and eventually to the stomach full of gastric acid, along with nasal mucosa, due to mucus secretion-based clearance from the submucosal glands and the olfactory cilium stroke that beats appropriate 1000 times every minute . As one There are two routes for substances to cross the epithelium: the transcellular and the paracellular pathway . The traAnother transportation route is the paracellular pathway through the extracellular route between epithelial cells. Epithelial cells form a cell sheet structure-based fluid compartment, control substance transport, and maintain the extracellular environment inside the epithelium. Therefore, epithelial cells tightly adhere to each other at the apicolateral borders to prevent material entry using size- and charge-selective pore filters. Tight junction components between adjacent cell membranes form a such barrier and are composed of transmembrane claudins and occludins, as well as cytosolic zonula occludens (ZO) proteins connecting such transmembrane proteins and actin filaments. In general, the pore diameters enabling paracellular flux range between approximately 0.5 and 1 nm. Permeation enhancers could open tight junction pores in the olfactory epithelium up to approximately 15 nm . Around The current substance transportation network in the brain is suggested to comprise micro drainage such as perineuronal space , perivasN-methylated cyclic peptide composed of 11 amino acids (1203 Da) and is produced by non-ribosomal peptide synthetases  and subsequently exocytosed to the brain . In. In76].  B-helix . 1Gly, 5 binding . Insulinsomerase , insulinIt has been known for long that a direct connection exists between the nose and the brain through the cribriform plate. Thus, direct nose-to-brain drug delivery is a promising potential strategy to ameliorate CNS diseases such as AD and PD. However, despite the expectation, intranasal administration to the brain depends not only on the olfactory nerve route but also on the respiratory route, which is probably due to the narrowness of the human olfactory region in the nose cavity, location at the top of the nasal cavity, and mucociliary clearance. This fact has to be accepted in the discovery and development of drugs delivered through the nose-to-brain route. Intranasally administered insulin was delivered to the brain through both routes . Nonethe


Rising incidence of extended- spectrum beta-lactamase (ESBL) induced urinary tract infections (UTIs) is an increasing concern worldwide. Thus, it is of paramount importance to investigate novel approaches that can facilitate the identification and guide empiric antibiotic therapy in such episodes. The study aimed to evaluate the usability of antecedent ESBL-positive urine culture to predict the pathogenic identity of future ones. Moreover, the study evaluated the accuracy of selected empiric therapy in index episodes. This was a retrospective study that included 693 cases with paired UTI episodes, linked to two separate hospital admissions within 12 month-period, and a conditional previous ESBL positive episode. Pertinent information was obtained by reviewing patients’ medical records and computerized laboratory results. Multivariate analysis showed that shorter interval between index and previous episodes was significantly associated with increased chance of ESBL-positive results in current culture . Additionally, cases with ESBL-positive results in current culture were more likely to have underlying urological/surgical condition . Investigations of the accuracy of current empirical therapy revealed that male patients were less accurately treated compared to female patients . Furthermore, surgical patients were treated less accurately compared to those treated in internal ward . Selecting an agent concordant with previous microbiologic data significantly increased the accuracy of ESBL-UTIs therapy (p<0.001). A quick survey of the previous ESBL urine culture results can guide practitioners in the selection of empiric therapy for the pending current culture and thus improve treatment accuracy. Escherichia coli (E. coli), and Klebsiella pneumoniae (K. pneumoniae) are usually the most commonly implicated species , while categorical variables were presented as numbers and percentages. Univariate analysis was conducted using the Mann-Whitney test for continuous variables and Chi-square (χ2) test for categorical variables. In order to determine factors (predictors) that were independently associated with urine culture identity results and accuracy of empiric therapy, multivariate analysis using binary logistic regression was performed including all variables with p<0.25 on univariate analysis. Odds ratios (OR) and their 95% confidence intervals (95% CI) were calculated. Statistical significance was set at p value less than 0.05. Statistical Package for Social Sciences  version 23 was used.E. coli was the most common causative uropathogen in previous culture (82.3%) and the results of more than half of index urine cultures were ESBL-positive (58.6%). Importantly, 61% of the empirical therapy was concordant with prior microbiological data. A detailed description of demographic and clinical data is presented in Out of the 2394 screened eligible hospitalized patients with at least one ESBL-positive UTI episode, 1424 cases were excluded since they failed to meet the inclusion criteria. During the study period, a total of 