Because acute small bowel ischemia has a high mortality rate, it requires rapid intervention to avoid unfavorable outcomes. Computed tomography (CT) examination is important for the diagnosis of bowel ischemia. Acute small bowel ischemia can be the result of small bowel obstruction or mesenteric ischemia, including mesenteric arterial occlusion, mesenteric venous thrombosis, and non-occlusive mesenteric ischemia. The clinical significance of each CT finding is unique and depends on the underlying pathophysiology. This review describes the definition and mechanism(s) of bowel ischemia, reviews CT findings suggesting bowel ischemia, details factors involved in the development of small bowel ischemia, and presents CT findings with respect to the different factors based on the underlying pathophysiology. Such knowledge is needed for accurate treatment decisions. Acute small bowel ischemia is an abdominal emergency that accounts for about 2% of gastrointestinal illnesses. Due to the aging of the population and the consequently growing rate of degenerative vascular diseases, its incidence has increased . Acute sCT findings suggestive of bowel ischemia can be specific or non-specific. As small bowel ischemia can be attributable to small bowel obstruction or mesenteric ischemia and CT findings depend on the underlying factors and pathophysiology, they must be known for the accurate interpretation of CT images.This review describes the definition and mechanism(s) of bowel ischemia, reviews CT findings suggesting small bowel ischemia based on its mechanism(s), and provides insight into factors involved in the development of small bowel ischemia by referring to specific CT findings. Such knowledge is needed for accurate treatment decisions.Bowel ischemia includes potentially life-threatening conditions that reduce the blood flow to the bowel. Acute bowel ischemia is characterized by three stages that reflect the extent of bowel-wall involvement. At stage I, the disease is reversible and pathologically characterized by necrosis, erosion, ulceration, edema, and hemorrhage localized to the mucosa. In stage II, necrosis extends into the submucosal and muscularis propria layers. In stage III, transmural necrosis involves all three layers . The morArterial or venous obstruction can result in bowel ischemia. In the presence of arterial occlusion eliciting ischemia or transmural infarction, the small bowel is dilated and presents a classic “paper-thin wall” appearance attributable to a loss of bowel-wall tissue, the vasculature, and the muscle tone due to nerve and muscle damage .As an interruption in the blood supply results in ischemic injury that rapidly damages metabolically active tissues including bowel tissue, effective reperfusion by re-establishing the blood flow is critical to salvage ischemic tissues. Ineffective reperfusion, on the other hand, can lead to ischemia–reperfusion injury  and exacVenous obstruction increases the hydrostatic pressure on the bowel wall because high-pressure arterial inflow may persist despite venous occlusion. Vascular engorgement and edema of the bowel wall lead to extravascular fluid leakage into the bowel wall and mesentery. Venous drainage impairment ultimately results in the loss of the arterial supply and subsequent ischemia and infarction. Mesenteric edema and extravascular fluid tend to be seen more often in venous than in arterial ischemia .Among patients with small bowel obstruction, 10% develop bowel ischemia  because Imaging studies are important for the evaluation of bowel ischemia. Multidetector CT (MDCT) is the most sensitive and specific diagnostic tool for bowel ischemia; it should be the first-line imaging modality. MDCT findings can help to exclude other factors producing acute abdominal pain. To include the entire course of the intestine, CT images should be acquired from the dome of the liver to the level of the perineum.Both non-enhanced and biphasic contrast-enhanced CT images are needed . NonioniAbnormal bowel enhancement confirms the suspicion of bowel ischemia. While conventional single-energy CT relies on a single-energy spectrum to obtain attenuation-based images, dual-energy CT (DECT) facilitates material decomposition such as iodine quantification by acquiring two sets of images of the same body site by applying different photon spectra (high and low kV). Because DECT allows the accurate estimation of contrast enhancement based on iodine quantification on an iodine map, it is preferable to conventional single-energy CT for the evaluation of bowel enhancement in patients suspected of bowel ischemia –20.On unenhanced CT images, the affected bowel wall is highly attenuated due to intramural hemorrhage and hemorrhagic infarction  Fig. 2)2). The aAir in the bowel wall on CT scans is indicative of pneumatosis intestinalis Fig. ; it ofteIn patients with arterial bowel ischemia, the involved bowel wall may be thin. Its paper-thin appearance is attributable to a loss in the volume of tissues and vessels and to the loss of the intestinal muscle tone in patients with serious arterial occlusive ischemia or bowel infarction (inflow obstruction) Fig. . MesenteBowel-wall thickening can be seen in the presence of arterial occlusion after reperfusion , 29. On Bowel-wall thickening and hyperenhancement due to hyperemia of the bowel are also seen in venous ischemia , 22 Fig. The preIn patients with bowel obstruction, the venous outflow and then the arterial inflow are obstructed . ConsequAcute mesenteric ischemia can be defined as a sudden interruption in the blood supply to a segment of the small intestine. If untreated, it can result in ischemia, cellular damage, intestinal necrosis, and eventual death. It is uncommon in patients reporting abdominal pain and its overall incidence is low; only 0.09–0.20% of all acute admissions to emergency rooms are due to acute mesenteric ischemia. A prompt diagnosis and intervention are essential to reduce its high mortality rate (50–80%) [Acute mesenteric arterial occlusion can be due to arterial embolism or thrombosis. Among patients with acute mesenteric ischemia, 50% present with arterial emboli . Emboli In most instances, emboli or thrombi in the mesenteric arteries are clearly depicted on contrast-enhanced CT images Figs.  and 9. OThe mortality rate is high (60–80%) . PeritonNon-occlusive mesenteric ischemia (NOMI), defined as intestinal hypoperfusion in the absence of vascular occlusion, is seen in 5–15% of all patients with acute mesenteric ischemia; its mortality rate is approximately 50%. NOMI is usually seen in patients with debilitating comorbidities such as shock, cardiac disorders, post-operative stress, pancreatitis, burns, dehydration, and hypovolemia . CardiacA 50% decrease in the blood flow through the SMA triggers autoregulation, and compensatory vasodilation develops in the splanchnic circulation. When hypoperfusion lasts for several hours, it ceases to be effective; the consequence is mesenteric vasoconstriction. However, this situation is reversible if the decline in the flow through the SMA is corrected promptly. When splanchnic vasoconstriction lasts more than 30 min, it is irreversible even after total reestablishment of the arterial blood flow . NOMI paInvasive digital subtraction angiography (DSA) is the gold standard for diagnosing NOMI because it demonstrates signs like a blood flow reduction, areas of vessel narrowing and spasm, and consecutive irregularities in the mesenteric vessels , 39. AltIn the management of NOMI, the underlying precipitating factor(s) are treated when possible to facilitate mesenteric perfusion. Fluid resuscitation, optimization of the cardiac output, and the elimination of vasopressors are important primary measures. Additional treatments may include systemic anticoagulation therapy and direct catheter infusion of vasodilatory and antispasmodic agents; the most commonly administered drug is papaverine hydrochloride . In patiIn 5–15% of patients with acute mesenteric ischemia, mesenteric venous thrombosis is observed. Mesenteric venous thrombosis is secondary to underlying diseases such as portal hypertension, hypercoagulation due to protein C- and protein S-deficiency, polycythemia, a factor V Leiden mutation, right-sided heart failure, abdominal trauma, abdominal infection, acute pancreatitis, malignancies, nephrotic syndrome, and cirrhosis which are contributing factors in 50–75% of all mesenteric venous thrombosis patients , 46, 47.As thrombi in the mesenteric veins are clearly visualized on contrast-enhanced CT images Fig. , such stIn general, the outcome in patients with mesenteric venous thrombosis, whose mortality rate is approximately 44%, is better than in patients with arterial ischemia such as mesenteric arterial occlusion and NOMI . Non-opeSmall bowel obstruction is an important etiology of bowel ischemia. It is due to intrinsic or extrinsic processes that can be benign or malignant. The most common factors are adhesion, hernia, and malignant tumors . AlthougSmall bowel obstruction can cause the material inside the bowel to back up into the stomach, resulting in nausea and vomiting of dark green bile (bilious vomiting). The bowel preceding the obstruction becomes large, dilated, and filled with the fluid and air that would otherwise move forward. This elicits bloating  . In patiStrangulation is observed in 5–42% of patients with small bowel obstruction ; their mAcute small bowel ischemia has a high mortality rate and requires immediate intervention to avoid unfavorable outcomes. As clinical and laboratory signs of bowel strangulation lack sensitivity for predicting bowel ischemia, CT imaging studies are important for treatment decisions. Small bowel ischemia can be induced by arterial ischemia such as acute mesenteric arterial occlusion and NOMI, venous ischemia including mesenteric venous thrombosis, and increased luminal pressure in the setting of small bowel obstruction. CT findings suggesting bowel ischemia vary depending on the factors eliciting arterial ischemia, venous ischemia, and increased luminal pressure. Reperfusion may alter the findings in patients with arterial bowel ischemia. Therefore, to reach accurate treatment decisions, the correct interpretation of CT images requires an understanding of the factor(s) implicated in the development of small bowel ischemia.


