The advancement of meta-omics and its integration into a variety of fields of biotechnology, environmental monitoring, and health over the last decade has been astounding and very promising. Our quest to develop personalized medicine and even improve human quality of life owes a great deal to meta-omics. Thus, the purpose of this Research Topic was to attract prospective and unique research trends and breakthroughs in meta-omics technologies relevant to biotechnological processes, environmental monitoring, and health. The six accepted articles delivered on this goal with intriguing discoveries that will shape our approach to applying meta-omics to the area of biomedical research in the coming years.Dave et al. revealed a novel approach, by applying single cell line model systems while capturing all represented populations and subpopulations, exposing their diverse and unique functional characteristics, which may have been lost with previous approaches. They provide an automated cell identification platform to be used before single-cell culturing that lowered cell stress and improved rare cell identification. These researchers’ pipeline can be utilized for patient samples to reduce processing time while retaining data quality and accuracy towards understanding the patient’s disease. This contribution to high-throughput single-cell multi-data assays makes it easier to quantify therapeutic responses, classify differentiation heterogeneity in generated models, and validate single-cell sequencing conclusions, which helps understand disease pathogenesis and tumor metastasis.Interestingly, cell line research work done by Li et al. noted that spatial transcriptomics’ localization-indexed gene expression data could help understand cancer’s complex heterogeneity. These researchers criticized single-cell sequencing for the limited information, it tends to provide. Remarkably, the previous research group of Dave et al. appeared to have been working on this challenge highlighted by Li et al. Both articles will be interesting to readers as they provide unintentional supporting contexts and contribute to breast cancer research in a great way. Li et al. also underline the importance of transcriptomics in tumor heterogeneity research and personalized therapy. Through comprehensive research, the authors demonstrate that intra-tumor heterogeneity hinders cancer diagnosis and personalized treatment. These scientists also note that novel technologies may be expensive, but they believe spatial omics, particularly spatial transcriptomics, will provide the most insights for screening tumor treatment targets and improving clinical diagnosis accuracy.In their review paper, Boileau et al. created the Single-cell Nanopore Spatial Transcriptomics (SCNAST), a new software suite that demonstrates the capability to analyze spatial gene expression from second- and third-generation sequencing platforms, to create a full-length near-single-cell transcriptional landscape of the tissue microenvironment. Their product development applied Visium Spatial to assign barcodes to long-read single-cell sequencing data for spatial capture technology. They successfully created a de novo transcriptome (long-read data) of a mouse heart after a myocardial infarction using four short axis heart sections. The successful assignment of 19,794 transcript isoforms revealed the spatial diversity of isoforms, a fascinating information that can help create new heart failure and cardiac problems treatments.Zhang et al. highlights the utility of RNA-seq platforms for investigating molecular pathways in Dermatofibrosarcoma protuberans (DFSP), a rare cutaneous sarcoma. These researchers used differential expression and enrichment analysis to tackle fourteen DFSP samples from Chinese patients. They were successful in identifying DFSP fusion genes, biomarkers, and microenvironment traits. Their study reveals the efficacy of RNA-seq as a viable technique for getting new insights into disease, leading to accurate diagnosis and specificity in therapeutic targets for certain diseases like DFSP.The study by Kasavi, who used this strategy to identify novel candidate biomarker signatures in three datasets of serous ovarian adenocarcinoma. The researcher discovered 439 common differentially expressed genes (DEGs) and created differential co-expression networks utilizing common DEGs in two situations, in this case healthy ovarian surface epithelia and serous ovarian cancer epithelia. This type of study, in particular, allowed for the likely identification of novel candidate predictive biomarkers for ovarian cancer. The latter is suggested to be potentially useful in ovarian cancer diagnosis and treatment advances.Differential co-expression network analysis continues to be useful in meta-omics research, as demonstrated by Zhou et al. investigated the link between metabolic reprogramming and malignant tumors. Their review focused on the non-essential amino acid serine and its role in cell proliferation. Their extensive literature survey highlighted the potential use of serine as a biomarker in tumor detection and pathology. They showed the importance serine metabolism plays in the network of glycolysis, folate cycle, and one-carbon metabolic pathways. These researchers, also suggest that future studies should target the fate of serine in these varied pathways for both normal functioning cells and diseased cells as this will be critical in the development of precision and personalized cancer therapy.Finally,


Purpose: The aim of this systematic review and meta-analysis is to evaluate the effect of mindfulness-based interventions (MBIs) on post-surgical pain in patients undergoing a total hip replacement (THR) or total knee replacement (TKR).Methods: We performed a systematic review and meta-analysis in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. A search of multiple databases, including PubMed and EMBASE, was performed for studies from database inception through March 2nd, 2022. Data were extracted, and pooled estimates of standardized mean differences in pain scores were calculated using a random effects model and inverse probability weighting.Results: Two randomized control trials were eligible for inclusion (299 patients). The average ages of participants in each study were similar at 65.5 and 64.8 years, and both studies were predominantly female at 72.4% and 61.9%. The mindfulness intervention ranged from an eight-week program to a 20-minute session. Both individual studies reported statistically significant reductions in postoperative pain for MBI groups. The pooled standardized mean difference in pain scores for the MBI groups compared to the control groups was -1.94 .Conclusions: There exists preliminary evidence for the beneficial effect of MBIs on reducing the postoperative pain experience in this patient population. Given the significant consequences of postoperative pain and the necessity for non-opioid forms of analgesia, this topic represents a promising area of research that warrants future randomized control trials to better understand the role of MBIs for postoperative analgesia. Total knee replacement (TKR) and total hip replacement (THR) are common orthopedic procedures for end-stage osteoarthritis and rheumatoid arthritis and are performed more than 1 million times per annum globally . If poorCurrent strategies to control postoperative pain after TKR and THR rely heavily on opioid medications. However, adverse effects are common within such analgesic regimes  and opioMindfulness is defined as “the awareness which arises from purposefully paying attention to present moment experiences non-judgementally” . MindfulMBIs have been utilized in the clinical setting as an effective non-pharmacological means of reducing musculoskeletal pain related to rheumatoid arthritis , fibromyCurrent studies on this subject include small sample sizes and some null results can be the result of a lack of statistical power. A meta-analysis can address these concerns and combine the results of individual studies to boost power. Further, a systematic review allows for the qualitative synthesis of the existing literature on this subject. The aim of this systematic review and meta-analysis is to evaluate, based on the existing literature, the effect of MBIs on post-surgical pain in patients undergoing a THR or TKR.MethodsProtocol and RegistrationWe performed a systematic review and meta-analysis in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) Guidelines . The proEligibility CriteriaStudies were considered eligible if they were randomized clinical trials or observational studies that investigated the impact of mindfulness-related interventions on post-surgical pain in patients undergoing a THR or TKR. Studies that employed interventions such as yoga, tai chi, qigong, and transcendental meditation or relaxation therapy without explicit reference to mindfulness were excluded. Studies that employed interventions that did not include formal mindfulness meditation practice, such as Acceptance and Commitment Therapy, were excluded. DatabasesClinicalTrials.gov for all articles published from database inception to March 2nd, 2022. We built the search strategy, without language restriction, for PubMed, EMBASE, PsycINFO, Web of Science, Cochrane, and Search StrategyA comprehensive search strategy was created with the support of a librarian at the Harvard Countway Library of Medicine. Our search terms included terms for mindfulness, meditation, mind-body therapies, breathing exercises, relaxation therapy, cognitive behavioral therapy, and arthroplasty. Our full search strategy is included in our appendix.Study SelectionSearch results from all sources were uploaded to Covidence. Duplicate studies were removed. Two reviewers (M.B. and J.G.) independently screened titles and abstracts and then reviewed full texts. Inconsistencies at each step were resolved by discussion and reaching a consensus. The reference lists of included studies were also reviewed for relevant articles. Data Collection Process and Data ItemsAs per the pre-study protocol, data extraction was performed by one author (M.B.) and checked by another (B.B.). Elements that were extracted from each study included the following: study reference, study design, study demographics, intervention details (length and type of training), control details , pain outcome data (mean and standard error for group scores or group differences), and the number of patients in each group (control vs. intervention). It was determined ahead of time that correspondence with study researchers would be sought via email to request any unreported data or details, if necessary.Risk of Bias in Individual StudiesFor our study-level quality assessment, we a priori specified that we would use the Cochrane RoB 2 (risk of bias 2) tool and the ROBINS-I (“Risk Of Bias In Non-randomised Studies--of Interventions”) tool for randomized control trials and observational studies, respectively, to assess the quality of our studies and risk of