970 cases with paired urine cultures; and a conditional initial ESBL-positive UTI episode were identified from hospital database. Of those, 277 cases were excluded because there was either less than 14 days (110/277) or more than 12 months (167/277) between the two episodes, leaving 693 cases that were included in the final analysis. A detailed summary of the identification and inclusion process of cases is presented in As shown in To investigate the accuracy of the current empirical therapy, we included cases with ESBL-positive culture whom received empirical antibiotic therapy in the analysis. Out of 406 episodes with current ESBL-positive culture, 121 cases were excluded due to one of either two reasons: (i) 4 cases did not have susceptibility data for the given empirical therapy and (ii) 117 cases were admitted to the hospital and given definitive therapy  as the results of urine culture were known before the admission (taken as outpatient). Of the remaining 285 cases included in the analysis of accuracy, the empirical therapy was accurate in 71.9%. When the empirical therapy was concordant with prior microbiological data, the rate of accuracy for treatment against the uropathogen improved from 7.3% to 92.7% (p<0.001). Importantly, all episodes (n = 190) that were given concordant therapy with previous culture were found to be accurate in the index episode (100%).As shown in methicillin-resistant Staphylococcus aureus (MRSA) to predict the subsequent MRSA bacteremia  with goal of achieving early adequate coverage. However, there are a few limitations to our study findings. Firstly, in retrospective studies, misclassification is a common concern but a clinical pharmacist in the current study had reviewed patients’ medical records for the purpose of validating exposure and outcome, rather than relying on diagnostic or billing codes. Secondly, there was no consistency in the selection of empiric antibiotics in our center, which might have impacted the clinical outcomes and study findings. Finally, our study was a single center study, and this can diminish the generalizability of the study results. A future research direction is to conduct prospective, multicenter studies to replicate our results in order to assess for potential addition as part of antibiotic stewardship program initiatives.Selecting an agent concordant with previous microbiologic data significantly increased the chance of accurate therapy for ESBL-UTIs. Clinician education is highly required to enhance the accuracy of empiric treatment of UTIs. Optimization of ESBL empiric antibiotic prescribing is a transdisciplinary approach; all members of healthcare team should be cognizant in dealing with potential ESBL-induced infections to balance the bar between both under and over prescription of broad-spectrum antibiotics. Improved ESBL treatment strategies is expected to limit cost burden on healthcare system, along with decreased mortality and morbidity rates and relieve associated pressure on hospital resources. Future prospective studies with a control arm might be helpful to confirm the results of present study. In addition, a research that focuses on test characteristics  would be useful for the physicians to make reasonable decision about the utility of a prior urine culture.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


Significance: Stimulated Raman scattering (SRS) and pump-probe microscopy are implementations of multiphoton microscopy that acquire high-resolution, label-free images of live samples encoded with molecular contrast. Most commercial multiphoton microscopes cannot access these techniques since they require sample illumination by two temporally synchronized ultrafast pulse trains. We present a compact and robust way of synchronizing an additional Ti:sapphire laser with a conventional single-beam multiphoton microscope to realize an instrument that can acquire images with enhanced molecular specificity.Aim: A passive optical synchronization scheme for a pair of commercially available, unmodified modelocked Ti:sapphire lasers was developed. The suitability of this synchronization scheme for advanced biomedical microscopy was investigated.Approach: A pair of modelocked Ti:sapphire lasers were aligned in master–slave configuration. Five percent of the master laser output was used to seed the modelocking in the slave laser cavity. The timing jitter of the master and slave pulse trains was characterized using an optical autocorrelator. The synchronized output of both lasers was coupled into a laser scanning microscope and used to acquire spectral focusing SRS and pump-probe microscopy images from biological and nonbiological samples.Results: A timing jitter between the modelocked pulse trains of 0.74 fs was recorded. Spectral focusing SRS allowed spectral discrimination of polystyrene and polymethyl methacrylate beads. Pump-probe microscopy was used to record excited state lifetime curves from hemoglobin in intact red blood cells.Conclusion: Our work demonstrates a simple and robust method of upgrading single-beam multiphoton microscopes with an additional ultrafast laser. The resulting dual-beam instrument can be used to acquire label-free images of sample structure and composition with high biochemical specificity. However, alternative nonlinear optical processes that require two spatially and temporally overlapped ultrafast pulse trains can be