For an improved understanding of disease progression, tissue formation during development, and tissue regeneration, labeling extracellular matrix (ECM) components secreted by cells persists is required. Bioorthogonal chemistry approaches offer solutions to visualizing and labeling ECM constituents without interfering with other chemical or biological events. Although biorthogonal chemistry has been studied extensively for several applications, this review summarizes the recent advancements in using biorthogonal chemistry specifically for metabolic labeling and visualization of ECM proteins and glycosaminoglycans that are secreted by cells and living tissues. Challenges, limitations, and future directions surrounding biorthogonal chemistry involved in the labeling of ECM components are discussed. Finally, potential solutions for improvements to biorthogonal chemical approaches are suggested. This would provide theoretical guidance for labeling and visualization of de novo proteins and polysaccharides present in ECM that are cell-secreted for example during tissue remodeling or  The visualization and labeling of ECM components secreted by cells are crucial for understanding tissue regeneration. Bioorthogonal chemistry approaches present a promising solution for labeling and visualizing ECM without interfering with other chemical or biological processes. This review focuses on recent advancements in using biorthogonal chemistry for metabolic labeling and visualization of ECM constituents. Moreover, histological staining is not the best option for quantification as the molecular weight of the stained ECM polymer, its charge state, and the ionic content of the explanted sample can interfere with achievable color intensity, leading to an incorrect conclusion  azide-alkyne cycloaddition reaction is a promising and noninvasive method for labeling settled and newly synthesized proteins in living systems. To enable the tagging of the proteome, non-canonical amino acids carrying reactive handles can be incorporated, allowing for subsequent detection of the labeled proteins ,78. One in vitro study by Amgarten et al. [in vitro without the need for the destruction of grafts. In this study, they seeded azido-proline treated cells onto 3D fluorescent poly (lactic-co-glycolic) acid (PLGA) scaffolds and placed constructs into a media containing Click-IT Alexa Fluor 488 DIBO Alkyne. Their findings demonstrated that the collagen produced by cells had been modified with azide-proline efficiently and the control group was shown to exhibit little background staining.Proline has been widely explored as a marker of protein biosynthesis in tissue engineering applications, especially for collagen which is the most abundant mammalian protein ,27. Azidn et al. , ovine on et al.  used simBesides, AHA has been employed in different research studies to label proteins containing methionine amino acids. For example, Loebel et al.  used AHA, and it also allows the evaluation of different parameters such as biophysical, biochemical, and mechanical cues on protein formation and deposition. Thus, this strategy is a powerful tool for monitoring matrix accumulation in regenerative medicine approaches and diagnosing diseases where abnormal collagen production is the main problem, such as diabetic nephropathy, cirrhosis, or idiopathic pulmonary fibrosis.This bioorthogonal approach using proteins-segment analogs enables type-specific protein visualization3.2The analysis of glycans has gained significant traction in the fields of biological research, clinical analysis, and pharmaceutical biotechnology production. This is due to the identification of distinct glycosylation patterns that are associated with a variety of health and disease states. As a result, glycan analysis has emerged as a valuable tool for investigating the molecular mechanisms underlying these conditions and developing novel therapeutic interventions . There aN-azidoacetylmannosamine (ManNAz), N-azidoacetylgalactosamine , N-azidoacetylglucosamine (GlcNAz), and fucose‐containing glycans are abiotic azide-derivatives of naturally occurring monosaccharides which are mostly used for metabolic labeling of glycans [Identification and quantification of glycans/polysaccharide formation can be also monitored by metabolic labeling with unnatural sugars followed by chemical reactions with imaging probes. Azide sugars such as  glycans . As demo glycans ,92. This glycans .Fig. 4Th4, and they cannot demonstrate the variation with time or the localization of the deposited matrix. Bioorthogonal chemistry enables noninvasive in situ monitoring of ECM, For instance, Loebel et al. [N-azidoacetyl-mannosamine to visualize proteins and proteoglycans, respectively, and then, harvested by first staining live cells using a fluorophore-conjugated cyclooctyne (DBCO-488) to fluorescently label AHA-containing proteins or N-azidoacetyl- mannosamine-tetraacylated -containing proteoglycans  polymer was synthesized through electrospinning. This polymeric scaffold was transplanted in mice in vivo and after 6 h macrophages that were labeled with tetraacylated N-azidoacetylmannosamine (Ac4ManNAz) were injected into the abdominal cavity of the same mice. They used this strategy to enable a controllable in situ cellularization of scaffolds to enhance their biological functions and tissue regeneration.In the field of tissue engineering and regenerative medicine, ECM plays a critical role. ECM formation along with temporal and spatial positioning of proteins and polysaccharide-containing structures deposited by cells can indicate tissue regeneration status. Different methods such as immunohistochemistry, histology, and microscopy techniques have been used for ECM visualization . Howeverl et al.  employedoglycans a. Fig. 5l-ascorbate. 1,3,4,6-tetra-O-acetyl-N-azidoacetylgalactosamine (50 μM) was added to the cell culture media and the flasks were incubated for 6 days  to produce an azide-modified ECM. Half of the culture medium was carefully removed every two to three days and replaced by fresh medium. The secreted azide-modified cells were fixed with formalin and later fluorescently labeled with an alkyne-modified fluorophore, employing a copper-catalyzed azide-alkyne cycloaddition reaction and detected using a confocal laser scanning microscope . One day after the last administration of the metabolic labeling reagents, organs were harvested from the animals and decellularized. For ex vivo labeling, freshly isolated porcine left lungs were cultured ex vivo in bioreactors for 24 h in the presence of GalNAz and decellularized. These experiments demonstrated that the azide-labeled acellular scaffolds can be immobilized of alkyne-modified biomolecules interest by click reaction (ECM-based biomaterial or cell-derived matrices have now emerged as a promising candidate to develop scaffold for tissue engineering applications . ECM crecroscope e. In ano process f and g. reaction h.Other research studies exist that utilized the bioorthogonal chemistry approach for labeling. Functionalization of ECM can be used to develop a bio-scaffold for different tissue regeneration approaches. Additionally, a similar strategy can be used to conjugate different molecules such as drugs and growth factors to the ECM scaffold for a more efficient regeneration and repair process. In 5in vitro and in vivo processes characterized by the relative abundance of the protein of interest or using additional technologies to demonstrate relative specificity [Even with great advancements in biorthogonal labeling techniques in recent years, noted limitations still exist with many forms of biorthogonal labeling chemistry. First, bioorthogonal labeling chemistry involving Copper or other metal-catalyzed reactions has shown cytotoxic effects on cells . Metal-fcificity . Biorthocificity .Further limitations include poor labeling due to the insolubility of some proteins, especially ECM proteins. Click chemistry reactions like CuAAC used in metabolic labeling have been shown to have poor efficiency with targets having limited solubility . To counOther concerns of note with metabolic labeling of ECM components are physiological relevance and how the introduction of foreign biorthogonal chemicals into the system will perturb the results. Whenever a foreign chemical entity is introduced into a biological environment, that environment will undergo some form of change or disruption in response to that foreign species regardless of whether that chemical species is bioorthogonal. The cell is a crowded place with a high density of many types of molecules of different sizes including proteins, sugars, and lipids. When a foreign chemical used as a metabolic label is introduced into the biological system, that chemical can physically hinder other molecules' and proteins’ ability to bind or react by simply being in the way of native molecules as found under normal physiological conditions. Furthermore, a foreign chemical species may interact non-chemically with any number of other molecules and disrupt diffusion or change the pH of the system for example. The unnatural chemicals can directly compete with native metabolites and proteins. Furthermore, depending on the method of metabolic labeling used, the unnatural chemicals may be less efficiently incorporated or metabolized compared to the natural chemicals present and may lead to unwanted and problematic metabolites . The sizChallenges to overcome with biorthogonal chemistry ECM labeling techniques in the future include improving upon the efficiency