Therefore, QT prolongation is a qualitative marker of proarrhythmic risk, and analysis of ventricular repolarization is therefore required for the approval of new drugs. To that end, the Thorough QT/QTc analysis evaluates QT interval prolongation to assess potential proarrhythmic effects. In addition, since diabetic patients have a higher risk to die from cardiovascular causes than individuals without diabetes, cardiovascular safety of the new antidiabetic drugs must be carefully evaluated in type 2 diabetic patients. These cardiovascular outcome trials reveal that some glucose-lowering drugs actually reduce cardiovascular risk. The mechanism of cardioprotection might involve a reduction of the risk of developing arrhythmia.Diabetes is a chronic metabolic disease characterized by hyperglycemia in the absence of treatment. Among the diabetes-associated complications, cardiovascular disease is the major cause of mortality and morbidity in diabetic patients. Diabetes causes a complex myocardial dysfunction, referred as diabetic cardiomyopathy, which even in the absence of other cardiac risk factors results in abnormal diastolic and systolic function. Besides mechanical abnormalities, altered electrical function is another major feature of the diabetic myocardium. Both type 1 and type 2 diabetic patients often show cardiac electrical remodeling, mainly a prolonged ventricular repolarization visible in the electrocardiogram as a lengthening of the QT interval duration. The underlying mechanisms at the cellular level involve alterations on the expression and activity of several cardiac ion channels and their associated regulatory proteins. Consequent changes in sodium, calcium and potassium currents collectively lead to a delay in repolarization that can increase the risk of developing life-threatening ventricular arrhythmias and sudden death. QT duration correlates strongly with the risk of developing  As a result, many type 2 diabetic patients may need exogenous insulin.Diabetes affected 422 million adults in 2014 , but theIn the last decades, available pharmacologic approaches have significantly improved the life span and the quality of life of diabetic patients. However, over time, diabetes-associated complications have emerged. Among them, cardiovascular disease stands out because is the major cause of mortality and morbidity in diabetic patients. In fact, the Framingham study reported that patients with type 2 diabetes were twice as likely as healthy people to die from cardiovascular diseases . CurrentBesides inducing cardiovascular disease, diabetes changes the myocardial structure leading to a cardiac dysfunction known as diabetic cardiomyopathy. Diabetic cardiomyopathy was first described by Rubler et al., in 1972 as heart failure in diabetics without hypertension, myocardial ischemia, congenital or valvular disease . It is cTorsade de pointes (TdP) is a ventricular arrhythmia characterized by a change in the amplitude and a twisting of the QRS complexes around the isoelectric line in the electrocardiogram. TdP usually ends spontaneously, but in some cases may degenerate into lethal ventricular fibrillation. Long QT syndrome is an inherited or drug-induced arrhythmia syndrome characterized by a prolongation of the QT interval  have become qualitative markers of proarrhythmic risk. The electrocardiogram of both type 1 and type 2 diabetic patients often shows prolonged QT  studies are performed with premarketing drugs in order to exclude those that prolong the QT interval (ICH E14). At the same time, concerns about cardiovascular risk in diabetic patients has guided the policy of the regulatory agencies to establish the safety of new glucose-lowering drugs. Therefore, cardiovascular outcome trials (CVOTs) that evaluate specific cardiovascular endpoints are being conducted on type 2 diabetic patients . Furtherβ-cells , the non-obese Goto-Kakizaki (GK) rat with defective β-cells, the obese Otsuka Long Evans Tokushima Fat rat (OLETF) or the Zucker diabetic fatty rats (ZDF). In addition, metabolic models that combine high fat and/or high-carbohydrate diet with an intraperitoneal low-dose of STZ  along with a reduction of the corresponding channel forming proteins  and diabetic patients have one-third greater risk of incidence of AF compared with unaffected individuals . Althougproteins .The most-studied electrical alteration of the diabetic myocardium is prolonged ventricular repolarization. Diabetes-induced lengthening of QT and QTc intervals associates with higher risk of developing ventricular arrhythmias and sudden cardiac death . ProlongAnother parameter that analyzes the repolarization is the QT dispersion or QTd, which reflects the difference between the longer and the shorter QT interval duration in the 12 leads electrocardiogram. An increase in the QTd is typically associated to an increased predisposition to ventricular arrhythmias. Diabetic patients show increased QTd and heart-rate corrected QT dispersion (QTdc) compared to non-diabetic subjects in the absence of other cardiovascular pathologies. Although the prevalence of severe prolonged QTc (>500 ms) and QTd (>80 ms) is low , the preThe basic electrical activity of the cardiac cells is an action potential consisting of a rapid depolarization phase followed by a repolarization phase with a plateau. In 1983, Fein et al. described for the first time a lengthening of the cardiac action potential duration (APD) in a rat model of T1D , an effeNa. Diabetic rabbits displayed a reduction in the density of the cardiac INa although the Nav1.5 protein levels were not significantly reduced  mice with prolonged QTc . There could also be intrinsic differences in the diabetic models, for instance, induced vs. genetic. The experimental conditions for current recordings are also crucial, since the amplitude of the L-type calcium current is very sensitive to the intracellular calcium content. In physiological conditions, L-type calcium channels inactivate via Ca2+-dependent inactivation, mainly by the calcium released from the sarcoplasmic reticulum . Diabetes prolongs the duration of both the contraction and the relaxation phases of the cardiac cycle  are reduced in diabetes and become fully restored after insulin supplementation (2+ leak to the cytoplasm (2+-ATPase (SERCA2) as well as its apparent affinity for Ca2+  expression. Although channel protein levels and current recordings were not assessed and the study was performed in few, aged and poly-medicated patients, this is the first work that directly compares the expression of cardiac channels between diabetic and non-diabetic humans  initiative works in a new paradigm for assessment of TdP proarrhythmic risk of new drugs that is not focused exclusively in hERG blockage and QT prolongation ; a positive control (a drug that prolongs the QTc); the drug under development at the maximum recommended therapeutic dose; and the drug at supratherapeutic dose to explore the “worst-case scenario”, for instance, in an impaired clearance of the drug. The TQT study is negative if the drug does not prolong the mean QT/QTc interval more than 5 ms, which is considered the threshold of regulatory concern . Howeverper se may correlate with prolonged QTc  has declined in both adults with and without diabetes over the last few decades, diabetic