used to provide biochemically specific contrast in nonfluorescent samples. Stimulated Raman scattering (SRS)Both SRS and pump-probe microscopy require precise control of the timing delay between the two pulse trains, and in particular a low pulse-to-pulse timing error, often referred to as timing jitter. Timing jitter on the order of the pulse duration has a deleterious effect on SRS and pump-probe contrast. Low timing jitter between ultrafast pulse trains can be achieved using frequency conversion devices, such as an optical parametric oscillator (OPO) that converts the pulsed input into two tightly synchronized, wavelength tunable output pulse trains. OPOs are widely used tools in biophotonics research laboratories that provide access to a broad range of wavelengths. However, many oscillators used for multiphoton microscopy cannot be easily combined with an OPO. This is because they have either insufficient power, or without using intracavity SHG which increases the burden on the pump power requirement due to conversion inefficiency, the resulting signal/idler tuning range is too far toward the IR. Thus in many cases, integrating an OPO into an existing single-beam multiphoton microscope would require the purchase of an OPO and the replacement of the original oscillator with one that is better suited to pumping an OPO. Turnkey, dual-wavelength synchronized ultrafast laser systems are now commercially available; however, the high cost of these systems represents a significant barrier to their broad adoption.,,–Integrating a second, tightly synchronized ultrafast laser into a single-beam multiphoton microscope is a cost-effective alternative as it works with, rather than replaces existing instrumentation. Active electronic cavity synchronization schemes have successfully realized few or subfemtosecond timing jitter between the pulse trains of two modelocked lasers.2–A pair of wavelength tunable, modelocked Ti:sapphire lasers  were aligned in master–slave configuration. The layout of the lasers and optics used for passive synchronization are shown in the green rectangle in To demonstrate the biomedical imaging applications for our passively synchronized, dual-femtosecond laser system, we integrated it into a custom laser scanning microscope setup configured for SRS and pump-probe microscopy. The full experimental setup is shown in To evaluate the quality of our dual-femtosecond laser synchronization scheme, we used SRS images of an olive oil droplet to air interface and compared our results with previously published measurements acquired using a OPO-based femtosecond SRS microscope.SRS is commonly used to image the molecular composition in thin samples such as cells. In Supplemental Fig. S1.To further investigate the stability of the passive synchronization scheme, we conducted imaging studies that place stringent demands on timing jitter of the two ultrafast pulse trains. First, we investigated the suitability of our setup for recording the ultrafast dynamics of ESA in the hemoglobin of fresh coverslip mounted RBCs. To acquire the hemoglobin transient absorption curves shown in Finally, we performed spectral focusing SRS in mixtures of PS and PMMA beads. A 35-mm long glass block was introduced into the common path of the master and slave beams to chirp the pulses. Following transmission through the glass block, the full-width at half-maximum of the slave laser at 727 nm had increased to 321 fs, and the master laser at 927 nm to 442 fs. The chirped pulses could now be used for spectral focusing measurements, where an adjustment of the master beam optical delay line resulted in a different vibrational resonance being driven in the sample. In 3,Passive optical synchronization of two (or more) commercial Ti:sapphire lasers presents a comparatively simple and robust approach to enhancing the imaging capabilities of existing multiphoton microscopes. In this work, we investigated the suitability of a dual-femtosecond laser system employing optical synchronization for biomedical imaging applications. We use image-based metrics from SRS and pump-probe microscopy to compare our synchronization technique against more complex and expensive OPO-based microscopes. In 4In this work, we present a compact, robust, and convenient approach to expand the label-free imaging capabilities of single-laser beam multiphoton microscopes. Our technique based on the passive optical synchronization of two modelocked Ti:sapphire lasers operates independently of the wavelength and polarization state of both lasers. We have shown that this approach realizes subfemtosecond timing jitter between two ultrafast pulse trains which can be used to excite a variety of nonlinear optical processes across the Ti:sapphire tuning range. In this work, we used two lasers with piezoadjustable cavity mirrors. However, in principle, this technique only requires one laser to have an electronically adjustable repetition rate which can be matched to the other. In future work, we aim to demonstrate passive optical synchronization of different types of ultrafast laser system and thereby demonstrate how readily this work could be used to upgrade existing single-laser beam multiphoton microscopes. We believe that this work will bolster the burgeoning interest in label-free, biochemically specific microscopy techniques in the biomedical research community.Click here for additional data file.