and precise quantifiability of labeling techniques while also reducing any cytotoxic or cell-compromising and physiologically detrimental effects. Even if the ECM labeling chemistry remains biorthogonal, some effects may prove to be detrimental over longer term labeling due to bioaccumulation over time from the cell's inability to degrade byproducts of biorthogonal reactions. Additionally, distinguishing between or applying differential labeling between already existing ECM components and newly formed ECM pieces remains a challenge. Research teams have already begun addressing these issues by incorporating multiple forms of bioorthogonal labeling simultaneously such as protein labeling and glycosylation-enabled labeling . With th6In conclusion, biorthogonal chemistry allows visualization and quantification of ECM component synthesis in living cells and tissues in real-time. By enabling quantification and visualization of the growing and living ECM, complex ECM dynamics can be more thoroughly investigated and understood. These techniques will help pave the way for better therapeutics that target ECM modalities and a wider understanding of how the ECM changes in various pathologies. Improving upon the challenges and limitations of bioorthogonal chemistry for labeling the ECM will further advance our understanding of the complexities involved in the ECM and related pathologies. Future directions in biorthogonal chemistry used in ECM labeling include combining different biorthogonal approaches simultaneously to deliver a more powerful understanding of the ECM with studies that combine glycomics, proteomics, and the interface between cells and biomaterials. Other possibilities include reducing the metabolic footprint of bioorthogonal chemistry introduced into living systems to improve upon the physiological relevance of the labeling method.Shima Tavakoli: Conceptualization, writing original draft, visualization and editing, Austin Evans: Writing original draft, reviewing and editing, Oommen P. Oommen: Reviewing original draft and editing, Laura Creemers: Reviewing and editing, Jharna Barman Nandi: Reviewing and editing, Jöns Hilborn: Reviewing and editing, Oommen P. Varghese: Supervision, conceptualization, reviewing original draft and editing.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.


Using advanced neuroimaging techniques, such as electroencephalogram (EEG), magnetic resonance imaging (MRI) and positron emission tomography (PET), researchers have been working on the mechanisms of human brain under both healthy and different disorders for decades. The development of robust neuroimaging analytical methods will fundamentally advance scientific understanding of the brain and facilitate numerous neuroscience and clinical research studies.This second volume of the Research Topic continues to provide a forum for researchers who are interested in the recent developments of neuroimaging methods and their applications in various domains. In the end, a total of six papers, including four original research articles, one method and one review, have been collected in this Research Topic. These included papers reported new neuroimaging findings and analytical methods helping to uncover the underlying brain mechanism.Wu et al. proposed a novel lightweight multi-scale aggregation mechanism for EEG pathology detection. They extracted the discriminative multi-scale features by aggregating local and global information, and further integrated these features with patients' age as multimodal features for raw brain signals. The experimental results prove the validity and feasibility of their proposed method. They also verified the significance when incorporating patients' age information in EEG pathology detection.EEG recordings contain vast amounts of physiological and pathological information, which can be useful in the diagnosis and analysis of brain diseases. To address the issue of feature redundancy when extracting EEG features and investigate the importance of patients' age information in EEG detection, Liao et al. investigated the alterations of dynamic intrinsic brain activity among 111 patients with Alzheimer's disease (AD), 29 patients with mild cognitive impairment (MCI) and 73 healthy controls (HC). They reported that patients with AD displayed altered dReHo variability in the right middle frontal gyrus and posterior cingulate gyrus compared to the MCI and HC groups. The dReHo variability in these two brain regions may serve as a potential biomarker for the diagnosis or prognosis of AD. Their study provides a new perspective to uncover the underlying neuropathological mechanisms associated with AD. In the study of Zhu et al., multiple rs-fMRI analyzing methods, including amplitude of low-frequency fluctuation, regional homogeneity, degree centrality and functional connectivity, were used to explore whether resting brain function differs between unaffected parents having Autism spectrum disorder (ASD) children and parents having typically development children. The findings revealed the presence of multiple aberrant brain regions and functional connectivity patterns in unaffected parents having ASD children. Notably, these anomalous rs-fMRI patterns exhibit similarities to the neuroimaging results reported in previous literature on individuals with ASD. Correlation analysis results further reflect that unaffected parents having ASD children may have subclinical elevations in ASD traits. Their study indicates that rs-fMRI metrics could be taken as biomarkers to reveal the neurobiological characteristics of unaffected parents having ASD children.The utilization of functional MRI (fMRI) technique allows for the investigation of underlying mechanisms of brain function, which has garnered widespread attention from researchers. Two resting-state fMRI (rs-fMRI) studies examined the altered intrinsic brain activity in patients with different disease. By using rs-fMRI and dynamic regional homogeneity (dReHo), Zhang et al. evaluated gray matter volume (GMV) alterations and the GMV alterations chronological process in patients with type 2 diabetes (T2D) over the course of their disease. They found that the right temporal pole could be the origin brain region affected by T2D and the GMV alterations in patients with T2D followed a progressive pattern, targeting regions in the limbic-cerebellum-striatal-cortical network. Their work provides a clue to the likely GMV progressive changing pattern of T2D, which may contribute to a better understanding of the progression and an improvement of current diagnosis and intervention strategies for T2D. In order to facilitate researchers to carry out surface-based measurement analysis, Long developed a user-friendly graphical user interface toolbox called “surface-based processing and analysis of MRI” (SPAMRI) based on MATLAB platform, which is compatible with different OS . This toolbox contains pipelines for data preprocessing, statistical analysis, multiple comparison correction, result reporting, extraction of surface measurements and seed-based structural covariance analysis. This toolbox enables a seamless surface-based image processing and MRI dataset analyses, thereby offering convenience to researchers, particularly clinicians and those new to MRI, in their exploration of structural morphologies.Structural MRI technique has attracted increasing attention in brain morphometric studies, allowing for the accurate measurement of brain structures, including volume and surface-based morphometric measurements. Using voxel-based morphometry and causal structural covariance network analyses, Huang et al. conducted a bibliometric analysis of the neuroimaging research on aphasia to reveal the current hotspots and cutting-edge trends of research in this field. By including 2,799 articles related to the neuroimaging of aphasia from 2004 to 2021, the authors summarized three research hotspots: the dual-stream model of language processing, the mechanism of injury and recovery of post-stroke aphasia, and the diagnostic criteria for primary progressive aphasia variants. They pointed out that future studies should expound the pathological diagnosis of primary progressive aphasia and its relationship with clinical features and MRI imaging by combining with new neuroimaging techniques.Based on the characteristics and advantages of neuroimaging techniques, a growing number of researchers have been focusing on neuroimaging techniques to conduct research on aphasia. A systematic review from In summary, the second part of this Research Topic presented innovative neuroimaging analytical methods and exciting achievements in clinical applications. These papers contribute to a deeper understanding of the physiological and pathological mechanisms of human brain from a neuroimaging perspective, and also promoting the development of new tools and techniques for processing neuroimaging data. We anticipate that this Research Topic will provide novel insights into neuroscience and clinical research. The advancement of sophisticated neuroimaging techniques and their applications will continue to attract more researchers' attention in the future.JD: Conceptualization, Funding acquisition, Investigation, Project administration, Supervision, Writing—original draft, Writing—review and editing. DZ: Conceptualization, Investigation, Project administration, Supervision, Writing—original draft, Writing—review and editing. WL: Conceptualization, Investigation, Project administration, Supervision, Writing—original draft, Writing—review and editing.