bias ,19. GiveSummary MeasuresStandardized mean differences were used as the main effect measure for analyzing pain differences for intervention vs. control groups. A negative mean difference reflects that the mindfulness intervention resulted in a lower pain score compared to the control group. If a mean difference was not reported, we calculated it ourselves using the difference in mean pain scores for intervention and control groups. In order to calculate standard errors (SE) for our effect measures, we utilized reported 95% confidence intervals (CI) and reverse-calculated standard errors using the equation: SE = (CI upper bound - mean)/1.96  . In antiSynthesis of ResultsWe created pooled estimates of our effect measures (standardized mean differences) using a random effects model utilizing inverse probability weighting . RestricGiven the limited number of studies and data, we were unable to perform other types of analyses, including publication bias analysis, sensitivity analysis, subgroup analysis, or metaregression. Statistical analyses were performed using the Metafor package in R statistical software, version 4.1.2 ,21. All ResultsStudy SelectionOur systematic search of articles published before March 3rd, 2022 and related to our study question identified 504 unique results. After title and abstract screening, we were left with 26 studies that we considered potentially relevant. Two studies remained after full-text review (reasons for exclusion included “wrong intervention” (13 articles), “wrong study type” (seven articles), “wrong outcomes” (three articles), and “wrong study design” (one article)). Two studies reported mean differences in pain scores and were included in the meta-analysis. For our full PRISMA flow diagram, please see Figure Study CharacteristicsTable Study population: Both studies were recently published open-label randomized control trials utilizing patients receiving either a total hip or knee arthroplasty. Hanley et al. 2021 recruited patients at the University of Utah’s Department of Orthopaedic Surgery in Salt Lake City, Utah, USA , while DIntervention: Preoperative MBIs varied considerably in their duration and content. Dowsey et al. 2019 utilized a preoperative MBSR program -a well-vControl: Dowsey et al. 2019 utilized a ‘treatment as usual’ (TAU) comparison group, where participants underwent THR/TKR without any additional non-pharmacological methods of analgesia. Hanley et al. 2021, on the other hand, used an active comparison group in the form of a 20-minute preoperative cognitive-behavioral pain psychoeducation (CB) intervention which consisted of education about the link between thoughts, emotions, and behavior and provided instruction on how to modify maladaptive thoughts and behaviors regarding pain that might otherwise exacerbate pain and distress. When Hanley et al. were contacted about their reasoning for choosing a CB control group, they stated they originally had planned to include CB as well as TAU control groups; however, they removed the TAU group at the direction of reviewers at a top-tier general medical journal (ultimately rejected for unrelated reasons). All groups, including the MBI groups, received comparable baseline standard medical care upon which psychological interventions were added or not (group-dependent). Standard baseline analgesia included intraoperative local infiltration, regional nerve blocks, Tylenol, non-steroidal anti-inflammatory drugs (NSAIDs), and opioids.Outcomes: The primary outcomes in Dowsey et al. 2019 were physical function and pain intensity-for which they utilized the knee pain component of the Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index to measure self-reported pain at three months and 12 months post-surgery. In Hanley et al. 2021, they measured pain intensity using a 10-point scale on postoperative days two, three, seven, 14, 21, and 28. The other primary outcomes in Hanley et al. 2021 were paint interference and opioid use. Dowsey et al. 2019 and Hanley et al. 2021 both reported mean differences in pain intensity between the MBI and control groups. This summary statistic was utilized as the effect size for both studies. For our full table of study characteristics, please see Table Risk of Bias in Individual StudiesThe risk of bias was assessed in both studies; please see Figure Mindfulness Interventions and Postoperative Pain: Individual Study ResultsTable Mindfulness Interventions and Postoperative Pain: Meta-AnalysisBoth studies reported mean differences in pain scores for the MBI groups compared to the control groups. Of note, Hanley et al. 2021 consisted of two MBI groups (MoP and MoB), and we included both groups in our analysis. These two groups plus the MoB group in Dowsey et al. 2019 made for a total of three groups for our meta-analysis. We utilized the three-month pain measurement timepoint from Dowsey et al. 2019 and the 28th postoperative day timepoint for Hanley et al. 2021 given they were the time points that were most similar to each other between studies (a strategy that was specified a priori). Overall, MBIs (MoP or MoB) were significantly associated with a decrease in standardized pain scores of -1.94  compared to control groups that received no MBI  and multimodal  analgesia has been recommended as a way to decrease complications while providing the best analgesia for patients . MBIs reMechanism of Action of MBIs for AnalgesiaThe experience of pain is complex, possessing both biological and psychological components. Pain is a protective somatosensory phenomenon first initiated by peripheral nociceptors at the site of tissue damage and then transmitted through the dorsal horn and spinothalamic pathway of the spinal cord into cerebral regions, which include the thalamus, somatosensory cortex, anterior cingulate cortex, and prefrontal cortex . It is cResearch suggests that mindfulness practices can alter the cognitive processes involved in pain processing, which leads to changes in pain perception . SpecifiThese cognitive schema modifications may lead to biological changes in pain processing, with evidence indicating that mindfulness practices attenuate pain by altering pain processing at the orbitofrontal cortex, anterior insula, anterior cingulate cortex, and thalamus ,12. ThisFuture DirectionsFurther evaluation of the relationship between MBIs and the magnitude and timing of their analgesic effect is needed. Whereas Dowsey et al. 2019 observed no significant pain reduction in the MBI group at three months, pain reduction was observed in the MoP group of Hanley et al. 2021 as early as postoperative day 14. Additionally, it would be helpful to better understand how the duration of MBIs might impact their level of analgesic effect. Dowsey et al. 2019 evaluated an eight-week intensive program while Hanley et al. 2021 utilized brief, 20-minute sessions undertaken three weeks before surgery. Both studies found significant pain reduction among their participants despite dramatically different program lengths and resource utilization. These results indicate that even brief interventions may be highly effective. In future years, it will be important for studies to establish whether a dose-response relationship between the duration of MBIs and postoperative pain reduction exists. Similarly, future research is needed to assess the analgesic effect of different mindfulness practices. Interestingly, Hanley et al. 2021 found the two mindfulness intervention groups studied produced different results as compared to the CB control. While the MoP practice was observed to produce a strong and consistent analgesic effect, the MoB practice failed to show any statistically significant reductions in pain versus the control group. Hanley et al. 2021 postulated that MoP intervention had more of an effect on cognitive factors that prolong postoperative pain, such as pain catastrophizing, thus allowing it to have the strongest analgesic effect postoperatively. Additionally, the two studies included in our review both reported combined data for patients undergoing THR or TKR. In future studies, reporting joint-specific subgroup analyses as well might also be useful to facilitate subsequent subgroup analyses at the meta-analysis level. Finally, further investigation is warranted to explore MBIs role in promoting other postoperative health and recovery outcomes. Dowsey et al. 2019, along with other studies, provide evidence that MBIs delivered preoperatively to TJA patients may result in enhanced postoperative physical function . FurtherStrengths and LimitationsA limitation of our study is the small number of published studies that had been conducted addressing our specific question. Given this, no definitive conclusions regarding the analgesic effect of MBIs for TKR/THR patients can be made currently. However, the results of the individual studies show the analgesic potential of MBIs for this specific application. Both studies were high-quality randomized control trials, and both demonstrated a statistically significant and clinically-relevant reduction in postoperative pain resulting from MBIs, as compared to the control condition, for THR/TKR patients. Furthermore, this study employed a detailed pre-study protocol, thorough search strategy, robust risk of bias assessment, and advanced meta-analysis techniques that included robust variance estimation. Another weakness of our study was the heterogeneity of our intervention and control groups across our individual studies. While it is true that the supervised mindfulness interventions across each study differed in terms of length (one session vs. eight weeks), additional self-directed practice of the mindfulness techniques after the teaching session may have made the actual intervention durations more similar than it would initially appear. Further, we believe that the interventions were similar in terms of content. Specifically, while there can be many definitions of mindfulness, both studies defined mindfulness in a similar way with an emphasis on awareness and acceptance. Additionally, while our control groups were also somewhat different , we felt they were similar to each other as well as both very different from the mindfulness interventions. Given all of this, we felt that a meta-analysis was appropriate, especially with the mixed effects model which allows us to account for the possible heterogeneity of effect sizes.While there is evidence of the benefits of MBIs for analgesia in various health conditions, this systematic review is the first of its kind to analyze the effectiveness of MBIs for the reduction of postoperative pain in THR/TKR patients. Our study suggests there may be a beneficial effect of MBIs on reducing postoperative pain in this patient population. Given the significant consequences of postoperative pain and the necessity for non-opioid forms of analgesia, this topic represents a promising new area of research that warrants future randomized control trials to better understand the role of MBIs for postoperative analgesia.