Existing studies have demonstrated that the integrative analysis of histopathological images and genomic data can be used to better understand the onset and progression of many diseases, as well as identify new diagnostic and prognostic biomarkers. However, since the development of pathological phenotypes are influenced by a variety of complex biological processes, complete understanding of the underlying gene regulatory mechanisms for the cell and tissue morphology is still a challenge. In this study, we explored the relationship between the chromatin accessibility changes and the epithelial tissue proportion in histopathological images of estrogen receptor (ER) positive breast cancer.An established whole slide image processing pipeline based on deep learning was used to perform global segmentation of epithelial and stromal tissues. We then used canonical correlation analysis to detect the epithelial tissue proportion-associated regulatory regions. By integrating ATAC-seq data with matched RNA-seq data, we found the potential target genes that associated with these regulatory regions. Then we used these genes to perform the following pathway and survival analysis.Using canonical correlation analysis, we detected 436 potential regulatory regions that exhibited significant correlation between quantitative chromatin accessibility changes and the epithelial tissue proportion in tumors from 54 patients (FDR < 0.05). We then found that these 436 regulatory regions were associated with 74 potential target genes. After functional enrichment analysis, we observed that these potential target genes were enriched in cancer-associated pathways. We further demonstrated that using the gene expression signals and the epithelial tissue proportion extracted from this integration framework could stratify patient prognoses more accurately, outperforming predictions based on only omics or image features.This integrative analysis is a useful strategy for identifying potential regulatory regions in the human genome that are associated with tumor tissue quantification. This study will enable efficient prioritization of genomic regulatory regions identified by ATAC-seq data for further studies to validate their causal regulatory function. Ultimately, identifying epithelial tissue proportion-associated regulatory regions will further our understanding of the underlying molecular mechanisms of disease and inform the development of potential therapeutic targets. Cancer heterogeneity results in tumors that exhibit distinct clinical features, therapeutic responses and patient outcomes. Understanding the factors involved in the onset and progression of cancers is pivotal for diagnosis and treatment. One of the main factors that contributes to the development of cancer is genetic changes –3. HowevHistopathology images are generally considered the gold standard for cancer diagnosis and grading in the clinic since they provide the distribution patterns of different tissues and cell types in the tumor microenvironment . PreviouThe systematic integration of histopathological studies and omics profiles is expected to provide further understanding of tumor molecular biology and potentially more accurate stratification of patient prognoses. Recent reports have highlighted the significance of the contribution of stromal gene expression and morphological structure as powerful prognostic determinants for a number of tumor types –13. HoweTo study the association between chromatin accessibility changes and tumor phenotypes, images of tumor sections and ATAC-seq data from matched tumor samples are needed. The Cancer Genome Atlas (TCGA) contains histopathology images along with clinical outcomes and has recently generated high-quality ATAC-seq data in tumor samples from 54 estrogen-receptor (ER)-positive breast cancer (BRCA) patients. These large-scale experimental datasets make comprehensive integrative and correlative analyses feasible.Most breast cancers are carcinomas that arise from the epithelial components of the lobules and ducts in mammary glands. Studies focused on developing tissue classification and segmentation algorithms have referred to tumors as epithelial tissues in image processing tasks , 22. FolIn this study, our aim was to identify key genomic regulatory regions that were associated with histological characteristics and thus, potentially impact clinical outcome. Such regions would be important for investigating the etiology of the associated disease and for identifying potential therapeutic targets.In this work, we systematically explored the relationship between chromatin accessibility changes and epithelial tissue proportion. First, we used our new computational pipeline to quantify the epithelial tissue proportion from each sample. By performing correlation analysis, we observed that the change in chromatin accessibility of some specific open regions were strongly correlated with