Protocols to differentiate human pluripotent stem cells have advanced in terms of cell type specificity and tissue-level complexity over the past 2 decades, which has facilitated human disease modeling in the most relevant cell types. The ability to generate induced PSCs (iPSCs) from patients further enables the study of disease mutations in an appropriate cellular context to reveal the mechanisms that underlie disease etiology and progression. As iPSC-derived disease models have improved in robustness and scale, they have also been adopted more widely for use in drug screens to discover new therapies and therapeutic targets. Advancement in genome editing technologies, in particular the discovery of CRISPR-Cas9, has further allowed for rapid development of iPSCs containing disease-causing mutations. CRISPR-Cas9 technologies have now evolved beyond creating single gene edits, aided by the fusion of inhibitory (CRISPRi) or activation (CRISPRa) domains to a catalytically dead Cas9 protein, enabling inhibition or activation of endogenous gene loci. These tools have been used in CRISPR knockout, CRISPRi, or CRISPRa screens to identify genetic modifiers that synergize or antagonize with disease mutations in a systematic and unbiased manner, resulting in identification of disease mechanisms and discovery of new therapeutic targets to accelerate drug discovery research. However, many technical challenges remain when applying large-scale functional genomics approaches to differentiated PSC populations. Here we review current technologies in the field of iPSC disease modeling and CRISPR-based functional genomics screens and practical considerations for implementation across a range of modalities, applications, and disease areas, as well as explore CRISPR screens that have been performed in iPSC models to-date and the insights and therapies these screens have produced. As CRISPR-based genetic screening techniques become more commonplace and stem cell technologies advance to recapitulate disease relevant biology with increasing level of detail, there is a need to advance implementation of human disease-relevant models in high-throughput screening platforms, which we aim to facilitate by outlining the potential use cases and advantages and disadvantages of these platforms, the practical considerations involved, and existing applications for these technologies.Pluripotent stem cells (PSCs) hold great potential in disease modeling due to their ability to differentiate into all cell types in the human body . There cWith the advent of CRISPR/Cas9 systems in 2012, genome editing and functional genomics screening platforms have likewise seen a rapid technological progression. While the first iteration of CRISPR-based screens relied on targeted single base insertions or deletions (CRISPR knockout), subsequent systems such as CRISPR interference and activation (CRISPRi/a) offer the ability to modulate endogenous gene expression. These CRISPR systems rely on the fusion of functional protein domains to catalytically inactive dCas9, and when run reciprocally in gain- and loss-of-function screens, for example, can identify more complex gene interactions than a knockout screen alone . There aAs the fields of iPSC modeling and CRISPR genome editing progress it becomes increasingly clear that harnessing the power of CRISPR-based screening in complex iPSC-derived models offers the potential to elucidate new and exciting biology in a high-throughput manner. Though the applications and scope of functional genomics screening platforms that utilize PSC-derived cells have been steadily increasing in recent years, several challenges remain that make these techniques more difficult to access than screening in standard cell line models. In this review we discuss the advantages and limitations of disease modeling in PSCs, including the ability to utilize them in more sophisticated model systems, considerations around genetic variation, and publicly available repositories with disease-relevant iPSC lines. Challenges also remain in terms of identifying, optimizing, and implementing a relevant screening assay for a particular biological question. To address these challenges, we discuss types of screening modalities and describe assay and readout capabilities that are compatible with iPSC-models, including strategies to overcome some of the current challenges in the field. In doing so, we outline the practical aspects to consider when designing a CRISPR screen in iPSCs as broken down by key steps within each category  and iPSCs can be differentiated into all the cell types originating from the three germ layers  in additAs noted in point 4 above regarding mutations in cell lines, while PSCs may also manifest with mutations, these can be minimized. Three broad sources lead to accumulation of somatic mutations in iPSCs: pre-existing mutations in the starting somatic cells, reprogramming-induced mutations, and passage-induced mutations, all of which can be avoided. A large proportion of iPSC lines were originally derived from skin fibroblasts, which could explain the higher mutational load in starting somatic cells, as skin cells are exposed to environmental stress and are therefore more prone to acquire mutations. However, iPSCs can also be derived from several other starting cells, such as peripheral blood mononuclear cells (PBMCs) , cells iThe reprogramming method can affect the number of mutations acquired during the reprogramming process. Studies have shown that the use of non-integrating vectors for iPSC reprogramming minimizes the genomic instabilities compared to the use of integrating vectors such as retroviruses . The lenin-vitro. Furthermore, advanced NGS techniques such as whole exome sequencing and whole genome sequencing should be used to estimate the mutational load and prioritize iPSC lines for use in disease modeling and drug discovery.Recent research comparing mutational burden of iPSCs with isogenic embryonic cells during embryogenesis revealed a similar mutational rate and burden , indicatiPSC-derived cells also offer advantages over animal models of disease. It has been reported that about 92% of drugs found safe and therapeutically effective in animal models fail in clinical trials either due to toxicity or inefficacy . AdditioMonotypic differentiation systems discussed above are good for modeling cell autonomous disease mechanisms. However, many diseases have non-autonomous multicellular contributions, and the disease relevance of functional genomics screens improves as the screening model becomes more like the cell type(s) affected by the disease. This represents another opportunity for PSC-derived cell model development, as PSCs can be expanded and differentiated prior to co-culture, making sophisticated multi-culture and assembloid-type platforms possible. Co-culture systems, where two or more PSC-derived cell types are cultured together, and organoid cultures, where PSCs are differentiated to self-organize into 3D structures, have shown great promise in studying cell non-autonomous mechanisms of disease. Several recent studies have shown that co-culture systems were critical for identifying disease phenotypes that arise from cell-to-cell communication. Neuron-astrocyte co-cultures have shown astrocyte contributions to neurological disorders such as ALS , Parkinsversus practicality, as co-culture or organoid systems are often able to do a better job of recapitulating complex disease biology but may be intractable as large-scale screening systems  . LND is Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by loss of upper and/or lower motor neurons. Ropinirole, Retigabine, and Bosutinib are three candidate anti-ALS drugs that have been identified in three separate iPSC-derived motor neuron-based drug screens  and are versus with CRISPR nuclease but may not be a complete loss of expression depending on the cell type and locus. This lack of complete repression can also be a benefit when the goal of the screen is to mimic the effects of small molecule therapeutics to identify candidate gene targets. CRISPRa screens can provide complementary information to CRISPRi screens, where genes are down- or upregulated at endogenous loci and hits can be compared to identify common hits that are enriched in opposing populations with the two screening modalities.Modern functional genomics screening strategies generally involve CRISPR/Cas9-based tools including CRISPR nuclease, CRISPRi, and CRISPRa , modalitOther domains that have been fused to elicit different effects on gene regulation include CRISPRoff/on  and otheCRISPR screens require expression of Cas9 machinery as well as the guide RNA targeting the gene of interest, which can be expressed on the same or different constructs. Most screens using iPSC-derived cells with stable expression of CRISPR/Cas9 machinery involve generation of iPSCs with genetically integrated Cas9 machinery and lentivirally expressed guide RNAs . AlternaLarge-scale screens can involve different types of manipulations, from screening across small molecule libraries