Nuclear-mitochondrial DNA segments (NUMTs) are mitochondrial DNA (mtDNA) fragments that have been inserted into the nuclear genome. Some NUMTs are common within the human population but most NUMTs are rare and specific to individuals. NUMTs range in size from 24 base pairs to encompassing nearly the entire mtDNA and are found throughout the nuclear genome. Emerging evidence suggests that the formation of NUMTs is an ongoing process in humans. NUMTs contaminate sequencing results of the mtDNA by introducing false positive variants, particularly heteroplasmic variants present at a low variant allele frequency (VAF). In our review, we discuss the prevalence of NUMTs in the human population, the potential mechanisms of de novo NUMT insertion via DNA repair mechanisms, and provide an overview of the existing approaches for minimizing NUMT contamination. Apart from filtering known NUMTs, both wet lab-based and computational methods can be used to minimize the contamination of NUMTs in analyses of human mtDNA. Current approaches include: (1) isolating mitochondria to enrich for mtDNA; (2) applying basic local alignment to identify NUMTs for subsequent filtering; (3) bioinformatic pipelines for NUMT detection; (4) k-mer-based NUMT detection; and (5) filtering candidate false positive variants by mtDNA copy number, VAF, or sequence quality score. Multiple approaches must be applied in order to effectively identify NUMTs in samples. Although next-generation sequencing is revolutionizing our understanding of heteroplasmic mtDNA, it also raises new challenges with the high prevalence and individual-specific NUMTs that need to be handled with care in studies of mitochondrial genetics. Nuclear-mitochondrial DNA segments (NUMTs) are fragments of the mitochondrial genome (mtDNA) that have been inserted into the nuclear genome of multiple organisms, including domestic cats, great apes, fruit flies, and humans ,2,3,4,5.NUMTs have been used to define phylogenetic outgroups and elucidate mtDNA evolution ,14. Rece4 births [NUMTs can originate from any part of the mtDNA and are found throughout the nuclear genome ,18. The NUMTs pose a challenge in studies evaluating mitochondrial genetic variation. Due to the high sequence similarity of NUMTs to the mtDNA within the individual, variants may be misidentified as being in the mtDNA rather than attributed to a NUMT. NUMTs interfere with the calculation of variant allele frequency (VAF), the number of copies of the mtDNA that contain a variant, used to define heteroplasmy . Early sNUMTs often do not encode functional proteins; however, some NUMTs are associated with disease . One stuNUMTs are found throughout the entire human nuclear genome ,15,26, bThe theory of mitochondrial endosymbiosis posits an evolutionary benefit of full-length mitochondrial gene transfer to specific loci in the eukaryotic nucleus that results in the production of a protein product ,28,29. TNUMTs are found across all nuclear chromosomes as insertions; hence, a biased mechanism of insertion towards any given locus seems unlikely . HoweverNUMTs may be preferentially inserted at points of double-stranded DNA breaks. The insertion of mtDNA fragments at double-stranded DNA breaks in the nuclear genome is thought by some to be an intentional process occurring during non-homologous end joining, a major double-stranded DNA break repair process in eukaryotic cells. Non-homologous end joining, while an effective way to repair double-stranded DNA breaks, results in deletion errors, possibly resulting in disease-causing frameshifts . EnvironA key question in the field is how the mtDNA fragments come to be inside the nucleus and available for insertion into double-stranded DNA breaks. Some literature suggests the involvement of mitophagy in the creation of freely available mtDNA fragments for insertion as NUMTs . AdditioIn addition to the computational approaches available for detecting NUMTs, several technical approaches can be used to limit the NUMTs in a sample. The most straightforward technical approach involves the isolation of mitochondria, thereby removing the nuclei, and hence, the nuclear genome and NUMTs, from the sample ,39. MitoFollowing mitochondrial isolation, a method called Mito-SiPE  can be employed to determine the true mtDNA sequences and heteroplasmic variants present in the mtDNA sample. After mitochondrial fraction enrichment from cells or tissues, ultra-deep sequencing is performed to gain >80,000× coverage of the mtDNA, thereby limiting the introduction of false heteroplasmic variants from PCR enrichment or NUMT contamination . SeveralTo prevent the interference of NUMTs in studies of mitochondrial genetic variation, some studies simply remove loci from existing catalogs of NUMTs from their analyses; however, this approach is likely not sufficient. Apart from common NUMTs that are widely shared, most NUMTs are rare and specific to individuals . The useBefore the advent of next-generation sequencing, NUMTs were primarily detected through a basic local alignment search (BLAST) of a PCR-amplified DNA sequence to reference genomes ,44. To dA novel NUMT discovery pipeline for paired-end whole genome sequencing (WGS) data has been developed and applied in multiple studies  1,48,49,4948,49.An alignment-free approach to detect NUMTs has also been developed . Local aNUMTs within WGS datasets can also be identified using mtDNA copy number . MtDNA cIf the mtDNA copy number is high enough (>500 copies) for a sample, as is the case for highly energetic tissues, NUMTs are likely not problematic for detecting heteroplasmic variants with a low VAF of 1–5%. In tissues with high mtDNA copy numbers, most NUMT-derived heteroplasmic variants will have a VAF below the commonly used VAF thresholds of 3–10% for defining heteroplasmic variants . HoweverTo distinguish low-level heteroplasmic variants due to mtDNA variants rather than variants found in NUMTs, software such as DREEP  has beenTo validate that the detected variants are truly from mtDNA, some bioinformatic pipelines generate a de novo mtDNA reference for each individual sample and conduct a second iteration of variant calling ,53. UsinNUMTs appear in all humans and across the entirety of the nuclear genome. Emerging evidence suggests that there may be a physiological and possible protective role of NUMT insertion into nuclear double-stranded break regions. However, false positive heteroplasmic variants due to the presence of NUMTs remains a challenge in confidently identifying low-level heteroplasmic variants in mtDNA sequencing datasets. Nonetheless, the methods for minimizing and identifying NUMTs, both technical and computational, are rapidly advancing. Mitochondrial isolation with subsequent sequencing represents an ideal scenario for removing the effect of NUMT contamination in mtDNA sequences. When mitochondrial isolation is not technically feasible, employing multiple computational methods can aid in identifying and removing NUMTs to facilitate studies of heteroplasmic variants with low VAFs. Ultimately, it is critical to run one or more of the aforementioned countermeasures to ensure accurate heteroplasmic mtDNA variant detection in mitochondrial genome sequencing.