patients still have 2-fold greater risk of CVD compared with the non-diabetic population . In addi; therefore, all of them are safe. The GLP1R agonist liraglutide and the SGLT2 inhibitors empagliflozin and, to a lower extent, canagliflozin, have yielded very positive results. Consequently, the American Diabetes Association recommended incorporating these drugs to the standard therapy in T2D patients with established atherosclerotic cardiovascular disease  inhibitors, sodium-glucose cotransporter 2 (SGLT2) inhibitors and glucagon-like peptide 1 (GLP-1) receptor agonists have undertaken CVOTs. Detailed summaries of completed and ongoing studies have been recently published . Althoug disease .Furthermore, very recent studies have focused on the potential protective effect of SGLT2 inhibitors against developing arrhythmias. In newly diagnosed diabetic patients, SGTL2 inhibitors associates with a reduced risk of new onset arrhythmia . SimilarAmong the proposed cardioprotective mechanisms are lowering blood pressure, which may lower cardiac afterload; improving cardiac metabolism increasing energy production; reducing inflammation and cytokine release; preventing adverse cardiac remodeling; reducing sympathetic nerve activity; improving renal function; improving mitochondrial dysfunction and improving ionic dyshomeostasis. Thus, the underlying cardioprotective mechanisms of SGLT2 inhibitors and GLP-1 receptor agonists remain unclear and might involve complementary systemic and direct cardiac effects (recently reviewed in +) was elevated in the diabetic myocardium, directly increasing the risk of sudden arrhythmic death  and (Ca2+) might reduce the risk of developing potentially lethal arrhythmias. Very interestingly, in insulin resistant rats dapagliflozin-treatment improved cardiac repolarization. Daplagliflozin supressed QT interval and AP prolongation, mainly by restoring the depressed potassium currents Ito and IK1, but also by reducing INa (Regarding ionic dyshomeostasis and arrhythmic risk, (Naic death . Empaglintration . Another/db mice . The rescing INa .Both T1D and T2D induce a cardiac remodeling that leads to mechanical dysfunction and/or cardiac arrhythmias. Experiments with animal models have consistently shown that diabetes alters the expression and regulation of cardiac ion channels and transporters, which impairs impulse generation, conduction, excitation-contraction coupling and myocyte contractility.Prolonged QTc interval duration persists in a number of treated diabetic patients, indicating that strict glycemic control is not sufficient to normalize the electrophysiological disturbances. Available glucose-lowering drugs that improve cardiovascular prognosis are crucial in the management of type 2 diabetic patients with established cardiovascular disease or at high cardiovascular risk. Further studies are needed to elucidate if cardioprotection includes electrical remodeling and prolonged repolarization. This could be of particular interest for patients with diabetes-associated complications that may increase the risk of arrhythmia.


Early diagnosis of brachial plexus injuries is crucial to prevent long-term morbidity and improve outcomes. We present a unique case of delayed onset of brachial plexus compression two months following a traumatic gunshot injury causing multiple injuries including a T1 vertebral body comminuted fracture and pneumothorax. The patient experienced significant pain and progressive neurological examination changes during follow-up visits, and thus duplex ultrasound and computed tomography (CT) angiography were performed, which demonstrated a left subclavian artery pseudoaneurysm. This was managed operatively by evacuation and interposition bypass. Injuries to the cervical and upper thoracic spine are complex, and when patients present with new-onset neurological findings, axillary swelling, or significant uncontrolled postoperative pain, secondary complications should be suspected. Patients at a high risk of vascular reinjury should be routinely monitored at follow-up to prevent the development of progressive neurological damage to the brachial plexus. Subclavian vessel injuries are extremely precarious as they can cause devastating neurological injury due to proximity to the brachial plexus . The mecA 20-year-old female was brought to the emergency department after sustaining multiple gunshot wounds Figure . Her pasThe patient was admitted to the intensive care unit and on hospital day 2. X-ray imaging demonstrated resolution of the pneumothorax. Additionally, the patient was started on a daily dose of aspirin 81mg. On hospital day 3, bilateral first rib fractures were noted on X-ray Figure . NeurovaFollow-up physical examination at one month post-injury was complicated by severe pain, which limited movement and caused significant emotional distress. At the three-month follow-up appointment with her primary care physician, the patient demonstrated new-onset decreased motor strength with a flaccid hand in extension and intact sensation; additional findings included left elbow pressure ulcer, swelling in the left axillary region, and increased pain. Presentation was concerning for possible deep vein thrombosis (DVT), which prompted a venous duplex ultrasound evaluation, which revealed a left ulnar vein thrombosis that was promptly treated with warfarin.The patient presented to the emergency department four days later due to increasing pain. CT angiography revealed a focal narrowing of the left proximal axillary artery associated with a large pseudoaneurysm in a distal segment of the left subclavian artery measuring 5cm x 1.8cm with compression of the brachial plexus  is indicated for vascular injury that requires surgical intervention ,13. In oOne limitation of this case report is an unclear timeline of pseudoaneurysm progression due to intermittent outpatient follow-up. The progression of a potential pseudoaneurysm is variable. The most practical recommendation would be extensive patient education on concerning signs and symptoms and frequent complete neurological examinations that lead to prompt follow-up imaging. Additionally, due to the intermittent follow-up and complicated patient presentation, the timeline and extent of compression on the brachial plexus is unclear.In penetrating traumatic cervical or thoracic spinal cord injuries, clinicians should have a high index of suspicion for further vascular or neurological compromise. These patients can have multiple confounders that decrease the sensitivity and specificity of physical examination alone. Accessible imaging modalities at follow-up visits include but are not limited to color Doppler ultrasound, with subsequent advanced imaging using MRI or CT angiography Table .Delayed onset brachial plexus nerve injuries can have a variety of etiologies. Patients with blunt or penetrating cervical or thoracic spinal cord injuries should be closely monitored at follow-up visits for evidence of any sort of vascular or neurological compromise. These patients can develop vascular injuries that can damage local structures including damage to the brachial plexus. Patient education about early warning signs can be coupled with high index of suspicion and early imaging modalities at follow-up visits to ensure timely detection and management.