the change in epithelial tissue proportion across all samples. Then we implemented a strategy of linking DNA regulatory elements to their target genes based on the correlation of ATAC-seq and gene expression data. Downstream pathway analysis demonstrated that those target genes enriched in breast cancer-specific biological processes were associated with well-known oncogenes. Furthermore, we showed that the identified target genes could effectively predict overall survival of BRCA patients. In summary, the integration of the multi-omics data and histopathological images can provide new insights to explore the drivers and the molecular mechanism of ER-positive breast cancer.The overall strategy of our integrative analysis comprises three stages as shown in Fig. n = 54) to identify the associated open chromatin regions. The epithelial tissue proportion data of the 54 TCGA ER-positive breast cancer cases is provided in Additional file We have previously developed an image processing pipeline that was used to classify and quantify epithelial tissue areas on histopathological images for all of the ER-positive breast cancer cases in TCGA. The image processing pipeline consists of three steps: 1) identification of a region of interest (ROI) on a whole-slide image; 2) patch-level segmentation of epithelial and stromal tissues in the ROI using a CNN model; 3) creation of a global tissue segmentation map by merging patch-level results followed by estimation of epithelial tissue proportions. For all 773 ER-positive patients, the previous image analysis results showed that these cases were enriched with stromal tissues, with a mean epithelial tissue proportion lower than 0.3 . Here, wFor each detected open chromatin region, we asked whether this potential regulatory region might have contributed to tumor development in ER-positive breast cancer patients. To begin to address this question, we implemented canonical correlation analysis between the quantitative chromatin accessibility measure and the epithelial tissue proportion across all 54 patients.p-value and the FDR of the correlation analysis, can be viewed on our RShiny website (https://yunlongliulab.shinyapps.io/omics-image/).The Spearman correlation coefficient, r, was used to evaluate whether the potential regulatory regions were significantly correlated with the epithelial tissue proportion. A total of 215,920 open chromatin regions were analyzed. Multiple testing correction (FDR < 0.05) is used to account for false positives. This analysis showed that 436 regulatory regions were significantly correlated with the epithelial tissue proportion, 111 of which were positively correlated and the other 325 were negatively correlated. The ATAC-seq peak signal data of 54 TCGA ER-positive BRCA cases is provided in Additional file Examples of the correlation between chromatin accessibility and spatial quantification of epithelial tissues are presented in Fig. We next asked whether the epithelial tissue proportion-associated regulatory regions identified by the correlation analysis could be related to elements of the breast cancer pathway. To address this question, we first identified candidate target genes for the regulatory regions that significantly correlated with the epithelial tissue proportion (see Methods). For putative promoter regions, the closest gene to each region was considered as the target gene. However, because enhancer regions can be far away from their target genes, we used the predicted distal peak-to-gene links obtained from TCGA .PARI, CCNE2 and RAD54B, were detected to have positive correlations with epithelial tissue proportion in our study. Previous studies have demonstrated that PARI overexpression was correlated with aggressive tumor cell proliferation and poor prognosis in breast cancer [CCNE2 in breast cancer is strongly predictive of shorter distant metastasis-free survival following endocrine therapy [RAD54B potentiates tumor growth and predicts poor prognosis of breast cancer patients [In total, 77 regulatory regions were in promoter regions, which we considered as potential promoter regulatory elements, while another 21 regulatory regions were detected by distal peak-to-gene links (< 500 kbp), which we considered potential enhancer regulatory elements. Many of these peak-to-gene links occurred in clusters that were predicted to be linked to the same gene, resulting in a total of 74 target genes that were selected for further downstream analysis. A complete list of these peak-to-gene links and target genes can be found in Additional file t cancer , high ex therapy  and RAD5patients .Estrogen-mediated S-phase Entry and Breast Cancer Regulation by Stathmin1, were significant findings from our integrative analysis. In addition, some tissue development and disorder associated functions were specifically enriched, such as Connective Tissue Disorders and Developmental Disorder. Altogether, these