Anxiety, posttraumatic stress, and depression markedly increased worldwide during the COVID-19 pandemic. People with these conditions experience distressing intrusive thoughts, yet conventional therapies often urge them to avoid suppressing their thoughts because intrusions might rebound in intensity and frequency, worsening the disorders. In contrast, we hypothesized that training thought suppression would improve mental health. One hundred and twenty adults from 16 countries underwent 3 days of online training to suppress either fearful or neutral thoughts. No paradoxical increases in fears occurred. Instead, suppression reduced memory for suppressed fears and rendered them less vivid and anxiety provoking. After training, participants reported less anxiety, negative affect, and depression with the latter benefit persisting at 3 months. Participants high in trait anxiety and pandemic-related posttraumatic stress gained the largest and most durable mental health benefits. These findings challenge century-old wisdom that suppressing thoughts is maladaptive, offering an accessible approach to improving mental health. Training people to suppress fearful thoughts improved mental health, especially for those with anxiety and post-traumatic stress. Suppressing upsetting thoughts may promote mental health during adversity, and this capacity may be trainable. Historically, thought suppression has been considered maladaptive because of the century-old Freudian proposal that suppressed content persists in the unconscious mind, resurfacing indirectly through symptoms and dreams  to changes in a control group that suppressed neutral events . Mental health changes in the Suppress-Neutral group provide a rigorous control against which to assess the unique effects of suppressing unpleasant content by quantifying general changes arising from other factors: from participants generating positive, negative, and neutral events at the study’s outset; from receiving thought suppression training in general; from placebo effects; or from socially interacting with an experimenter. If suppressing feared events harms or benefits participants, then mental health changes for the Suppress-Negative group should exceed those in the Suppress-Neutral group. In a complementary manipulation, we juxtaposed the mental health effects of suppressing fearful thoughts with those induced by positive imagery about hopeful future events. On Imagine trials, half of the participants imagined positive future events (Imagine-Positive), and half imagined neutral events , within each of the two Suppress groups . We quantified such changes by measuring these clinical features before and after thought suppression training and after 3 months. To detect mental health effects unique to suppressing distressing thoughts, we compared changes in our indices for a group that suppressed feared events , and this effect did not vary across the Suppress-Negative and Suppress-Neutral groups . We also tested whether suppression altered events’ phenomenological quality by quantifying changes in reported vividness observed after training compared to before training. Suppressed events showed larger reductions in vividness (relative to pretraining) than did Baseline events , and this effect did not vary across the Suppress-Negative and Suppress-Neutral groups . Thus, as with laboratory stimuli, suppression reduced access to feared and neutral personal events, at least on a retention test immediately after suppression.We first tested how thought suppression training affected memory for the suppressed events. We sought evidence for ironic increases in accessibility of suppressed content to quantify the risk of training people to suppress their fears. In healthy individuals, suppressing retrieval typically reduces later recall of the suppressed content, at least for simple words and images  and reduced vividness  after, compared to before, training. Only 1 person of 120 showed higher detail recall for suppressed items after training. Of the 61 participants that suppressed fears, 6 reported increased vividness for No-Imagine items after training .We considered the possibility that ironic rebound effects only arise for emotionally intense fears of great concern to participants. To address this, we examined whether the degree to which suppression training reduced vividness or detail recall for No-Imagine fears varied according to participants’ initial emotional intensity ratings for those fears. Contrary to such concerns, greater fear intensity was associated with larger suppression-related reductions in vividness after training compared to before training . One interpretation of this pattern is that suppression dissipated over the delay, causing an ironic rebound in fear accessibility. Contrary to this rebound effect, both memory for key details of No-Imagine items  and the vividness of No-Imagine items  remained far lower, not higher, after 3 months than they were before suppression, with no indication of any special resurgence of these suppressed items. For example, after 3 months, in the Suppress-Negative group, numerically fewer participants showed increased vividness for No-Imagine events  than showed such increases for Baseline events (N = 21); the Suppress-Neutral condition showed no difference . As a result, for fears, the relative risk of ironic increases in vividness of suppressed events, relative to Baseline events suggested decreased, not increased risk, although this reduction was not significant (table S5A). Even when we examined individual events, the number of fears (per participant) that increased in accessibility did not differ between No-Imagine and Baseline conditions for any measure or group . These findings suggest that suppression does not trigger rebound processes.The foregoing findings only address suppression’s impact immediately after training. Suppression might briefly benefit participants yet trigger hazardous hyperaccessibility of suppressed thoughts at longer delays. SIF was not detected after 3 months: At the follow-up, No-Imagine items were no longer recalled more poorly than were Baseline items, nor did they show greater declines in vividness compared to Baseline  and the higher the reported depression  with the effect of depression surviving statistical correction  nor depression  predicted smaller declines in recall, and higher posttraumatic stress scores marginally predicted larger, not smaller, declines in recall . Similar though weaker relationships arose after 3 months , providing no evidence of increased vulnerability.Last, we tested whether people reporting higher trait anxiety or pandemic-related posttraumatic stress suppressed event details or imagery less well. Participants reporting mental health symptoms may be especially vulnerable to rebound effects. We tested whether indices of mental health collected before training predicted declines in vividness and detail memory for No-Imagine fears in the Suppress-Negative group. Contrary to rebound concerns, suppression-related declines in vividness were greater, not smaller, the higher the reported trait anxiety , and this affective SIF did not vary across Suppression groups . Consistent with our memory indices, suppression-specific reductions in affective responses were not detected at 3 months, with no differences observed for No-Imagine and Baseline items . However, at 3 months, affective ratings of No-Imagine events remained lower compared to pretraining, , establishing that time’s passage did not ironically increase subjective emotions to suppressed future fears. For feared events, suppression-related reductions in affect and vividness were robustly associated, suggesting that rendering thoughts less accessible weakens their capacity to instill anxiety  reported more anxiety about suppressed fears after training, and the number was the same for nonsuppressed baseline fears (N = 15). Given this pattern, the relative risk of a rebound in anxiety about suppressed fears was 1.0, indicating that the risk was no different from when no suppression had occurred , and both higher trait anxiety  and depression  before training showed trends toward steeper declines in affect for suppressed fears, not smaller declines. Pretraining anxiety and depression were not reliably related to the decline in affect that fears showed after 3 months . Last, the average emotional intensity of a participant’s fear at the study’s outset  did not predict the suppression-related affect reduction measured on immediate or delayed tests . Together, these findings show that suppression training caused no measurable rebound effects on emotional responses to feared events regardless of their initial intensity and often reduced affective responses on immediate tests. Suppression-related reductions in perceived fear were more prominent for people reporting higher trait anxiety and depression.In the Suppress-Negative group, few participants .Mental health improved on most measures after training people to suppress distressing thoughts . In contTo be sure that overall training benefits did not disguise ironic effects in the Suppress-Negative group, we quantified how often mental health declined after training for individual participants. In relative risk analyses, no mental health index showed significantly greater risk of ironic effects after suppressing fears than after suppressing neutral thoughts, with most measures indicating numerically less frequent adverse changes for the former . This fiN = 60) to those after training participants to imagine neutral events (N = 60). Because half of each group suppressed negative and half suppressed neutral events, positive imagery’s impact can be examined with suppression valence held constant. Although practice at imagining positive events reduced worry and negative affect after training, comparable benefits arose when people practiced imagining neutral events, suggesting that improvements did not stem from positive imagery. Our PCA latent variable for mental health change yielded no reliable advantage for the Imagine-Positive over the Imagine-Neutral group . The lack of specific benefits of positive imagination cannot be attributed to an ineffective manipulation: Imagined events showed robust increases in detail recall , vividness , and subjective ratings of affect . These increases in vividness, memory, and affect for specific positive future events, however, did not uniquely improve participants’ mental health beyond imagining neutral events.Unlike suppressing fears, training participants to imagine positive and joyful future events conferred little unique benefit. We compared mental health changes after training participants to imagine hopeful future events , although this did not survive statistical correction. Participants who reported greater pandemic-related posttraumatic stress showed similar though weaker relationships for most mental health indices, and only the benefits to well-being  survived statistical correction (fig. S3B). Higher trait anxiety  predicted greater improvement (more negative scores) on our PCA-derived mental health change latent variable. These correlations did not occur in the Suppress-Neutral group: Trait anxiety or posttraumatic stress did not correlate with most individual measures  or with our mental health change latent variable  for No-Imagine and No-Imagine Baseline items. We judged SIF as having occurred if participants reported a larger change in vividness for No-Imagine than for No-Imagine Baseline items. We performed an analogous analysis to detect suppression-induced changes in affective intensity. We computed the impact of imagination on vividness and affective intensity in analogous fashion but using Imagine and Imagine-Baseline scores instead of No-Imagine and No-Imagine Baseline scores. We conducted similar analyses on