to genetic screens using siRNA, cDNA-based overexpression, or CRISPR guide libraries, among other possibilities. The readouts can also range broadly, with two main categories: arrayed screens, which are generally performed in multi-well plates with different perturbations tracked in each well, and pooled screens, which are generally performed in large culture vessels with all perturbations combined and read out downstream . GeneralThe appropriate readout and assay to use for a screen depends on the biological question of interest. Typical pooled screen readouts include survival/dropout of cells over time, FACS-based sorting or selection of a dye, protein, or reporter of interest, or single cell sequencing readouts . ArrayedThe utility of PSC-derived cells for disease modeling requires identification and optimization of differentiation protocols to generate the relevant cell type, as described above. Equally important is adequate testing to ensure that the cellular phenotype and/or function of interest is exhibited. Examples of cases where this has been applied include iPSC-derived macrophages, which express macrophage markers, secrete pro-inflammatory and anti-inflammatory cytokines, and exhibit phagocytic activity . IPSC-deOnce an assay and readout are chosen, they need to be optimized and validated to ensure suitability for large-scale functional genomics screening applications. The specifics will depend on the assay, but, in general, it is important to ensure that an assay provides a sufficient dynamic range to capture the signal of interest above noise for the largest possible perturbation window. If the assay involves a stimulus and subsequent readout , then it is important to ensure that the stimulated condition is clearly separated from the control with minimal signal overlap. For genetic validation, it can be helpful to test manipulate some control genes that are expected to demonstrate specific phenotypes in your assay to ensure they give the appropriate phenotypic shift, which provides greater confidence that the screen will adequately capture the biology of interest.The final step in a screening workflow is analysis and hit calling, which again will vary based on assay and readout. The available analysis tools for pooled screens are described and summarized elsewhere , but in Large-scale functional genomics screens implementing iPSC models have been used across several disease areas to gain insights into molecular mechanisms underlying disease biology and to identify new therapeutic targets or pathways of interest. Several cell types and disease areas are highlighted below. .Doxorubicin is a common chemotherapy compound used to treat various cancers but results in severe side effects, including heart failure. A genome wide pooled CRISPR KO screen in iPSC-derived cardiomyocytes was performed and identified two new targets, SLCO1A2 and SLCO1B3, which are both human-specific transporters whose loss of function protects cardiomyocytes against doxorubicin-cardiotoxicity but does not affect cell death in cancer cells .Stem cell-derived islets generated by directed differentiation from hPSCs are a great source for pancreatic β cell replacement therapy to treat insulin-dependent diabetes. In recent years there has been promising clinical trial data using progenitor cells  or fullyExamples of screens performed in the context of neuronal disease include a genome-wide pooled CRISPR-Cas9 knockout (KO) screen in human neural progenitors to identify molecular therapeutic targets that disrupt the host-dependent mechanism of Zika virus infection . AnotherSome of the challenges with traditional neuronal differentiation methods include multi-step protocols that take several weeks, can be difficult to scale up, and usually yield heterogeneous populations of differentiated cells. One promising alternate method for hiPSC-derived differentiation, however, is transcription factor-directed differentiation of hiPSCs. For example, overexpression of neurogenin 2 (NGN2) transcription factor from AAVS1 safe-harbor locus in hiPSCs directs the differentiation into glutamatergic neurons with high efficiency and homogeneity . Tian etPFN1 and INPP5D as novel modulators of phagocytosis in microglia, and variants in these genes can possibly be associated with neurogenerative diseases.Another example of transcription factor-directed differentiation of hiPSCs is generation of microglia-like cells based on the inducible expression of six transcription factors in hiPSCs following an 8-day efficient protocol . In thisLeng et al performed a pooled CRISPRi screen in hiPSC-derived cells, identifying cellular pathways that control cytokine-induced inflammatory astrocyte reactivity . To geneThe rise of CRISPR-based screening techniques over the past decade has drastically increased the power of genetic screening to probe biological questions across a wide range of models. We anticipate that CRISPR screening in iPSC-derived models will expand this capability by allowing for the ability to interrogate disease biology in more relevant contexts. In addition to the CRISPR screening systems discussed in this review, there are several new CRISPR modalities and emerging screening technologies that have the potential to further drive biological discovery.Of particular interest is the emergence of epigenetic remodeling systems that not only offer the ability to cause reversible epigenome-level silencing but could allow us to study how epigenomic regulation plays a role in disease biology and cell identity. One such system is CRISPRoff/on, which deposits targeted methylation marks via its DNMT3A/3L methyltransferase domains and can likewise reverse this modification with the demethylase activity of TET1. Through transient expression of the CRISPRoff system, robust epigenetic silencing can be achieved and has been shown to be maintained in iPSCs through differentiation . Similarin vivo counterparts.Other capabilities that show future promise involve screens in complex organoid and co-culture models, however, there are several current limitations that need to be overcome for these systems to substantially reduce the need for animal experiments, as outlined in a prior review . Organoiin vitro population genetics used 68 iPSC lines to identify metabolic and transcriptomic phenotypes of a SNP for metabolic disease (The rapid increase in the number of GWAS has led to the identification of numerous single nucleotide polymorphisms (SNPs) and eQTLs associated with a variety of disorders. Most eQTLs are identified using expression data from bulk tissue samples or easily accessible cell types, but numerous studies have shown that a considerable proportion of eQTLs are cell-specific, tissue-specific and sometimes even specific for a particular region in the tissue .This pre disease . Future An additional application of GWAS data is to design experiments that would functionally validate disease-associated eQTL and SNP loci to identify the disease-relevant target genes. This is complicated by the fact that the majority of the disease-associated eQTL and SNP loci lie in non-coding regions of the genome, which can regulate their target genes over long genomic distances. A few CRISPR based approaches have shown immense promise in accurately mapping enhancers to their target genes. In one such experiment the authors developed CRISPRi-FlowFISH where the gRNAs were targeted to cis-regulatory elements and the effect on expression of target genes was assessed using RNA FISH . The autversus sufficiency. In addition, the ability to robustly utilize multiple Cas variants in PSCs would allow for multiplexing across modalities, for example, combining a dCas9-CRISPRa system with a dCas12a-CRISPRi system. This type of parallel multiplexing could be used, for example, to dynamically modulate transcription factor up- and downregulation to understand differentiation pathways and deterministically drive differentiation toward lineages of interest.While this review focused largely on Cas9-based CRISPR systems, other Cas enzymes provide additional opportunities, including Cas12a which is more amenable to multiplexing due to the mechanism by which its gRNA arrays are processed. Recent advances in dCas12a approaches for transcriptionally modulating genes without introducing double stranded breaks will allow for more sophisticated studies of pathway redundancy and protein cooperativity . For PSCRecent advances in iPSC model systems combined with our rapidly expanding databases of human genetic and genomic data and our ability to functionally perturb genes and non-coding regions of the genome have allowed for incredible advances in our understanding of human disease biology. Researchers have uncovered novel mechanisms underlying disease predisposition, initiation, and progression and, in doing so, have identified new therapeutic targets. As these fields continue to co-evolve it will be exciting to see how they combine in new ways with new models, screens, and readouts to continue to drive our understanding of human development and disease.