results underscore the ability to utilize our integrative analysis of image and chromatin accessibility data to identify genes that play a role in breast cancer development.For these 74 target genes, we performed pathway and function enrichment analysis using Ingenuity Pathway Analysis (IPA). We further observed that these genes were enriched in breast cancer-related pathways and functions, especially genes that were positively correlated with epithelial tissue proportion . The log-rank test results of all survival-related variables are listed in Additional file BCL3 had significantly worse overall survival than those with low BCL3 expression . It is noteworthy that the patients with relatively low epithelial tissue proportions showed longer survival  of the target genes identified in this study were also identified in our previous work, which directly analyzed the relationship between the epithelial tissue proportion and gene expression data . Importa/74 of thr > 0.5) and the multiple testing correction (FDR < 0.5) that were used to minimize false positive findings. Furthermore, the distribution of the epithelial tissues in the 54 cases used in this study were consistent with those in the entire TCGA ER-positive breast cancer cohort, which further supports the generalizability of our findings.While the integrated analysis approach used in this study revealed a relationship between image morphological and genomic features, there are limitations to this study. First because obtaining matched ATAC-seq and image datasets is challenging, only 54 ER-positive breast cancer samples were used in the correlation analysis. As a result, pairwise correlations between specific chromatin accessibility changes and the image-based epithelial tissue proportion could not be validated in other datasets, which could introduce dataset-specific bias such that the detected open chromatin regions may not represent all regulatory regions in this tumor type. A second limitation is that only the epithelial tissue proportion was used as an image feature in this study. The tumor microenvironment is a complex system and histopathological images demonstrate a complicated distribution of different tissues and cell types. Other histopathological image features have proven to be important for the diagnosis and prognosis of breast cancer, such as the textural features of epithelial tissue , 33 and Our future studies will extend this framework to incorporate additional representative image features to further investigate the intrinsic relationship between genotypes and clinical phenotypes. Future comparative ATAC-seq experiments with normal samples are needed to verify our findings. Additionally, to further understand the genetic basis of this disease, future studies could integrate our algorithm with rare variant or eQTL analysis.Our analysis demonstrates the ability to integrate chromatin accessibility signals and histological images for exploring the drivers and molecular mechanisms of ER-positive breast cancer. This integrated analysis will enable efficient prioritization of gene regulatory regions identified by correlation studies for further studies to validate their causal regulatory function. Ultimately, identifying regulatory regions and their target genes will further our understanding of the underlying molecular mechanisms of breast cancer. Furthermore, the entire pipeline can be easily applied to different diseases.Chromatin accessibility data, gene expression data, H&E-stained whole-slide histopathology images and matched clinical information were obtained from TCGA and can be downloaded from the link provided in the Data Availability section. Both the ATAC-seq and mRNA expression data were obtained from frozen tissue sections in proximity to the sections that were used to generate the H&E-stained tissue slides . The 74 The TCGA BRCA cohort provides two types of H&E stained whole-slide images: tissue slides and diagnostic slides. Tissue slides are sections from frozen tumor specimens that are typically used to determine whether the tumor borders are clean. Diagnostic slides are formalin-fixed paraffin-embedded (FFPE) sections, which typically have better preservation of cell morphology; however, these sections frequently show areas with tissue damage. Only tissue slides were used for the histopathological images in this study. Histopathological images were downloaded in the native image format as Aperio SVS files. Each image was acquired at a 40X objective lens using Aperio Scanscope, with each pixel corresponding to a 0.24 × 0.24 square micron area.Proportionepi represents the epithelial tissue proportion and Areaepi and Areastro represent the epithelial and stromal tissue area identified by the CNN model, respectively.We used our previously described whole-slide image-processing framework  to calcur between each quantitative chromatin accessibility and epithelial tissue proportion were