As exemplified by prone positioning, regional variations of lung and chest wall properties provide possibilities for modifying transpulmonary pressures and suggest that clinical interventions related to the judicious application of external pressure may yield benefit. Recent observations made in late-phase patients with severe ARDS caused by COVID-19 (C-ARDS) have revealed unexpected mechanical responses to local chest wall compressions over the sternum and abdomen in the supine position that challenge the clinician’s assumptions and conventional bedside approaches to lung protection. These findings appear to open avenues for mechanism-defining research investigation with possible therapeutic implications for all forms and stages of ARDS. Considered independently, these interlocked components of the respiratory system have different innate mechanical properties . Quite aVertical alterations of body position that occur along the sagittal plane  influence the gravitational forces acting on the chest and abdomen. As a result, a marked increase in lung volume normally occurs when transitioning between the supine and fully upright positions, especially within peri-diaphragmatic zones . ConversProne positioning reversibly stiffens the relatively compliant anterior portions of the chest wall—peri-sternal area and ventral abdomen, relieves the weight of the heart from the lungs and lessens the cephalad thrust of abdominal hydrostatic pressure on dorsal lung sectors . In combincrease the apical to caudal gradient of pleural pressures and to encourage atelectasis to form in peri-diaphragmatic zones [The physiological effects of regional chest wall restriction have been explored in non-critical settings by the use of chest strapping and abdominal binders , 8. Almoic zones .2O and predictably increased plateau pressure during volume-controlled ventilation without PEEP [2O PEEP was simultaneously applied to the airway opening.Tethering of the diaphragm by its costal attachments to the inner chest wall imposes a physical limit on the extent to which the diaphragmatic dome can be pushed cephalad by increasing abdominal pressure. External pressures have been applied in the supine position using weights placed on the rib cage or abdomen. –12. At aout PEEP . Notably2O, a value that approaches the upper limit of the normal range for non-morbidly obese humans. In healthy obese patients, however, resting IAP when supine may exceed 10 cmH2O [The transmission of controlled intra-abdominal pressures to the pleural space has been studied in both normal and lung-injured large animals –16. Most10 cmH2O . If IAP 10 cmH2O . As expe10 cmH2O .sustained regional applications of external chest wall pressure. Studies of sustained sternal compression with pleural monitoring have neither been carefully conducted nor reported in acutely ill patients. It is reasonable to assume, however, that such an unrelieved positive bias of pleural pressure would hinder venous return, raise small airway closing volumes, and encourage sustained collapse in any dependent zones that harbor unstable alveoli. Modified distribution of intra-pulmonary perfusion by sustained sternal pressures must be also assumed, but scientific data are lacking.The transmission of external sterno-xiphoid pressure to the heart has long been a focus of CPR physiology and effectiveness, with standard CPR generating transient intrapleural pressures increases of 25–40 mmHg during the downward compression phase . In the The beneficial consequences of stiffening the anterior aspect of the chest wall and abdomen by prone positioning include more uniform distribution of transpulmonary pressures and improved ventilation to perfusion (V/Q) ratios, generally without compromising total respiratory system compliance or end-expiratory volume (FRC) , 20. TheSustained external pressure applied to the rib cage or abdomen reduces its local compliance at the point of compression, together with the global compliance of the integrated respiratory system. With regard to selective abdominal compression, stiffening of the lung’s enclosure from below occurs in the supine position as a nonlinear function of intra-abdominal pressure, as already noted . On the phasic chest wall compression timed to occur during tidal expiration as a rescue maneuver to reduce hyperinflation, improve ventilatory mechanics and enhance hemodynamics [Transient, intermittent application of external pressure during expiration has been reported to have diagnostic value as a maneuver to demonstrate the presence (or absence) of expiratory flow limitation during tidal breathing in patients with diffuse airflow obstruction , 24. Altdynamics , 26. To From the preceding discussion it is should be clear that external compression of the chest wall reduces chest wall compliance, so that the expected response to sustaining such maneuvers with tidal volume and PEEP unchanged would be to increase tidal plateau and driving pressures, indicating reduced tidal compliance of the respiratory system. That expectation rests, however, on the assumption that lungs’ compliance is not simultaneously improved by the imposed change in chest wall stiffness. Indeed, such assumptions do usually apply. However, extensive recent experience with one specialized form of ARDS, COVID-19 pneumonia (CARDS), has demonstrated that violations of that expectation do occur under some conditions, with important implications for ventilator management , 28.COVID-19 has challenged the clinician’s understanding of certain aspects of ARDS and in the process has broadened perspectives regarding the underlying physiology that should guide its ventilation. For example, despite severely impaired oxygenation, the gas volume and respiratory system compliance of patients with C-ARDS initially may be quite well preserved. However, unresolving late phase C-ARDS may be characterized by impressive loss of aeratable lung units, in part due to fibroblastic proliferation and organization within the parenchyma. In this latter setting, recent reports indicate that late phase patients unexpectedly experienced improved respiratory system compliance and gas exchange in response to compressive sternal  or abdomreduction of volumes in the unusually small ‘baby’ lungs of C-ARDS. As the total aerated space of the C-ARDS lung shrinks to a very low capacity, its remaining lung units operate closer to their non-compliant upper range during tidal inflation, while the elastance of the highly compliant chest wall changes comparatively little.  