the immediate assessment and after the 3-month delay. For delayed vividness and affect analyses, we computed change scores by comparing participants’ follow-up ratings after 3 months to their pretraining ratings (follow-up − pre).One hundred and twenty-seven people took part initially, of whom we excluded seven participants . The final sample included the 120 participants for whom we had complete data in the immediate and follow-up sessions .f = 0.27 , each of which further differed in events that participants needed to imagine during training . We assigned participants via blocked randomization, such that we ran each condition once for every four participants, ensuring that we matched the number of participants in groups as we ran the study. We determined the sample size for each of the four groups based on a priori power analysis with G*Power 3.1 software (We recruited participants from the MRC Cognition and Brain Sciences Unit (CBU) participant panels with the constraint that they had no prior participation in research involving the Think/No-Think (TNT) or the INI paradigms. We also recruited a small set of participants via online study advertisements on Facebook and Twitter and via word of mouth from previous participants. We paid participants according to the rates set for behavioral experiments at the MRC CBU (£6/hour). Exclusion criteria included a history of attention deficit disorder, reading disability, or lack of English fluency. Non-native English speakers could participate if they were fluent in English from early childhood and scored above 90% on the Cambridge General English Assessment , 20 positive events , and 36 neutral events . Participants completed four cells for each event: (i) a tagline in less than eight words describing the essence of the event, (ii) a more elaborate description of details that the experimenter used to verify the event’s compliance with the rules, (iii) a Cue Word, an obvious reminder that was used to evoke the event during training, and (iv) a Key Detail, a single word expressing a central event detail not mentioned in the tagline and obvious only to the participant, which was used to assess event recall during the immediate and delayed tests.Although all participants generated the same amount and types of events at the outset, we drew selectively from these events to construct the stimuli set that differed in the four groups. Specifically, we used only 40 of the 76 events generated initially as stimuli for each participant . The reasons why people generated more material than we used for their training were that we (i) wanted to be certain that all participants generated an equal number of events in the negative, neutral, and positive conditions (avoiding any biases) and (ii) the INI task for each of our four groups required materials with different valences. Thus, we selected a subset of critical events according to the condition to which participants had been assigned. When we assigned participants to suppress negative events and imagine positive events, we set neutral events aside and used only the positive and negative events. When we assigned them to suppress negative and imagine neutral events, we set aside positive events and used negative events and neutral events. When we assigned them to suppress neutral events and imagine positive events, we set aside negative events, selecting positive and neutral events, and when we assigned participants to suppress and imagine neutral events, we set aside all positive and negative events, focusing exclusively on neutral events. For each participant, we allocated eight events to each of four conditions: Imagine, No-Imagine, Baseline Imagine, and Baseline No-Imagine. For example, in the Suppress-Negative, Imagine-Positive group, Imagine and Baseline Imagine would each be composed of eight positive events, and No-Imagine and Baseline No-Imagine would be composed of eight negative events. Within a condition pair , we assigned events to conditions algorithmically to match a given manipulation with its baseline as closely as possible. For example, through an automated matching algorithm, we maximized the average similarity of six ratings of event characteristics  across the No-Imagine and Baseline No-Imagine conditions and between the Imagine and Baseline Imagine conditions.We administered mental health questionnaires either once or multiple times  using the secure online survey tool Qualtrics . Last, wThe experiment includes a set of initial training sessions and a follow-up session 3 months later. Here, we describe the procedures of these two segments. The initial stage of the study was conducted in three phases conducted over 5 days: introduction, training, and testing see . In the Each event needed to comply with six rules that were clearly explained to participants. Specifically, events needed to (i) be genuinely positive , negative , or neutral ; (ii) be something that the participant can and has vividly imagined from their first-person perspective; (iii) concern possible developments in the participant’s life that could take place within the next 2 years; (iv) be a specific episode with a particular time and place; (v) only last between a few minutes to 1 day; and (vi) be current concerns or recurrent fears (i.e. thought of at least three times in the past 6 months) in the case of negative events. Upon completion of each template, the experimenter checked to ensure that (i) each event followed the six rules, (ii) no repeats of words are present for Cue Word and Key Detail across all templates, and (iii) Key Detail words do not appear in the tag lines and gave feedback by email if any edits were required. Once all events complied with the rules, the experimenter emailed the participant the ratings task in html generated via PsyToolkit , whereasAfter participants completed the event generation phase, they proceeded to the first day of training ] in either green or red font depending on whether it was an Imagine or No-Imagine trial, respectively. For Imagine trials, we instructed participants to do the following for as long as the green Cue Word remained on the screen: (i) Read the Cue Word silently and recognize the event to which it referred. (ii) Recall the associated Key Detail to mind silently. (iii) Imagine the future event in vivid detail. (iv) Add a novel feature to the event each time the Cue Word repeated. For the two groups that imagined positive events on Imagine trials, we further asked them to create a positive and hopeful feeling each time they imagined the event. For No-Imagine trials, we instructed participants to do the following for as long as the red Cue Word remained on the screen: (i) Read the Cue Word silently and recognize the event to which it referred. (ii) Stop any further imagination of the event by blocking the event and any associated details out and keeping them out of mind. To ensure that participants engaged direct suppression and not thought substitution  three times, yielding 192 trials. Thus, across the whole session, the eight Imagine and eight No-Imagine Cue Words repeated 12 times. We determined the random ordering of conditions (I or NI) over trials once at the study’s outset, ensuring that no more than two consecutive trials came from the same condition; all participants followed this order. Cue Words were presented in a pseudorandom order across the blocks following the same rule as the practice phase. In between runs, we provided a short (up to 60 s) break for the participant to rest their eyes. Diagnostic questionnaire was administered again verbally after the second block . Trial timings matched the timings in the practice phase: fixation cross for 5 s and colored Cue Word for 4 s, with 600-sms isi.At the beginning of the training phase, the experimenter always turned off her video camera, whereas the participants kept their cameras on. After a training session and before ending the activities on the first day, we told participants to not intentionally engage with the materials outside of the training sessions . On the second and third training days, participants followed the same procedure, except that we omitted the criterion test. We conducted the three training days consecutively.After the INI task on the final training day, we tested participants on all 40 of the main target events in the experiment. The testing phase comprised of (i) a recall test in which a correct verbal response of the corresponding Key Detail to the Cue Word presented constituted an accurate response and (ii) a rating task in which vividness and affect ratings were given through html files sent by the experimenter for each of the events. During these tests, events were ordered such that no more than two consecutive trials were drawn from the same condition. After the tests, we included a questionnaire probing participants’ out-of-experiment activities, as well as a series of mental health questionnaires.For the follow-up session, we contacted participants via email to schedule the follow-up session 3 months following the date of their introduction session (±1 week). The follow-up session lasted around 1 hour and consisted of a recall test, vividness and affect ratings, and follow-up questionnaires. The Key Word recall test was the exact same one as was used in the testing phase on the last day of training, with only the time of stimulus display extended from 4 to 5 s.t tests were performed using JASP  to POMP scores, which are calculated as POMP = 100


Digital pathology (DP) is being increasingly employed in cancer diagnostics, providing additional tools for faster, higher-quality, accurate diagnosis. The practice of diagnostic pathology has gone through a staggering transformation wherein new tools such as digital imaging, advanced artificial intelligence (AI) algorithms, and computer-aided diagnostic techniques are being used for assisting, augmenting and empowering the computational histopathology and AI-enabled diagnostics. This is paving the way for advancement in precision medicine in cancer. Automated whole slide imaging (WSI) scanners are now rendering diagnostic quality, high-resolution images of entire glass slides and combining these images with innovative digital pathology tools is making it possible to integrate imaging into all aspects of pathology reporting including anatomical, clinical, and molecular pathology. The recent approvals of WSI scanners for primary diagnosis by the FDA as well as the approval of prostate AI algorithm has paved the way for starting to incorporate this exciting technology for use in primary diagnosis. AI tools can provide a unique platform for innovations and advances in anatomical and clinical pathology workflows. In this review, we describe the milestones and landmark trials in the use of AI in clinical pathology with emphasis on future directions. Some important milestones in computational pathology are as follows:1950: Alan Turing conceived the idea of using computers to mimic intelligent behavior and critical thinking [1956: John McCarthy coined the term artificial intelligence (AI) , 3.1959: Arthur Samuel coined the term machine learning (ML) as “the ability to learn without being explicitly programmed” .1960: Prewitt and Mendelsohn scanned images from blood smear and reported a method to convert optical data into optical density values –7.1965: Computerized image analysis of microscopy images of cells and chromosomes by Judith Prewitt and Mortimer Mendelsohn .1986: Term deep learning (DL) coined by Rina Dechter [1988: Convolutional neural network (CNN) invented by Yann LeCun .1990: Whole slide scanners introduced , 12.1998: Tripath becomes the first company with an automated PAP smear screening product to receive FDA approval –15.2003: Cytyc received FDA approval for their ThinPrep Imaging System –15.2013: Development of photoacoustic