calculated across all ER-positive BRCA cases. Given the correlation coefficient r and the sample size, the P-value for the correlation coefficient was calculated using the exact permutation distributions for the two-tailed test. The FDR was calculated following the Benjamini–Hochberg procedure [Associations between chromatin accessibility changes and the epithelial tissue proportion were determined using canonical correlation analysis. Specifically, for each detected open chromatin region, chromatin accessibility was quantified using the normalized count from that specific region for each case. The Spearman correlation coefficients rocedure . FinallyTo associate regulatory regions with the genes they are predicted to regulate, we adopted the same procedure as TCGA . SpecifiThe distal peak-to-gene link was based on the correlation of ATAC-seq accessibility and gene expression across all samples. All peaks whose summit were located within 500 kbp from a gene’s TSS were considered. A conservative FDR cutoff of 0.01 was used to avoid false positives. Putative enhancer peaks were further filtered if (i) the correlation with gene expression was strongly driven by DNA copy number amplification, or (ii) links involved an ATAC-seq peak that overlapped the promoter of any gene.Canonical Pathways and function analysis was derived from Diseases & Functions.Ingenuity Pathways Analysis  was used to explore possible signaling pathways and functions for genes whose regulatory region was correlated with epithelial tissue proportion. IPA core analyses was conducted for each identified gene using experimentally observed knowledge in the Ingenuity Knowledge Base. Pathway analysis was conducted using For the univariate survival analysis, we used the median value of each feature to stratify cases into low-risk and high-risk groups. The Kaplan-Meier method and log-rank test were used to fit the survival data and test for survival difference between the two groups.For the multivariate survival analysis, a previously described method utilizing a k-means clustering algorithm  was implAdditional file 1: Supplemental Figure 1. Representative H&E stained histopathology tissue image of TCGA breast cancer cases. A) Original H&E stained histopathology image and paired tissue segmentation result of a high-epithelium case. B) Original H&E stained histopathology image and paired tissue segmentation result of a low-epithelium case. The tissue segmentation results were derived from our previous work, with the red, green and black regions corresponding to epithelial and stromal tissue and background in the original image, respectively.Additional file 2: Supplemental Table 1. The epithelial tissue proportion data of 54 TCGA ER-positive BRCA cases. Supplemental Table 2. The ATAC-seq peak signal data of 54 TCGA ER-positive BRCA cases. Supplemental Table 3. Significant epithelial tissue proportion-associated peaks and their target genes. Supplemental Table 4. Promoter and distal enhancer peak-to-gene links and their target genes. Supplemental Table 5. Expression data of the identified genes and clinical information for 663 TCGA ER-positive BRCA cases. Supplemental Table 6. Results of the univariate survival analysis.


Clinical trials have demonstrated that antihypertensive treatment (AHT) in older adults is beneficial. Longitudinal studies, in contrast, have shown that low blood pressure is associated with higher all-cause mortality, especially in frail older adults. Despite the high quality of the available evidence, its translation into clinical guidance for the heterogeneous older population is challenging. To give a systematic overview of blood pressure targets for older adults recommended in clinical guidelines, we searched PubMed, Embase, Emcare, and five guideline databases. We selected guidelines with numerical thresholds for the initiation or the goal of non-disease-specific AHT (January 2008-October 2019). Guidelines with advices concerning AHT in older adults were analyzed. We appraised the guideline quality with the AGREEII-instrument. Of the 44 guidelines containing a numerical threshold for the initiation or the goal of AHT, 33 (75%) provided recommendations concerning AHT for older adults. Nineteen advised a higher target of systolic blood pressure (SBP) for older adults in comparison with the middle-aged population and 3 more recent advised a lower target. Over half (19/33) recommended to treat hypertension in the oldest old to a SBP <150 mmHg, while others advised intensive treatment to targets <120 mmHg. Although many guidelines mentioned frailty, only three gave specific thresholds and targets for frail older adults. The quality of the guidelines was not related with the recommended targets. Targets of AHT in older adults in international guidelines range from less strict to more intensive in comparison with the middle-aged and are set on chronological rather than biological age.