dropping tidal volume is key to avoiding end-tidal overdistention and, paradoxically, to improving tidal mechanics and lung protection. If so, such lessons may apply not just to CARDS, but to other forms of very severe ARDS, as well.Perhaps the most likely explanation for better tidal compliance during external compression, however, stems from the predictable consequences of compression-induced le. Fig. . By thisle. Fig. . As detadiagnostic value for adjusting the ventilatory prescription: lower PEEP, lower tidal volume, and more horizontal or head-dependent body positions. If the local compression maneuver increases rather than reduces respiratory system compliance, plateau and driving pressures decline during weighted volume control  has To our knowledge the physiology of different methods for applying local chest compression in the clinical setting of severe acute lung injury has not been defined nor have the characteristics of alternative compression maneuvers been compared head-to-head; whether ‘belly push’ (27) or sternal depression (28) is to be preferred as the compressive diagnostic technique also has not been explored. Yet, we speculate that because the abdomen is relatively compliant, the ‘belly push’ might prove to be more efficient in eliciting any benefits from reducing lung volume. On the other hand, at most lung volumes the diaphragmatic surface yields more easily than the rib cage, perhaps favoring its caudal displacement by sternal pressure. In theory, sustained pressure differentially applied to the abdomen runs a lower risk of impairing venous return than a supra-diaphragmatic pressure that produces a similar lung volume change and increase of pleural pressure. Moreover, because downward sternal pressure is better aligned with gravitational forces in the supine posture, extensive collapse of dorsal lung units would seem more likely to occur with downward sternal pressure than with a ‘belly push’ directed cephalad.What is the incidence of ‘paradoxical response’ among ARDS patients of all severities? Do they correlate with lung compliance?What are the effects of focal compression of ventral surfaces when applied in the prone position?How does perfusion re-distribute in response to focal compression of sternum or abdomen?How best to titrate applied chest wall pressure at varied levels of PEEP and body angulation ?Does therapeutic benefit or hazard accrrue from sustained external pressure in compression maneuver ‘responders’?What are the effects of sustained local compressions on gas exchange?Does a paradoxical response of mechanics to external pressure when supine reliably predict better mechanics and/or gas exchange during prone positioning?Numerous unanswered areas and questions remain regarding local compression of the chest wall in acute respiratory failure, many with immediate clinical implications. Prominent among these are:Recent observations made in late-phase patients with severe ARDS caused by COVID-19 have revealed surprising mechanical responses to local chest wall compression that challenge the clinician’s assumptions and conventional bedside approaches to lung protection. These findings appear to open several avenues for mechanism-defining research investigation—not just for C-ARDS, but perhaps for all forms and stages of ARDS. At the bedside, the response of Pplat to manual compression of the sternum or upper abdomen in the supine position may provide a useful indicator of the safety margin necessary to avoid unsuspected end-tidal lung overdistention. Whether sustained focal chest wall compression holds therapeutic value or risks harm is yet to be determined.


ABSTRACT IMPACT: The implementation of DPYD and UGT1A1 pharmacogenetic testing, a promising tool of precision medicine, translates evidence-based research into clinical oncology practice with personalized dosing to better predict interpatient variability in chemotherapy tolerability. OBJECTIVES/GOALS: Patients with DPYD and UGT1A1 genetic variants are at risk for severe toxicity from fluoropyrimidines and irinotecan, respectively. We propose that providing clinicians with the option to order a pharmacogenetic (PGx) test with relevant dose recommendations will increase test uptake to guide pharmacotherapy decisions and improve safety outcomes. METHODS/STUDY POPULATION: We plan to conduct a non-randomized, pragmatic, open-label study in 600 adult patients with gastrointestinal (GI) cancers initiating a fluoropyrimidine- and/or irinotecan-based regimen at three cancer centers within a health system. Implementation metrics of a new, in-house laboratory developed PGx test will be measured, including feasibility of returning results within one week, fidelity of providers following dose recommendations, and penetrance via test ordering rates. Clinical aims will include assessing severe toxicity during the first six months of chemotherapy. Outcomes will be compared to a historical control of GI cancer patients enrolled in a biobank and treated with standard dose chemotherapy. RESULTS/ANTICIPATED RESULTS: We anticipate that there will be an increase in PGx test uptake given its shorter turnaround time to facilitate clinical decision-making prior to the first dose of chemotherapy. Through integration of test results in the electronic health record (EHR) and clinical decision support tools for patients with actionable genotypes, we also expect that providers will have a high level of agreement to the recommended dose adjustments. We anticipate a decreased incidence of severe (Grade >3) toxicity among prospectively genotyped patients in the first six months of chemotherapy compared to DPYD and UGT1A1 variant carriers in the historical control group. Exploratory clinical utility data on costs of hospitalizations, chemotherapy treatment, PGx test, and medical services will also be reported. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study aims to address barriers identified by key stakeholders to implementing PGx testing to better tailor chemotherapy dosing to the genetic profiles to patients. This may prevent adverse event-related hospitalizations, improve quality of life for patients, and reduce health system resource utilization costs.