microscopy imaging technique .2014: Ian Goodfellow introduced generative adversarial network .2016: MUSE microscopy technique invented to enable high resolution imaging without tissue consumption .2017: Philips receives approval for a digital pathology whole-slide scanning solution (IntelliSite) .2018: FDA permits first medical device using AI to detect diabetic retinopathy in adults (IDx DR) [2021: FDA authorizes the first AI-based software to detect prostate cancer (Paige Prostate) [Machine learning (ML)-based approaches are based on the machine “learning” to make predictions based on the input data and algorithms and falls within the broad ambit of AI , 23. DeeML algorithms can be developed either using intrinsic domain knowledge of pathologists and oncologists (domain-inspired features) or without this inherent domain knowledge (domain-agnostic features). This process is called feature engineering , 58, 59.Image characterization across several disease and tissue types can be better depicted using domain-agnostic features. Examples include nuclear and gland shape and size, tissue texture and architecture. Automated Gleason grading of prostate pathology images has been arrived at using a series of wavelet and tissue texture features enabling machine-based separation of low and high Gleason grade prostate pathology images. Hence both domain-agnostic and domain-specific hand-crafted feature-based approaches have been used for the diagnosis, prognosis, grading, and prediction of response to therapy for various cancers such as breast, prostate and brain tumors .Handcrafted and unsupervised features have several advantages and limitations. Handcrafted features are more transparent and intuitive to the pathologist or oncologist. Domain-inspired features require a strong foundational knowledge of the pathological process and its manifestation within the tissue and thus are more challenging to develop. The unsupervised feature generation-based approach of deep learning strategies lacks feature interpretability though it can be applied quickly and seamlessly to any domain or problem , 61, 62 Recently, promising strides in AI have opened new vistas for significantly altering the way cancer is diagnosed and classified . SeveralDiagnostic algorithms can be incorporated into digital pathology workflow as independent reporting algorithms, as diagnosis-aided tools, and as automated quantifiers of specific features. Independent reporting algorithms can provide diagnosis and automated reports without any input from the pathologists. Screening algorithms can identify normal tissue like colonic, gastric, breast etc. from biopsies. However, it is important to consider the wide varieties of normal tissue during the algorithm development pathway, to avoid missing rare microlesions (such as benign mimickers of cancer) or focal lesions which are rare variants of cancer. Diagnosis-aided tools include algorithms that assess one of the various histological features such as tumor grade, type, and extent. Accurate pathological diagnoses involve assessment and combination of multiple features by the trained human eye. The utility of these AI algorithms is determined by its ease of incorporation into the diagnostic workflow and the added value it brings to the pathologists’ diagnoses. This can be assessed based on the features assessed and the time required to provide results as well as on its accuracy. As an example, breast cancer grading algorithms have the potential advantage of objectivity, inter-reader reliability and prognostic clarity compared to the inter-observer variability seen in clinical practice. Hence in this case, the added value of such an AI algorithm would be better reproducibility rather than efficiency , 72. TheMany digital image analysis (DIA) platforms have been adopted to aid pathologist assessments especially for quantitative biomarker evaluations . AmongstA milestone study in computational pathology is The CAMYLEON16 challenge, the first major challenge on computer-aided diagnosis in histopathology using whole slide images. H&E images from sentinel lymph nodes of breast cancer patients were used with the aim of identifying metastasis. With no time constraint, DL algorithms showed comparable performance to a pathologist in detection of micrometastasis. To simulate a clinical practice setting, time constraint was imposed, which demonstrated an outperformance by algorithm over manual evaluation by 11 pathologists . DiffereAnother key milestone in the field of DP/AI has been the PANDA challenge, the largest histopathology competition thus far. Nearly 1,290 developers joined hands and used 10,616 digitized prostate biopsies for the development of AI algorithms for Gleason grading. The algorithms submitted were selected based on the level of accuracy achieved compared to pathologist on independent cross-continental cohorts. In United States and European external validation sets, the algorithms achieved agreements of 0.862 , 0.840–0.884) and 0.868  with expert uropathologists. However, in order for these algorithms to be applied clinically, across different patient populations, laboratories, and reference standards, prospective clinical trials evaluating AI-based Gleason grading is warranted .To date, the most widely used DL algorithms in pathology applications is convolutional neural networks (CNNs). Defined as a type of deep, feedforward network, CNN consists of multiple sequential layers  that can calculate an output from an input (such as an image), by hierarchically deconstructing the image into low-level cues. Aggregation of these low-level cues, such as edges, curves, or shapes results in the construction of a high-order structure to identify features of interest , 82–87. AI can be used to predict prognosis and therapeutic responses based on histological features , 90. DirThe prognostic implications of AI based tools were also highlighted in 2018 by Saltz et al. who used a convolutional neural network (CNN) to augment pathologist feedback for the automatic detection of spatial organization of tumor-infiltrating lymphocytes (TILs) in images from The Cancer Genome Atlas. Their findings revealed this feature to be prognostic of outcome in 13 cancer subtypes . A similA seminal multi-institutional study published by Beck et al. used a staggering number of morphological and spatial features (6642) to train a prognostic model in breast cancers and demonstrated these features to be associated with overall survival (OS) . A similRecent advancements entail using H&E images to predict genetic alterations by deep learning algorithms. AI tools can be used to derive information about tumor genetics/genomic profiles from morphology and thus help in understanding underlying cancer biology . MoleculWhile the identification of association between morphological patterns and tumor genetics seems to be straightforward, integrating mega volumes of genomic data such as next-generation sequencing (NGS) can be challenging. Studies highlighting the impact of combining NGS data with other features are warranted before implementing such algorithms in clinical diagnostic practice. What further complicates the picture is the lack of in-depth knowledge about the interaction between imaging and genomic features. Although integrating the imaging and molecular features can provide a comprehensive view of individual tumors, however, development, training, and validation of models capable of tackling such sophisticated multidimensional data remains a challenge .AI tools provides critical tools to enhance pathologists’ training, provide helpful annotations and other interactive functions to create a dynamic teaching environment for trainees. This can help integrate a strong knowledge of morphology with the use of novel approaches and advanced technologies in enabling the practice of high-quality personalized and precision medicine. Whole-slide imaging is already used for teaching at conferences, virtual workshops, presentations, and tumor boards , 102. ThImmune checkpoint inhibitors (ICIs) have led to a paradigm shift in treatment of various cancers over the past few years. However, many patients receiving ICIs do not respond to this therapy, and this has resulted in the potential need for combining AI with digital pathology to stratify patients based on likely therapeutic benefit . A studyIn 2016, Wilkinson et al. highlighted the need to improve the infrastructure supporting the reuse of scholarly data and came up with the FAIR guiding principle for scientific data management and stewardship. Data should be easily accessible, operator-independent, and reusable to ensure stringent data management. This is essential for knowledge discovery and innovation, and ensures reusability of data by the community after publication . Buying cloud storage platforms might be costly as well as have challenges in in the massive bandwidth required to transmit gigapixel-sized WSI images into data clouds. Additionally, cloud storage requires uninterrupted fast wi-fi communication between end-users and the cloud. Universal adoption of 5G would improve speed and address some of these difficulties in the future . The cos2 at × 40 magnification resulting in a file size of 48 megabytes! To overcome this, most WSI platforms resort to image compression algorithms  to reduce size significantly. The disadvantage of this compression is the introduction of image artefacts which can compromise overall pixel quality , the European Medicines Agency (EMA) and other regulatory agencies lay down stringent guidelines and frameworks for ensuring scientific rigor of the reported metric. The FDA approval of medical devices is based on a three-class system with Class I devices supposed to have the lowest risk and Class III devices deemed to have the highest risk. AI-based models fall within class II or III, with class III requiring a rigorous premarket approval. 510(k)-approval pathway and De Novo pathway are some other ways of getting AI algorithms approved. Be as it may, the process is highly rigorous and comprehensive –121. In A key roadblock for the incorporation of AI in clinical practice comes from an apprehension about the change in workflow. This is partly because of lack of interpretability and partly due to the somewhat unclear question of performance thresholds of AI algorithms. While there is evidence of decreased error rates and improved performance using a combination of DL-based model predictions with pathologist diagnoses, replacing human evaluation entirely by assessment by machine is met with considerable cynicism . A studyIn the past few years, there has been an increase in the development of AI tools for detection of cancer by various companies like Visiopharm, Halo, Proscia, DeepLens, Inspirata and PAIGE.AI. Of these, Inspirata and PAIGE.AI are actively involved in creating large WSI repositories for training DL-algorithms , 127. FDThe last few years have seen a tremendous growth in the development of novel AI approaches in pathology. These tools, when used intelligently, can improve diagnostic workflows, eliminate human errors, increase inter-observer reproducibility, and make prognostic predictions. While there has been an increase in the development of AI tools, the integration into clinical practice has somewhat lagged owing to several issues related to interpretability, validation, regulation, generalizability, and cost. As the need for personalized cancer care increases, AI applications may be implemented and used appropriately in conjunction with human pathologists, after standardized usage recommendations, and harmonization with current information systems. A multimodal approach using proteomics, genomics, and AI-based multiplexed biomarker quantifications, might be necessary for comprehensive patient-specific tumor precision therapy , 130.