The field of endocrinology, reproduction, and metabolism has long been fascinated by the intricate interplay of cellular dynamics that underlie complex physiological processes. Exploring the complex cellular dynamics that govern these processes has been a pursuit of scientists and researchers for decades. Recent advancements in single-cell analysis techniques have propelled us into a new era of understanding, allowing us to dissect the intricate molecular underpinnings of these systems at unprecedented resolution. In this Editorial, we delve into the implications and potential of single-cell analysis in elucidating the complexities of these fields, focusing on the remarkable insights garnered from three seminal articles.Yang et al. have illuminated the diverse cell subtypes and their subtype-specific marker genes within the placenta. This comprehensive cell atlas not only enhances our understanding of the placenta’s cellular function but also offers insights into GDM’s molecular underpinnings. The implications of this research extend beyond diagnosis, potentially informing novel strategies for GDM treatment and prevention.Gestational diabetes mellitus (GDM) presents a significant challenge to maternal and fetal health. Increasing evidence has suggested that the placenta, a central player in pregnancy, is intricately involved in GDM’s pathogenesis. However, unravelling the complexities of the human placenta has proven elusive until the advent of single-cell RNA sequencing (scRNA-seq). By examining cellular signatures and transcriptomes, Fang et al. have delved into the cellular tapestry of the corpus cavernosum in patients with severe ED. This meticulous analysis has unveiled a myriad of cell populations, revealing the complex cellular heterogeneity inherent to this tissue. Furthermore, this study has illuminated potential contributors to ED, including fibrosis and inflammation. By comprehensively unravelling the cellular landscape, this research not only advances our knowledge of ED’s cellular basis but also lays the foundation for individualized treatment strategies.Erectile dysfunction (ED), a condition often rooted in the intricate connections between reproduction and metabolism, has remained a clinical challenge. The corpus cavernosum, a critical component of the male reproductive system, harbours crucial insights into ED. Employing single-cell RNA sequencing, Fan et al. have probed the role of histone phosphorylation in HCC prognosis. By constructing a prognostic risk signature based on the expression of specific genes, they have provided insights into the intricate interplay between histone modifications, the tumor microenvironment, and disease progression. This not only aids in prognostic assessment but also deepens our understanding of the molecular mechanisms underlying HCC’s complexity.The intricate interplay between metabolism and malignancy is exemplified in hepatocellular carcinoma (HCC). Epigenetic regulation, particularly histone modifications, influences gene expression patterns and contributes to HCC’s development. Leveraging the power of single-cell RNA sequencing, As these three articles collectively showcase, single-cell analysis is revolutionizing our understanding of the intersections between endocrinology, reproduction, and metabolism. The insights garnered from scrutinizing individual cells transcend traditional methodologies, uncovering nuances that were previously hidden. These studies collectively emphasize the significance of dissecting cellular heterogeneity, unravelling gene expression patterns, and deciphering the dynamic interactions between cells. Also, these studies underscore the value of dissecting cellular heterogeneity and interactions to glean novel insights into diseases that profoundly impact human health. Such a comprehensive approach holds immense promise in the realm of personalized medicine, offering tailored interventions for different conditions and diseases.In the broader landscape of scientific progress, these endeavours represent more than individual studies. They are pieces of a larger puzzle that, when integrated, illuminate the complex web of cellular interactions underlying health and disease. As the field of single-cell analysis continues to evolve, the depth of knowledge gained will undoubtedly inform clinical approaches and therapeutic strategies. From unravelling the mechanisms of pregnancy complications to deciphering the cellular basis of sexual health and probing the epigenetic intricacies of cancer, single-cell analysis is a powerful tool that propels us toward a future of personalized, precision medicine.In closing, the symbiotic relationship between science and technology continues to reshape the boundaries of human understanding. The uncovering of endocrinology, reproduction, and metabolism through the lens of single-cell has revolutionized our ability to dissect the intricacies of these fields, shedding light on previously uncharted territories and offering profound insights into various physiological and pathological phenomena. Single-cell technologies have enabled us to investigate the interactions and functions of a single cell in a way that was unthinkable a decade ago. Single-cell technologies are still in their infancy and require further breakthroughs to understand molecular and metabolic interactions. Various emerging technologies will pave ways to investigate intracellular signalling which may lead to novel discoveries.FD: Writing – original draft, Writing – review & editing. CB: Writing – original draft.


HIV prevalence among people who use drugs (PWUD) in Baltimore, Maryland, is higher than among the general population. Pre-exposure prophylaxis (PrEP) is a widely available medication that prevents HIV transmission, yet its usefulness is low among PWUD in Baltimore City and the United States. Community-level interventions to promote PrEP uptake and adherence among PWUD are limited.We describe the development of a capacity-strengthening intervention designed for frontline harm reduction workers (FHRWs) to support PrEP awareness-building and promotion among PWUD.Our study was implemented in 2 phases in Baltimore City, Maryland. The formative phase focused on a qualitative exploration of the PrEP implementation environment, as well as facilitators and barriers to PrEP willingness and uptake, among cisgender women who use drugs. This work, as well as the existing literature, theory, and feedback from our community partners, informed the intervention development phase, which used an academic-community partnership model. The intervention involved a 1-time, 2-hour training with FHRWs aimed at increasing general PrEP knowledge and developing self-efficacy promoting PrEP in practice . In a separate paper, we describe the conduct and results of a mixed methods evaluation to assess changes in PrEP-related knowledge, attitudes, self-efficacy, and promotion practices among FHRWs participating in the training.The pilot was developed from October to December 2021 and implemented from December 2021 through April 2022. We leveraged existing relationships with community-based harm reduction organizations to recruit FHRWs into the intervention. A total of 39 FHRWs from 4 community-based organizations participated in the training across 4 sessions . FHRW training attendees represented a diverse range of work cadres, including peer workers, case managers, and organizational administrators.This intervention could prevent the HIV burden among PWUD by leveraging the relationships that FHRWs have with PWUD and by supporting advancement along the PrEP continuum. Given suboptimal PrEP uptake among PWUD and the limited number of interventions designed to address this gap, our intervention offers an innovative approach to a burgeoning public health problem. If effective, our intervention has the potential to be further developed and scaled up to increase PrEP awareness and uptake among PWUD worldwide. HIV prevalence among people who use drugs (PWUD) remains high, including both people who inject drugs (PWID) and those who use drugs by other routes of administration -3. DespiThere are high numbers of PWID in Baltimore, many of whom engage in polysubstance use -8. BaltiHIV infection occurs when viral particles enter the bloodstream, most commonly via sexual or injection-mediated exposure, and infect the cells of the immune system. If left untreated, HIV infection may progress to AIDS; people with HIV who receive effective treatment can live long healthy lives and prevent onward transmission. Although there is not yet a vaccine to prevent HIV infection, medication exists to prevent successful infection of the immune system even in the event of exposure to the HIV virus. This medication, pre-exposure prophylaxis (PrEP), is a user-controlled pharmaceutical method that provides an empowering and highly effective means of preventing HIV in vulnerablized populations when taken as prescribed. PrEP reduces the risk of HIV acquisition via sexual transmission by approximately 99% and from injection drug use by at least 74% . HoweverIn 2014, the CDC released clinical guidance recommending the use of PrEP for persons at increased risk of acquiring HIV . HoweverInterventions to address PrEP awareness, uptake, and adherence among PWUD are limited . PrelimiTo address this gap, the Optimizing PrEP Engagement Among Women Living in Baltimore City (OPAL) study was initiated with the goal of developing a PrEP intervention for WWID by (1) conducting formative research with service providers and WWID to guide intervention development and (2) developing, piloting, and evaluating an intervention that addressed emergent barriers to PrEP uptake in this population. The study was expanded to encompass PWUD of all gender identities.The OPAL study was a multiphase, sequential mixed methods formative research study  conducteUnderstanding the complex lived experiences and challenges faced by WWID to inform intervention development requires a combination of methods drawing from different perspectives and epistemologies. A hybrid inductive-deductive qualitative approach is ideally suited for such an endeavor. In the formative phase, we facilitated 4 web-based focus group discussions with 20 HIV and substance use service providers who served PWUD to characterize the PrEP implementation environment in Baltimore City . The OPAAll study activities were conducted in Baltimore, Maryland, which is a midsize urban center on the Eastern coast of the United States.The intervention development approach centered a combination of community participatory ,33 and hFurthermore, intervention component development was theory driven. For example, the role-play activities included in the trainings were informed by the social learning theory, which posits that human behavior is learned observationally , and theFinally, the process and team members were motivated by harm reduction philosophies, which promote nonjudgmental orientations toward stigmatized behaviors, respecting personal autonomy, practicality, and innovation . For exaThis project was initially conceived of as a way to investigate and meet the needs of WWID, given their unique risk factors ,39 and eThe intervention development team used an academic-community partnership model. The academic research team comprised faculty and student researchers from Johns Hopkins Bloomberg School of Public Health. The research team had experience with relevant research methodologies , the study topic, and the population of focus. Student members of the team received skills development training to make meaningful contributions in their respective roles. The team ranged in gender, race, sexual orientation, age, and educational preparation.The community partners included staff from Charm City Care Connection, a community-based harm reduction organization in Baltimore City that works to connect those impacted by drug use to high-quality health care services and address any obstacles that might threaten that connection. Their mission is to promote health, self-determination, and self-advocacy for individuals and communities affected by drug use, stigma, poverty, and inequities, with a primary focus on serving PWUD. Provided services include, but are not limited to, safer use supplies , overdose education and prevention materials , safer sex supplies, peer recovery services, case management, miscellaneous services for basic life necessities , a drop-in center for PWUD, street-based outreach, and a small housing program. Charm City Care Connection also operates a Harm Reduction and