To the Editor: We previously proposed a nomenclature for human respiratory syncytial virus (HRSV) to standardize the sharing of viral isolates and sequences (https://www.gisaid.org). One situation not covered in our proposal was when subtypes HRSV A and HRSV B coexist in the same clinical sample. Although this situation appears relatively infrequently, usually in <1% of HRSV-positive respiratory samples  and long-read approaches  might also be employed to ensure correct generation and assignment of HRSV A and B sequences from dual infections. Whereas co-infections with other respiratory pathogens are clearly recognized and well-studied, dual infections with HRSV A and B remain less so, but we are now well-positioned to identify these infections.We also note that dual infections may raise technical difficulties, such as assignment of sequence reads to the correct subgroup. However, algorithms such as IRMA  (


Due to complicated anatomical and physical properties, targeted drug delivery to ocular tissues continues to be a key challenge for formulation scientists. Various attempts are currently being made to improve the in vivo performance of therapeutic molecules by encapsulating them in various nanocarrier systems or devices and administering them via invasive/non-invasive or minimally invasive drug administration methods. Biocompatible and biodegradable lipid nanoparticles have emerged as a potential alternative to conventional ocular drug delivery systems to overcome various ocular barriers. Lipid-based nanocarrier systems led to major technological advancements and therapeutic advantages during the last few decades of ocular therapy, such as high precorneal residence time, sustained drug release profile, minimum dosing frequency, decreased drug toxicity, targeted site delivery, and, therefore, an improvement in ocular bioavailability. In addition, such formulations can be given as fine dispersion in patient-friendly droppable preparation without causing blurred vision and ocular sensitivity reactions. The unique advantages of lipid nanoparticles, namely, solid lipid nanoparticles, nanostructured lipid carriers, nanoemulsions, and liposomes in intraocular targeted administration of various therapeutic drugs are extensively discussed. Ongoing and completed clinical trials of various liposome-based formulations and various characterization techniques designed for nanoemulsion in ocular delivery are tabulated. This review also describes diverse solid lipid nanoparticle preparation methods, procedures, advantages, and limitations. Functionalization approaches to overcome the drawbacks of lipid nanoparticles, as well as the exploration of new functional additives with the potential to improve the penetration of macromolecular pharmaceuticals, would quickly progress the challenging field of ocular drug delivery systems. The complex anatomy, physiology, and biochemistry of the human eye make it nearly inaccessible to foreign particulates, including drugs. As a result, developing an ocular drug delivery system remains a fascinating and difficult issue facing formulation and development experts. The key objective behind the design and development of an ocular drug delivery system is to offset the protective barriers of the eye to provide high therapeutic efficacy without inducing permanent tissue damage. However, the performance of many ophthalmic preparations is often restricted by short retention time, restricted permeability of corneal epithelium, high pre-corneal clearance rate due to rapid blinking rates (6–15 times/min), high tear turn over 0.5–2.2 μL/min), nasolacrimal discharge and non-productive conjunctival uptake .2 μL/min. The curThe aqueous humor, cornea, conjunctiva, iris, ciliary body, and lens are all found in the anterior chamber of the human eye, whereas the vitreous humor, retina, sclera, choroid, and optic nerve are all found in the posterior chamber . The corSchematic representation of various ocular barriers to drug absorption in the human eye is depicted in Topical delivery of the drug into cul-de-sac may be absorbed through either a non-corneal route by diffusion across sclera and conjunctiva or via corneal membrane into the intraocular tissues. Due to certain beneficial properties, such as avoidance of systemic absorption, as well as hepatic metabolism and ease of drug administration, the topical route is preferred to treat superficial infections and inflammation, besides conditions such as glaucoma or uveitis that affect the anterior segment of the eye ,10. It hp value between 1–3, which is also comparable with other biological membranes [In comparison to the non-corneal route of absorption, transcellular transport through the corneal epithelial membrane and stroma represents the major mechanism of absorption for most therapeutic agents. The corneal permeability depends on physicochemical properties of actives such as surface area, diffusivity, concentration gradient, pKa, optimum log embranes . Though embranes . Hence, embranes , variousembranes . Investiembranes . Static embranes . Lipid tThe intracameral injection represents drug administration directly to the anterior chamber of the eye, but prior administration of general anesthesia and potential damage to intraocular structures often restricts this mode of delivery . The intDue to submicron particle size and peculiar physicochemical characteristics possessed by the nanocarriers, they can act as an efficient delivery vehicle to transport actives to site-specific targets. Nanocarriers are typically constituted of particulate, soluble, or are attached with target-specific ligands for various drug delivery applications . They arIn this context, various efforts are being attempted to improve the pre-corneal contact time and trans-corneal permeability properties that could potentially enhance intraocular bioavailability . In receSLN emerged as a dominant lipid-based nanocarrier in diverse drug delivery systems. The nano-sized (10–1000 nm) particles of SLN are conventionally prepared by dispersing a solid lipid matrix in an aqueous phase comprised of surfactant as stabilizing agent . SLNs haSLNs have the potential ability for rapid diffusion across the corneal membrane and are largely distributed in ocular structures. Furthermore, enhanced interaction and adhesion between SLNs and the corneal endothelial membrane barrier would allow them to be considered as an attractive delivery tool for ocular drug transport . Due to The main ingredients typically used in the preparation of SLNs are solid lipid, emulsifier, and water. The most commonly used solid lipids as a structural component of SLN are triglycerides (tristearin), partial glycerides (glyceryl monostearate), fatty acids (stearic acid), fatty acid esters (glyceryl behenate), steroids (cholesterol), and waxes  . To reduStabilizing agents such as surfactants are included to lower the interfacial tension formed at the boundary between the lipid and the aqueous phase of the SLN formulation . They teOne of the important parameters influencing the adequate dispersibility of drugs in the lipid matrix in SLNs and NLCs is significant lipophilicity (log P > 2) . In geneThe preparation techniques of SLN are mainly based on the utilization of either high energy, low energy, or organic solvents. The most extensively used high energy processes are high shear homogenization and/or ultrasonication, microwave-assisted, high-pressure homogenization, and hot and cold homogenization techniques. An overview of various SLN preparation methods, procedures, advantages, and limitations are given in A novel method for the treatment of diabetic retinopathy with SLNs containing siRNA to silence HuR expression has been described . It was Rs1h-deficient mice, green fluorescent protein and retinoschisin expression were observed in all retinal layers indicating a partial recovery of the retina. SLNs have been also used as nonviral vector carriers for cell-specific gene delivery employing retinoschisin specific photoreceptors, murine opsin promoters. It was found that hyaluronic acid-SLN resulted in a significantly higher increase in the thickness of both retina and outer nuclear layer, which can be interpreted as the higher transfection capacity of murine opsin promoter [A successful RS1 gene transfer to Rs1h-deficient animals using SLN embedded with non-viral vectors such as hyaluronic acid or dextran has shown promising results for the treatment of X-linked juvenile retinoschisis . Fifteenpromoter .In the coacervation technique, a mixture comprising salts of fatty acids and aqueous phase constituting polymeric stabilizing agent is heated to Kraft temperature point until a transparent alkaline micellar salt solution of lipid is obtained . To allo® is limited by side effects due to slow metabolic degradation. Nevertheless, SLN prepared from Compritol® are recognized as being appropriate for intravenous use since the administered dose is very less during therapy. It is worthwhile to note that most of the lipids at a concentration utilized for the fabrication of SLN are physiologically compatible and biodegradable. Toxicity evaluation of risperidone SLN formulations using Caco-2 cells by 2,5-diphenyl-tetrazolium bromide assay disclosed that all formulations are biocompatible and well-tolerated [In vitro degradation and in vivo toxicity studies have been carried out on various lipids typically used to fabricate SLNs. The literature suggests that SLNs comprised of cetyl palmitate are well tolerated for parenteral administration though it is not a physiological compound . In vitrolerated .During the last few decades, great advancements have been made in various techniques utilized for in vitro characterization of SLNs. The lipids in SLNs can undergo crystallization tendencies and polymorphic changes during formulation and storage and can affect the system’s stability. Therefore, the characterization of lipids in SLN formulation is significant to compare compared to other lipid-based nanoparticles. Particle size is a critical evaluation parameter that is typically determined using dynamic light scattering or photon correlation spectroscopy, the laser diffraction technique, coulter counting, scanning ion occlusion sensing, and flow field fractionation methods . Shape aThe main objective of the SLN in the ocular drug delivery is to extend the retention time of applied formulation with the ocular epithelium, thereby enhancing corneal permeation via various transport mechanisms. Further, SLN dispersed in mucoadhesive polymeric gel formulation has the additional benefits of controlling the drug release and extended stability. Interpenetration and entanglement of polymer chains with mucin are responsible for mucoadhesion to ocular epithelia. Polymer hydration, swelling, and mucin dehydration are the main mechanisms underlying mucoadhesive strength.Mucins have many critical functional roles in the ocular tissues, such as hydration, lubrication, and management of tear flow to facilitate smooth blinking, act as protective cell surface barrier, trap and eliminate allergens, pathogens, and debris; they also aid in the diffusion of essential nutrients and oxygen . Two majIt was reported that spatial charge distribution within mucin matrices have a critical role in selective mucosal transport, design, and development of drug delivery carrier with modifiable transport characteristics . The capCationic lipid nanoparticles are presumed to enhance ocular bioavailability because they enable electrostatic interactions with the anionic ophthalmic mucosal surface resulting in prolonged retention time of the drug. Epigallocatechin gallate embedded positively charged SLNs (EGCG-SLNs) were prepared by multiple emulsion techniques using various cationic surface-active agents such as CTAB and dimethyl dioctadecyl ammonium bromide (DDAB). Drug-loaded SLNs were evaluated for modified release and site delivery properties . DynamicFunctionalized chitosan-based SLNs were also used for enhanced corneal permeation and efficient ocular delivery . A modifThe thiolated conjugate of cysteine-PEG monostearate was utilized for fabricating NLCs loaded with cyclosporin A for ocular delivery . The in 2/h; p < 0.0001) through goat corneal membrane as compared to control (10.94 µg/cm2/h). Pharmacokinetic evaluation of selected formulation (CL10) in New Zealand Albino rabbits indicated significant enhancement of clarithromycin bioavailability (p < 0.0001) confirmed based on a 150% increase of Cmax (~1066 ng/mL) and a 2.8-fold rise in AUC in comparison to control solution. In distinct bacterial endophthalmitis, the mean drug concentration observed in the aqueous humor  of intraocular pressure was demonstrated with NLCs in rabbits.Depending on the preparation process and lipid constitution of the matrix, there are imperfect, amorphous, and multiple types of NLC. The NLCs possess better characteristics as a drug delivery system overcoming typical formulation constraints associated with SLNs, such as high lipid crystallinity and improved long-term stability. Further, combining solid and lipid matrix in NLCs leads to less ordered lipid matrix structure with enhanced drug entrapment and minimum drug expulsion during storage. Imparting mucoadhesive to nanocarrier by providing surface retentive properties could potentially increase their precorneal contact time and ocular bioavailability. An improved mucoadhesion has been demonstrated with an NLC surface coated with cationic, chitosan oligosaccharide designed for ocular drug delivery applications . The surRecently, a smart drug delivery system created from a nanohybrid system that combines the beneficial properties of each material was described. NLC can be immobilized in a hydrogel matrix covalently or noncovalently with adequate crosslinking density to prevent the untimely release of nanoparticles. In NLC-based hydrogel, it was reported that rehydration and re-dissolution of hydrogel films could lead to the recovery of NLC. Surprisingly, the structure and size of nanoparticles were restored even after reconstitution due to hysteresis. NLC-based hydrogel is expected to release the drug slowly since the drug must cross an additional barrier due to encapsulation within nanoparticles. In vitro study performed on dexamethasone-NLC