Maternal smoking during pregnancy has been associated with adverse effects on foetal development, including congenital limb anomalies. This systematic review aimed to provide an updated assessment of the association between maternal smoking during pregnancy and the risk of congenital limb anomalies. A systematic search was conducted to identify relevant studies published up to February 2023. Studies reporting on the relationship between maternal smoking during pregnancy and congenital digital anomalies or congenital limb reduction defects were included. Two independent reviewers screened the studies, extracted data, and assessed the quality of the included studies. Meta-analyses were performed to estimate the pooled odds ratios with 95% confidence intervals using fixed and random-effects models. In total, 37 publications comprising 11 cohort and 26 case-control studies were included in the systematic review. The meta-analysis demonstrated a significant increased risk of congenital limb reduction defects  in infants born to mothers who smoked during pregnancy. Similarly, a significant relationship was observed for the development of polydactyly/syndactyly/adactyly when considered as a single group . Yet, in contrast, no significant association was observed when polydactyly  or syndactyly  were considered individually. This systematic review provides updated evidence of a significant relationship between maternal smoking during pregnancy and increased risk of congenital limb anomalies. These findings highlight the potential detrimental effects of smoking on foetal limb development and underscore the importance of smoking cessation interventions for pregnant women to mitigate these risks. Congenital limb defects refer to structural abnormalities that occur during foetal development and affect the limbs of newborns. These abnormalities can vary in severity, ranging from minor deformities like extra fingers or toes, to more severe malformations like complete absence of a limb. Despite being relatively rare, with an estimated prevalence of 5–27 per 10,000 live births ,5,6,7,8,It has long been demonstrated that environmental exposure of the mother to various aetiological agents during pregnancy can result in significant birth defects, including limb anomalies. Among these include teratogenic medications such as thalidomide or anticonvulsants and many others ,17,18,19A previous systematic review published by Hackshaw et al. in 2011 suggested an association between maternal cigarette smoking during pregnancy and the risk of congenital limb defects in children [This systematic review adhered to the Preferred Reporting in Systematic Review and Meta-Analysis (PRISMA) guidelines  39]. A . A 39]. Studies were included in the systematic review if they met the following predefined criteria: Firstly, study designs were limited to original randomised control, case-control, or cohort studies that evaluated the association between maternal smoking during pregnancy and congenital limb defects in children. The included outcomes were restricted to two main subgroups: congenital digital anomalies  and congenital limb reduction defects. These studies could be conducted retrospectively or prospectively, allowing for a wide range of study designs to be included in the analysis. Secondly, only studies that provided sufficient data to calculate odds ratios (ORs) or relative risks (RRs) with 95% confidence intervals (CIs), or provided these statistics in the study, were eligible for consideration of quantitative analysis. Studies reporting outcomes which had insufficient information to obtain quantitative data were included in the presented tables and study but were unable to be considered for meta-analysis. Lastly, this review was limited to studies with human subjects, as the review focused on the impact of maternal smoking during pregnancy on congenital limb defects in human children.On the other hand, studies were excluded from the systematic review if they were case reports, reviews, conference presentations, cross-sectional studies, editorials, letters to the editor, animal studies, or if they did not report relevant outcomes. These exclusion criteria were applied to ensure that only original research studies with relevant and reliable data were included in the analysis, while excluding studies that were not primary research or lacked relevant outcome data. The inclusion and exclusion criteria were applied in a systematic and rigorous manner to select studies that were most relevant to the research question and ensure the robustness of the systematic review. Titles and abstracts of all identified articles in the search were reviewed by two independent reviewers (JC/OS) who conducted the study selection process, and any discrepancies were resolved through discussion and consensus or the involvement of a third reviewer where necessary (JG). Data were extracted into data extraction tables into a shared spreadsheet in Microsoft Excel. A comprehensive and systematic approach was employed to gather relevant information from each included study. Multiple data points were carefully extracted to provide a comprehensive overview of the included studies. These data points included the title, authors, publication year, country of the study population, study design, study period, data source, details of exposed group and controls, period of smoking, cases of congenital limb defects among each group, confounding factors adjusted for, and study outcomes measured. Smoking status was considered as a binary variable, where studies had stratified patients by numbers of cigarettes per day; where possible, numbers were totalled to compare smokers and non-smokers dichotomously. Similarly, for congenital digital anomalies, the presence of any of polydactyly, syndactyly, or adactyly was considered as a case given that these were the consistent variables reported amongst included studies. The presence of other digital anomalies such as clinodactyly, brachydactyly, macrodactyly, and others were not reported amongst any found study and therefore were not assessed. The quality of the included studies was assessed using the Newcastle–Ottawa Scale (NOS) for case-control and cohort studies . The NOSWhen assessing the comparability of each study, stars were awarded to studies if they had adjusted their analyses for one or more of maternal age/paternal age, maternal diabetes, maternal obesity, family history of limb anomaly, and maternal alcohol consumption. These confounders were chosen as previous studies have displayed evidence that these factors may potentially influence the formation of congenital limb anomalies in children ,43,44,452 statistic and pooled odds ratios with 95% confidence intervals were calculated using fixed-effects models when heterogeneity was low (I2 < 50%) or random-effects models when heterogeneity across studies was high. Meta-analyses were performed separately for the two subgroups of congenital digital anomalies and congenital limb reduction defects. Studies were deemed of sufficient quality to be included in the meta-analysis if they were assessed to be of “fair” or “good” quality as determined using the Newcastle–Ottawa Scale. Publication bias was assessed using funnel plots and visually inspected for asymmetry. All statistical analyses were conducted using statistical software using the Review Manager program [A meta-analysis was conducted to estimate the overall effect size of the association between maternal smoking during pregnancy and congenital limb defects in children. Heterogeneity was assessed using the I program .A total of 4552 (2406 from PubMed + 481 from EMBASE + 127 from CINAHL + 1538 from Web of Science) studies were initially identified through a comprehensive search of electronic databases and additional sources. After removing duplicates, 4515 studies were eligible for initial review. Following screening of study titles and abstracts, 106 studies were selected for full-text review. Five studies did not have available full texts. Following the full-text review, 37 publications were finally included in this systematic review based on the predefined inclusion and exclusion criteria .The included studies were published between 1978 and 2022 and were conducted in various countries, including the United States, Sweden, Denmark, China, Japan, Finland, Hungary, Norway, Poland, and Australia. In total, 38 studies from 37 publications, comprising 11 cohort and 27 case-control studies, investigated the association between maternal cigarette smoking during pregnancy and the risk of congenital limb anomalies (one publication included descriptions of two case-control studies from different populations and methodologies ). This iThe quality of the included studies was assessed using the Newcastle–Ottawa Scale for cohort and case-control studies. In total, six studies were considered of “good” quality, 14 of “fair” quality, and 18 of “poor” quality . Most stThe funnel plot  for congAs previously mentioned, meta-analyses in this study were performed only on studies that were deemed of good or fair quality as assessed by the Newcastle–Ottawa Scale.In total, 12 studies provided data on the association between maternal cigarette smoking during pregnancy and congenital digital anomalies, including polydactyly, syndactyly, and adactyly. Nine were of sufficient quality to be included in the quantitative analysis. A meta-analysis was conducted to estimate the pooled odds ratio (OR) and 95% confidence interval (CI) for the association between maternal cigarette smoking during pregnancy and the risk of congenital digital anomalies.2 of 46% and 39%, respectively, for polydactyly and syndactyly. The meta-analyses of the various outcomes showed no significant association between maternal cigarette smoking during pregnancy and an increased risk of polydactyly . However, this analysis was limited by the fact that it only included two studies.In contrast, the meta-analysis of studies that reported the outcome of polydactyly/syndactyly/adactyly was found to be significant, indicating an increased risk of children developing polydactyly or syndactyly or adactyly among mothers who smoked during pregnancy .In total, 31 studies provided data on the association between maternal cigarette smoking during pregnancy and congenital limb reduction defects, and 13 were of sufficient quality for quantitative analysis. The meta-analysis of these studies showed that maternal cigarette smoking during pregnancy