Anti-Oppression Training Program, where PWUD can volunteer at the drop-in center and participate in workshops that focus on personal success planning, anger management, conflict resolution, trauma and crisis management, healthy relationship building, and self-advocacy. Once these trainings are completed, trainees are offered the opportunity to work as part-time employees, providing the same services that they once received to their peers.The activities of the formative research phase were designed, developed, and implemented by the academic research team. Logistical support during this phase was provided by the community partner and included linking clients with study staff for interviews. Upon completion of the formative phase, findings were shared with the partner organization, who emphasized that FHRWs felt underequipped having conversations about PrEP with PWUD due to a lack of knowledge and low self-efficacy associated with the sensitive nature of HIV-related discussions. Ongoing conversation regarding formative findings, interpretation, and implications within the academic research team led to the conceptualized idea to develop a PrEP promotion training (PPT) for FHRWs. This concept was presented to representatives of the partner organization, who endorsed the concept and agreed to participate in the development and implementation process.To guide the intervention development process, the academic research team developed a logic model for the intervention, which included the formative research and project work (inputs), ultimately with an aim of reducing HIV incidence among PWUD (impact) by increasing PrEP awareness and relevant skills among FHRWs (outputs), which would subsequently increase PrEP awareness, knowledge, and linkage to services among PWUD (outcomes) .The training content was developed by members of the academic research team in consultation with the partner organization representatives, who provided input and feedback to ensure that content was relevant to their work, representative of their experiences with clients, and digestible by prospective training participants. For example, feedback from the partner organization spurred the academic research team to include role-play exercises and other opportunities for active learning and engagement in the training design.Findings from focus group discussions  underscoInterviewed WWID exhibited moderate PrEP awareness overall, with most having been introduced to PrEP through television commercials or advertisements. Few women reported ever discussing PrEP with a health care provider. Many WWID reported that HIV risk management did not feature as prominently in their everyday lives as other health and survival priorities, such as managing withdrawal symptoms or earning money. However, many women described engaging in HIV risk reduction through condom and sterile syringe use, which they received from FHRWs. Furthermore, many WWID reported routinely accessing harm reduction services in outreach settings (mobile- or street-based) or from drop-in centers.Taken together, these findings highlight the existing proximity of FHRWs to WWID in Baltimore City and their potential to facilitate PrEP promotion among WWID. FHRWs already support these women and are well situated to promote reflection on HIV risk, initiate conversations about PrEP, and support clients in accessing PrEP if and when ready.The intervention was tailored to the experiences, knowledge, and client interactions typical of FHRWs at community-based organizations in Baltimore City that offer both outreach and drop-in services and do not have standing PrEP programs onsite. FHRWs were chosen as the study population due to their proximity to and strong relationships with PWUD, developed through existing motivational interviewing skills and regular client interactions. Furthermore, many FHRWs have lived experience with drug use and other adversities faced by PWUD, which facilitates connection and helps to overcome issues establishing trust and rapport frequently reported by PWUD when accessing services in traditional health care settings ,23. ThisThe PPT was delivered as a 2-hour training divided into 2 sections: the first section was didactic, presenting basic information about PrEP  and steps for making referrals to local PrEP services . PrEP indidactic)Pre-exposure prophylaxis (PrEP) 101 Raising awareness and promoting PrEP (The continuum of PrEP awareness and readiness (mosaic)Introducing and reviewing client personasStrategies for introducing PrEP into client dialoguesRole play exercises: (1) raising awareness, (2) answering questions, and (3) supporting PrEP uptakeSupporting continuity in PrEP dialogue with clientsQuestions and feedbackThe second portion of the training involved several active learning activities designed to enhance FHRWs self-efficacy promoting PrEP in practice. First, we introduced the concept of a “PrEP awareness mosaic” , an inteWe developed 5 client “personas” (or archetypes), loosely informed by the composite lived experiences and PrEP willingness of interviewed WWID in the study’s formative research phase  44]. Th. Th44]. After discussing client personas, we discussed strategies for introducing PrEP into dialogues with clients encountered during drop-in services or during mobile outreach. This included identifying both simple entry points  and more ambitious targets  for introducing PrEP during client interactions.We then facilitated a roleplay activity in which training participants were paired up and practiced skills for 3 different types of PrEP conversations: identifying clients’ knowledge of PrEP, answering clients’ questions about PrEP, and linking clients to PrEP services. During each roleplay exercise, participants played the role of either a FHRW or 1 of the 5 client personas. After each roleplay exercise, select pairs volunteered to present their roleplay dialogues to the other training attendees, allowing all participants to observe, reflect upon, and troubleshoot strategies for PrEP-related conversations.At the end of the training, we facilitated a short debrief session to elicit feedback on the content and delivery of the PPT, identifying favorable attributes of the training as well as opportunities to modify training content and delivery.In addition to sharing training resources , we provided PrEP information flyers containing basic information about PrEP and local venues where PrEP information and services could be accessed, adapted from the Maryland Department of Health’s PrEP toolkit . We instGiven the novelty of the intervention, we conducted an evaluation to assess changes in PrEP-related knowledge, attitudes, self-efficacy, and promotion practices among FHRW participating in the training . Using 3The Johns Hopkins Bloomberg School of Public Health Institutional Review Board reviewed and deemed the study protocol as nonhuman subject research. The study team took all appropriate cautions to maintain data confidentiality by redacting all personally identifiable information from in-depth interview transcripts and generating unique study identifiers, which were linked to participant information only through a secure, password-protected file.The intervention development occurred from October to December 2021. Intervention piloting began in December 2021 and was completed in January 2022.The academic research team leveraged existing relationships with community-based harm reduction organizations to recruit FHRWs to attend the training and participate in the evaluation. We engaged 4 nonprofit organizations in Baltimore City offering low-threshold harm reduction services to various populations. The populations served by these organizations’ missions included transgender individuals, PWUD, people with unmet mental health needs, and women who sell sex. Community partners all offered drop-in as well as outreach  harm reduction services, which included syringe and overdose prevention services, HIV and Hepatitis C testing, linkage to health care and safety net programs, among others. Shared technical expertise among staff at organizations included case management, service navigation, motivational interviewing, and harm reduction promotion.A total of 39 FHRWs from 4 community-based organizations participated in the training across 4 implementations . FHRW training attendees represented a diverse range of work cadres, including peer workers, case managers, and organizational administrators. Attendees ranged in terms of racial and gender identities. Prior to the training, only two-thirds of participants were familiar with PrEP.We initially designed the PPT to be delivered in person. In December 2021, we successfully facilitated our first in-person training with a community organization. However, due to surges in COVID-19 cases in January 2022, we pivoted to online delivery of the remaining trainings via Zoom. This transition required retooling of the practice-based training portion. To approximate the active participation and partner-based work involved with the in-person training, we used an integrated polling feature, conducted short knowledge checks, and encouraged the use of response and feedback tools  to facilitate participant engagement throughout the web-based sessions. Web-based delivery also enabled us to deliver the training to more than 1 community organization in the same session, facilitating cross-organizational learning. A consequence of the shift to web-based delivery was hampered accessibility; less technically proficient FHRWs had difficulty in joining the videoconference session and using the web-based tools. To address this, we created a recording of 1 training delivery session and made it available to FHRWs who were not able to participate in live sessions.Evaluation activities began at the onset of the intervention pilot (December 2021) and were completed in March 2022. Of the 39 FHRW participants, 37 met the eligibility criteria and were included in the evaluation. The evaluation results were published in a companion article .This intervention offers a novel approach to HIV prevention among PWUD, supporting FHRWs in initiating PrEP conversations and supporting client advancement along a PrEP continuum, as appropriate. The subsequent evaluation provides information on intervention effectiveness—preliminary feedback is promising . Given tThis intervention takes a community-situated approach to HIV prevention among PWUD by focusing on and expanding the robust skillsets of FHRWs, which is a cadre of the health care and harm reduction workforce that has been underutilized in PrEP promotion approaches. FHRWs are particularly well situated and skilled to support PWUD in adopting additional harm reduction approaches, namely increasing PrEP-related general knowledge and services, given their role as trusted and accessible allies ,23. In fThe proposed intervention is not without potential obstacles and limitations. For example, although capacity building and skills development are integral to the ongoing success of harm reduction organizations, the addition of new services may be burdensome to existing operations. Harm reduction organizations are already working tirelessly, frequently with insufficient resources, to address overdose prevention and syringe exchange needs of PWUD. Although HIV and other infectious disease prevention is an implicit goal of this work, the addition of PrEP promotion can add additional operational considerations to an already-taxed workforce. However, harm reduction organizations are already adept at remaining nimble to policy changes and environmental shifts to ensure that they are meeting their mission. This is evidenced by the pivoting and rapid service adaptations exemplified by harm reduction organizations during the COVID-19 pandemic ,49. Our This intervention has the potential to be further developed and expanded to increase PrEP awareness and uptake among PWUD in a range of settings. After the achievement of conclusive results, we plan to refine and scale up the intervention. Our long-term vision is to develop this intervention with modifiable content units that can be tailored to specific subgroups of PWUD as needed, such as PWUD who exchange sex, of different gender identities, from different regions or political contexts, and residing in jurisdictions with variable PrEP service delivery infrastructure. Furthermore, such an intervention could be adapted to address additional HIV prevention and care services, such as HIV counseling and testing, HIV care and treatment, and emerging new technologies such as long-acting injectable PrEP.