hybrid hydrogel provide a cumulative drug release of 88.65% demonstrating sustained release up to 72 h while dexamethasone loaded in NLC showed a faster drug release profile with 93.10% of total dexamethasone delivered within 48 h . The stuDifferent formulation techniques typically utilized for the preparation of NLCs are closely similar to SLNs, such as high-pressure homogenization, solvent emulsification-evaporation, phase inversion, high-speed homogenization, and/or ultrasonication, and solvent injection . High-prNanoemulsions are thermodynamically unstable, kinetically stable, optically clear, or translucent submicron (20–200 nm) isotropic colloidal dispersion system typically comprised of an aqueous and oil phase, surfactant as a primary emulsifying agent, intermediate-length alkanols as an auxiliary emulsifying agent, and, infrequently, an electrolyte . It can ® (Allergan) and Cationorm®  by the European Union (2008) for the treatment of dry eye. Recently, cyclosporin A-based nanoemulsion, Ikervis®, was approved for treating severe keratitis [Typically, in situ nanoemulsions are positively charged and are preferred particularly for lipophilic drugs targeted against various ocular bacterial, fungal, viral infections, dry eye disease, and immune-mediated inflammatory anterior ocular disease ,121,122.Nanoemulsions are typically prepared by either energy-intensive processes namely, ultrasonication, high-pressure homogenization, high-shear mixing, microfluidic and membrane methods, or low energy methods such as phase inversion emulsification techniques . In highNanoemulsion can also be prepared by mixing an organic phase containing the dissolved drug, surfactant, and cosurfactant and then injecting it into a continuously stirred aqueous medium. Though this method is feasible for encapsulating thermolabile actives, the lack of emulsion stability limits the favorable outcome . The ultThe first step in the vitro characterization technique is visual inspection or light transmittance technique to check the clarity to examine potential physical instability issues during processing and storage. An ideal pH and osmolarity are compulsory for ophthalmic formulation to avoid tissue irritation, retain corneal integrity and maintain clinical performance. The antimicrobial efficacy of the nanoemulsion is evaluated by incubating it with probable pathogens at a specific concentration; viable microorganisms are tested by culturing them in suitable media, as per the protocol and procedure mentioned in ISO 11930 and USP Chapter <51> . A partiThe low viscosity of the nanoemulsions posed a novel obstacle to the formulation scientist to prolong the contact time with the ocular epithelial surface. Nanocarrier formulation can be converted to in situ forming gels based on the type of polymer and change in pH , temperaVarious studies indicated that the chemical properties of the excipients, such as lipid, surfactants and polymers, play a key role in the stability as well as the sustained release profile of the nanoemulsions. Blood–retinal barriers can be effectively permeated by nanoemulsions as shown by the extended-release pattern of lutein up to 12 h, detected with fluorescence in the retina from penetratin-modified lutein nanoemulsions dispersed within in situ gel . The ele2/h versus ~31.53 μg/cm2/h) for both optimized formulation (MM3) and control, respectively. Ocular tolerance of MM3 indicated good tolerance and storage in a refrigerator for 3 months indicated good physical stability. High aqueous humor moxifloxacin level  and decreased Tmax value (2 h) exhibited by MM3 propose a decreased dosing frequency, enhanced therapeutic efficacy and, hence, an improved patient compliance compared to control . The aqueous humor AUC0–8 h of MM3 (1859.76 ± 424.51 ng·h/mL) was ~2 fold higher (p < 0.0005) than the control, thus, demonstrating a major improvement in ocular bioavailability , surfactant (Tween 80), cosurfactant (Soluphor P), and water . Ex vivolability .® as the lipid phase, and Tween® 80 and Poloxamer 188 as surfactants. Selected drug loaded-NE formulation demonstrated nanosized, uniformly distributed globules indicated by polydispersity index, and zeta potential, respectively. In vitro drug release and ex vivo, trans-corneal diffusion investigations demonstrated controlled release, as well as a 2.1-fold increase in penetrability, respectively, in comparison with marked ciprofloxacin eye drops. Moist heat sterilized nanoemulsion formulation was found to be stable at refrigerated and room temperature for one month. The investigation disclosed that nanoemulsion could provide an efficient ocular delivery carrier for ciprofloxacin and could enhance therapeutic outcomes in bacterial keratitis.Bacterial infections such as ocular keratitis can cause visual impairment; fluoroquinolone derivatives have been recommended as the drug of choice by the US FDA. An investigation was conducted to develop a nanoemulsion loaded with ciprofloxacin to facilitate ocular drug penetration . Ciprofl® technology platform is utilized to deliver cationic nanoemulsion to negatively charged corneal and conjunctival cells lining the ocular surface at a physiological pH [It was reported that positively charged nanoemulsion can be considered as a feasible ocular delivery vehicle in the prevention and probable treatment of ocular neovascular diseases . The thegical pH . FurtherBecause of the amphiphilic characteristic of the corneal membrane, versatile nanocarriers such as liposomes can act as an efficient and safe ocular transporting agent for various bioactive. Liposomes have spherical vesicular structures that allow polar medications to be loaded into the aqueous core and lipophilic pharmaceuticals to be intercalated into the phospholipid bilayer . Due to Liposomes can adhere to the corneal cell surface, which increases the ocular residence time, as well as permeation of poorly absorbed drugs. Intracellular delivery of liposomes can be explained by four different mechanisms, namely, adsorption facilitating passive diffusion or transport; endocytosis resulting internalization into endosomes, degradation in lysosomes and release of drug to the cytoplasm ; fusion Based on the size and number of bilayers, liposomes can be classified broadly into multilamellar vesicles and unilamellar vesicles. Unilamellar vesicles are further classified into small unilamellar vesicles and large unilamellar vesicles. Almost all liposome preparation methods generally involve the following stages, such as extraction of lipids from organic solvent and dispersing them in aqueous solvent or buffer, purification of lipids in the formed liposomes, and analyzing the final product . The druSuitable in vitro characterization techniques should be performed for the prepared liposome to evaluate their reproducibility and to ensure the specificity of their desired function . The key®) was approved by FDA for the treatment of non-infectious intermediate, posterior, and panuveitis [The surface charge, lipid composition, physicochemical nature of the encapsulated agents, and the interplay between the drug and the vesicles are the factors that