was significantly associated with an increased risk of congenital limb reduction defects  among offspring of smoking mothers compared to non-smoking counterparts. In contrast, the results for individual congenital digital anomalies were not significant, with meta-analyses of studies reporting outcomes of polydactyly and syndactyly individually not being found to show any significant association . Yet, when studies that reported the outcome of any one of polydactyly/syndactyly/adactyly were meta-analysed, a significantly increased risk of congenital digital anomalies among smokers was observed . However, these results must be interpreted with caution as only two studies were available for quantitative analysis for the latter meta-analysis. Overall, there seems to be an association between maternal smoking and the development of limb reduction defects among offspring; however, the results are less clear for congenital digital anomalies. It is unclear why this disparity exists, perhaps the relative paucity of the literature pertaining to congenital digital anomalies has limited the ability of this review to find a clear association if one does in fact exist.These results are consistent with a previous meta-analysis conducted over a decade ago, which also reported a similar positive association between maternal smoking during pregnancy and the risk of congenital digital anomalies and limb reduction defects . This prSmoking during pregnancy has been shown to increase the risk of birth defects through various mechanisms. The toxic chemicals in tobacco smoke, such as nicotine and carbon monoxide, can cross the placenta and directly damage developing foetal tissues. Nicotine, a vasoactive agent, can also constrict blood vessels, reduce blood flow, and impair oxygen delivery to the foetus, causing disruption in development and growth . FurtherIn addition to lifestyle factors such as maternal cigarette smoking or alcohol consumption, numerous other risk factors for congenital limb differences among offspring exist. Genes play a crucial role in the development and patterning of limbs during embryonic development. Mutations or alterations in specific genes can disrupt the normal limb development process, leading to congenital limb anomalies. For example, mutations in genes such as the HOX gene family, SHH gene, and TBX genes (among many others) have been associated with limb development abnormalities . FurtherThis systematic review has several strengths. Firstly, this review employed a broad and comprehensive search strategy to identify relevant studies, which can help minimize the risk of missing important evidence. Additionally, the study selection process was rigorous, involving careful screening of studies based on predefined inclusion and exclusion criteria. Furthermore, the review included a methodical assessment of study quality, ensuring that only studies of sufficiently high quality were included in the analysis.However, there are several limitations to consider. One limitation is the lack of high-quality studies assessing the relationship between cigarette smoking and congenital digital anomalies. While we found 12 studies assessing this relationship, the various outcomes reported in each study differ, making meaningful meta-analysis of these outcomes difficult. Hence, the relationship observed between maternal smoking and the development of polydactyly/syndactyly/adactyly was based on only two studies in the meta-analysis, making this relationship less reliable. Another limitation is the potential for recall bias in case-control studies that used interviews as a method of investigation. Interviews rely on self-reporting by participants, which can be subject to recall bias and may not always accurately capture exposure information, such as smoking during pregnancy. Additionally, despite efforts to only include studies in the meta-analysis that had adjusted for confounding variables, residual confounding may still be present in the included studies. For example, while the review mentioned adjustment for significant confounding variables such as maternal age, diabetes, obesity, family history of limb anomaly, and alcohol consumption, there may be other unmeasured or unknown confounding factors that could impact the relationship between maternal smoking and congenital limb anomalies. A study protocol was not registered prior to the conduct of this study. Furthermore, publication bias may be a limitation, as indicated by the asymmetric funnel plot in congenital limb reduction defects. Additionally, unfortunately we were unable to assess a dose–response relationship between studies due to inconsistent reporting of number of cigarettes smoked by mothers in the included studies. Lastly, there was no consideration of the effect of passive smoking or second-hand smoke exposure in the meta-analysis. Maternal exposure to second-hand smoke during pregnancy has been shown to be connected with congenital defects and limb deficiencies, and could potentially confound the relationship between maternal smoking and congenital limb anomalies as the non-smoking mothers may experience similar detrimental effects of cigarette smoke from second-hand smoking ,57,87. This systematic review provides updated evidence of the relationship between maternal cigarette smoking during pregnancy and congenital limb anomalies, specifically congenital digital anomalies and congenital limb reduction defects. The detrimental effects of smoking on foetal development are well-established, and our review further reinforces the harmful impact of maternal smoking on limb development. Further research is warranted to better understand the underlying mechanisms linking maternal smoking and congenital limb anomalies, particularly congenital digital anomalies, and to explore potential strategies to mitigate these risks. This review underscores the critical need for effective smoking cessation interventions for pregnant women to ensure optimal foetal development.


Although the biliary tract is a small organ, it is diverse in location  and disease , thus a multidisciplinary approach involving endoscopists, surgeons, oncologists, radiologists, and infectious disease specialists is often required to treat biliary tract disease. In particular, obstructive jaundice and cholangitis associated with biliary stasis are common complications, and biliary drainage by endoscopic retrograde cholangiopancreatography (ERCP) is an indispensable procedure. In the past, percutaneous transhepatic biliary drainage (PTBD) has been performed in cases of difficult ERCP, such as surgical altered anatomy (SAA) or duodenal obstruction, but there have been many incidents and problems with external drainage, which reduce the patient’s quality of life. Recently, new endoscopic techniques such as balloon endoscopy-assisted ERCP (BE-ERCP) and endoscopic ultrasound-guided biliary drainage (EUS-BD) have been developed and are widely used as alternatives to PTBD and ERCP.Among EUS-BD procedures, EUS-guided hepaticogastrostomy (EUS-HGS) has traditionally been performed only in high-volume centers because of its high technical difficulty and the possibility of serious complications such as biliary peritonitis and stent migration, but recent advances in devices and the accumulation of knowledge on techniques ,2 have mBoth EUS-BD and BE-ERCP are useful in cases of SAA, but each procedure has its own advantages and disadvantages, and it is important to select the method according to the patient’s condition and the endoscopist’s experience . EspeciaFor the management of combined malignant biliary and duodenal obstruction, double stenting is required. Traditionally, transpapillary biliary drainage with ERCP and duodenal stenting have been used, but the technical difficulty of transpapillary drainage and duodenobiliary reflux have been a problem. Recently, EUS-guided novel methods such as EUS-BD- and EUS-guided gastrojejunostomy (EUS-GJ) for duodenal drainage have been developed; moreover, new metal stents, such as an anti-reflux metal stent (ARMS) and lumen-apposing metal stent (LAMS), have been developed, which are expected to provide better treatment results .The gold standard of treatment for acute cholecystitis is laparoscopic cholecystectomy, but gallbladder drainage is performed in patients with a high surgical risk. In recent years, many reports have demonstrated the usefulness of endoscopic gallbladder drainage including transpapillary drainage and EUS-guided drainage as alternatives to percutaneous transhepatic gallbladder drainage (PTGBD); however, the long-term preventive effect on recurrent cholecystitis has not yet been fully clarified . FurtherThe current standard of care for unresectable hilar malignant biliary obstruction is bilateral drainage with an uncovered self-expandable metal stent (UCSEMS). However, recent advances in chemotherapy have prolonged survival and increased the chance of stent occlusion, and unremovable UCSEMS have become a problematic barrier to reintervention. A fully covered SEMS (FCSEMS) can be removed and is expected to have a good patency period due to its larger diameter compared to a plastic stent , but theThe standard treatment for ampullary tumors is pancreatoduodenectomy, but adenomas and carcinomas in situ can be cured by local resection without lymph node dissection. Local resection includes endoscopic papillectomy (EP) and surgically transduodenal ampullectomy, but the less invasive EP is considered the first choice when possible .Although transpapillary drainage is the gold standard for the treatment of acute cholangitis, there is no settled opinion regarding the duration of antibiotics administration. Recently, shorter durations have been recommended to prevent the emergence of resistant organisms, shorten hospital stays, and reduce costs. For mild or moderate cholangitis, two days of antibiotics administration may be sufficient  if an apIn recent years, evidence regarding chemotherapy for biliary tract cancer has been increasing. In addition to conventional cytotoxic agents, many clinical trials have been conducted on molecularly targeted agents and immunotherapy . As for This Special Issue contains various articles on the recent advances in the clinical aspects of biliary tract diseases. Although the COVID-19 pandemic had a major impact on healthcare worldwide, it was possible to diagnose and treat biliary tract diseases without delay by taking appropriate exposure prevention measures . It is h