Non-small cell lung cancer (NSCLC) is the most common type of lung cancer with a 5-year survival rate of less than 25% . CurrentThis Research Topic focuses on the latest research findings and insights into the immunological and molecular mechanisms that underpin NSCLC drug relapse and therapy resistance. The articles in this Research Topic cover a range of topics, including the identification of novel biomarkers to predict resistance, tumor heterogeneity in metastatic disease, metabolic vulnerabilities, and the design of new strategies to improve treatment outcomes. Together, they provide a comprehensive overview of the current state of research into NSCLC drug resistance, and highlight promising new directions for future studies.Rangamuwa et al. describe “immune phenotyping” which characterizes the immune milieu of the tumor, and may provide insights into response to immune-modulating therapy. A range of techniques may be employed to examine the tumor microenvironment, including immunohistochemistry, cytometry, and transcriptomic analysis. The utility of such modalities, particularly with small-volume tissue samples obtained from patients with advanced stage disease, is an area of growing interest  is recognised as potential biomarker of responsiveness to immunotherapy. While there is evidence of genomic heterogeneity between tumor regions in the individual patient , there iBain et al. discuss strategies to target minimal residual disease in EGFR-mutant NSCLC by targeting senescence-like cell dormancy and enhancing apoptosis by combining EGFR TKIs with other therapeutic agents. These strategies are aimed at achieving deeper responses and potentially preventing/delaying drug resistance. However, biomarkers that can accurately detect minimal residual disease in solid tumors to identify patients at risk of relapse, escalate or de-escalate treatment, and determine optimal treatment duration remains elusive.Minimal residual disease in NSCLC is defined as micro-metastatic disease that persists after initial therapy, representing a potential source of subsequent metastatic relapse at distant sites . In the EGFR mutations  is variable. Effective strategies to target these alterations is a clinically unmet need. Zhang et al. reviewed the potential of using EGFR-targeting monoclonal antibodies in combination with TKIs to treat this patient population. This strategy has shown to produce synergistic antitumor effects albeit high toxicity rates. Novel therapies such as the EGFR–MET-targeted bispecific antibody, amivantimab, and tyrosine kinase inhibitors that specifically target uncommon EGFR mutations , have shown promise and continue to be evaluated in this patient population in combination with other therapies.In the metastatic setting, the third-generation tyrosine kinase inhibitor, osimertinib has been approved in NSCLC with common EGFR mutations (L858R or Exon 19 deletions). Despite striking benefits, resistance invariably occurs against osimertinib and the efficacy of EGFR-TKIs in uncommon Tang et al., the therapeutic potential of glutamine metabolism has been explored in lung cancer. In addition to anti-cancer benefits of targeting the glutamine pathway, this review additionally highlights that glutamine supplementation during radiation therapy could mitigate the severity of complications during radiotherapy for lung cancer patients.Altered metabolic properties of cancer cells can also play a driving role in drug relapse and therapy resistance. By exploiting metabolic pathways to increase antioxidant production to bolster cellular protection and harness glucose-independent energy production using glutamine, cancer cells can become dependent on this non-essential amino acid. Reviewed in this Research Topic by Chang et al. found that an Aldolase A (ALDOA) and Phospholipase D1 (PLD1) axis drives a more aggressive phenotype and may represent a target for inhibition. The ALDOA/PLD1 axis is crucial for enhancing the proliferation of lung cancer cells, autophagy, and DNA repair capabilities after exposure to alkylating agents and radiation. The study also found that ALDOA can inhibit the activity and enzyme function of PLD2 through direct protein-protein interaction to enhance the activity of PLD1 and additional carcinogenic features. Together, these two papers provide insights into the metabolic vulnerabilities in lung cancer cells and the potential of targeting specific metabolic pathways as a personalized therapy approach.In response to radiation and alkylating agents in lung cancer, Collectively, the articles in this Research Topic provide an important snapshot of the current challenges and ongoing efforts to understand the mechanisms of drug relapse and therapy resistance in NSCLC. Additional studies that build on these findings will be critical to advance the development of more effective treatment strategies to improve patient outcomes and survival.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


The regeneration capacity after an injury is critical to the survival of living organisms. In animals, regeneration ability can be classified into five primary types: cellular, tissue, organ, structure, and whole-body regeneration. Multiple organelles and signaling pathways are involved in the processes of initiation, progression, and completion of regeneration. Mitochondria, as intracellular signaling platforms of pleiotropic functions in animals, have recently gained attention in animal regeneration. However, most studies to date have focused on cellular and tissue regeneration. A mechanistic understanding of the mitochondrial role in large-scale regeneration is unclear. Here, we reviewed findings related to mitochondrial involvement in animal regeneration. We outlined the evidence of mitochondrial dynamics across different animal models. Moreover, we emphasized the impact of defects and perturbation in mitochondria resulting in regeneration failure. Ultimately, we discussed the regulation of aging by mitochondria in animal regeneration and recommended this for future study. We hope this review will serve as a means to advocate for more mechanistic studies of mitochondria related to animal regeneration on different scales. Mitochondria are highly specialized and dynamic double-membrane bound organelles in eukaryotic cells. As the powerhouses of the cell, mitochondria enable the eukaryotes to efficiently generate ATP from energy-rich molecules Chan . Over anMitochondrial networks display dynamic morphologies under various physiological and pathological conditions  act as secondary messengers, with calcium balance being regulated by interactions among mitochondria, ER, and lysosomes  production  can be released from dying cells in injured tissues. These mitoDAMPs can serve as initiation factors for an early immune response to injuries  activity  can induce mitochondrial fragmentation by translocation of a mitochondrial fission-promoting protein DRP1 to mitochondria in normal primary human keratinocytes  cycle to generate the energy for cellular processes. In contrast, stem cells rely on glycolysis to provide energy regardless of oxygen availability. This phenomenon is termed “aerobic glycolysis”, and called the “Warburg effect”. Accumulating evidence indicates that the metabolic shift to glycolysis plays a vital role in regeneration. For instance, extracellular flux (XF) analysis indicated that metabolic changes occur with increased glycolysis during regeneration in planarians . Genetically/pharmacologically increasing or reducing the bioactivity of these metabolites promoted or impeded CNS axon regeneration  which thereby increases mouse life span , as a vital regulator in axonal damage signaling has been proven to be located at axons in response to damage in nematodes and mice  injury in the brain happens during the reperfusion of blocked blood due to the restoration of oxygen-rich blood. The main strategy to mitigate ischemic stroke injury is revascularization, which may lead to I/R injury  and mitochondrial ubiquinone (MitoQ)  show an increased level of S-adenosyl-L-methionine (SAM), which is sustained in mitochondria as the methyl source of nicotinamide N-methyltransferase (NNMT) enzyme. Knockout of NNMT increased SAM levels and H3K27me3 marks and promoted the transition from naïve to primed stage (Sperber et al. mt, protein translation and epigenetic modification are three major pathways to regulate downstream gene expression. Additionally, mitochondrial shape and dynamics can serve as essential inputs in determining cell fate and function (Chen and Chan UPRAs much evidence has shown mitochondrial direct or correlated function in animal regeneration, aging factors have also been noted to influence mitochondria-regulated animal regeneration. Mitochondria have been under extensive study in aging research. It is well-accepted that regeneration ability decreases in aged animals and the accumulation of mtDNA mutations has been demonstrated in tissue during aging and cultured conditions (Kujoth et al.