determine the effectiveness of liposomes in drug delivery . Variousnuveitis . In vitrnuveitis . The incnuveitis .In vitro corneal permeation and in vivo ocular absorption in rabbits reported that liposomal surface charge is a crucial factor that impacts the performance of ocular drug delivery systems. Cationic liposomes entrapped with acyclovir demonstrated higher drug entrapment efficiencies, rapid drug delivery rates, the enhanced penetration rate in comparison to negatively charged and neutral acyclovir encapsulated liposomes . The proChitosan-coated liposomes showed an increased precorneal residence time and decreased drug metabolism at the epithelial surface of the corneum. Employing liposomes alone has limited applicability due to nano-size and surface potential, which leads to low entrapment efficiency, minimum residence time, and limited penetration. A comparative investigation was conducted between chitosan-coated liposomes and conventional liposomes encapsulated with triamcinolone acetonide based on drug entrapment and release properties. The film hydration method was used to prepare liposomes that were subsequently dispersed in chitosan under stirring at room temperature to provide a coating. Chitosan-coated liposomes demonstrated enhanced drug loading efficiency (74%), a high positive zeta potential (+41.1 mV), and prolonged residence time . The maxPseudomonas aeruginosa and Staphylococcus aureus for 24 h. It was proposed the antibacterial activity is contributed by the electrostatic attraction between the cationic chitosan and negatively charged microbial cell membranes. Though this investigation revealed that medium molecular weight chitosan is more effective for liposome coating, results from other research indicate the benefits of using aqueous-soluble chitosan of low molecular weight as a suitable coating polymer.The antimicrobial efficacy of chitosan-coated liposomes embedded with ciprofloxacin was also investigated . It was Functionalization of liposomes can often decrease the particle aggregation tendency of the liposomes and, thereby, improve the physical stability. Surface modification of diclofenac-loaded liposomes with hydrophilic PVA and corresponding derivatives carrying a hydrophobic center was found to improve the stability. Enhanced physical stability and decreased particle agglomeration were noticed in addition to strong mucoadhesion characteristics because of increased chain flexibility and better dispersion capability. The retinal transport of diclofenac was greater with PVA-coated liposomes in comparison to non-liposomal formulation .Dry eye disease is typically characterized by the absence of adequate homeostasis of the tear film resulting in inflammation, soreness, and visual impairment. Lactoferrin, a glycoprotein with anti-inflammatory, antibacterial, antiviral, antifungal, and immunomodulatory properties has been studied for different ophthalmic conditions. Lactoferrin loaded into liposomes coated with hyaluronic acid was prepared by the conventional lipid hydration method followed by high-pressure homogenization . The nan50 values reported for distamycin A-solution (DA-Sol) against Herpes simplex virus (HSV) ocular infections without any indications for corneal penetration [min = 0.364 ± 0.018) and DA-Sol (321 ± 0.038 mg/mL). The drug concentration in the lachrymal fluid was substantially more at 3, 5, and 10 min (p < 0.05) for distamycin A-liposome formulations when compared to the reference formulation. However, after 30 min, the concentration of distamycin A was found to be more than IC50 values reported for HSV1 from both preparations, but for HSV2 the results indicating IC50 and distamycin A concentration were identical only for distamycin A-liposome. These results were demonstrative of a slow clearance of distamycin A after topical applications of the liposome preparation. The pharmacokinetic analysis reported the half-life of distamycin A for DA-Sol and distamycin A- liposome in lachrymal fluid as 1.82 and 2.75 min, respectively. The AUC values conformed to higher ocular bioavailability (1.73 fold) from distamycin A- liposome formulation compared to DA-Sol. A large quantity of distamycin A was present in the ocular structures 30 min following application with DA-Sol (1.579 ± 0.087) and distamycin A- liposome formulations (2.028 ± 0.063 ng/mg), confirming the strong binding of the liposomal and the corneal structures. It was proposed by the investigators that small nanoparticles may remain within the corneal and/or the scleral layers promoting the corneal permeation of the drugs while macro-sized nanoparticles might be remained beneath the eyelids or in the inner canthus, thereby extending the contact time of the drug. The suggested hypotheses along with cytocompatibility of distamycin A- liposome may elaborate the extension of the precorneal residence time of distamycin A and the two-fold increment of ocular bioavailability of distamycin A- liposome formulation  in intraocular pressure compared to pure drug solution [HSV keratitis has been recognized as the frequent cause of corneal blindness or severe mononuclear visual impairment, affecting nearly 1.5 million people worldwide . Drug-losolution .A targeted drug delivery system is beneficial since it can avoid typical undesirable effects and potential drug interactions. Liposomal fluconazole formulation (2 mg/mL) administered through topical route three times per day in one month was shown to be highly effective in patients suffering from an ocular infection caused by Candida keratitis . The lipStealth liposomes or long-circulating liposomes are surfaces coated with inert, biocompatible hydrophilic polymers such as PEG to avoid identification by opsonins and subsequent clearance via mononuclear phagocyte system . The cov2, IL-6, and TNF-α secretion in lachrymal fluid in tears and aqueous humor in a rabbit conjunctivitis model. To avoid the loss of drugs by enzymatic degradation during endocytosis, fusogenic lipids or peptides are typically used. This will destabilize the membrane after conformational activation and subsequently deliver the drugs directly to the cytoplasm at the low pH of endosomes. Thus, pH-sensitive liposomes have the potential ability for high cytoplasmic drug delivery. Many investigations are currently being conducted to evaluate the targeting ability of pH and temperature-sensitive liposomes and stealth liposomes [To achieve site-specific delivery of the drug, targeting ligands such as immunoglobulins and their fragments are frequently attached to the liposomal surface for targeted delivery without affecting their integrity. To deliver non-viral vectors to endothelial surface receptors, immunoliposomes have been developed . Non-viriposomes .®) displayed better therapeutic efficacy to retinal occlusion as demonstrated by a reduction in retinal central subfield thickness and enhancement of visual acuity [Due to the beneficial property of overcoming the various physiological barriers of the eye, lipid nanoparticles are considered as a novel formulation strategy for the treatment of posterior segment eye diseases. Liposomal formulation loaded with triamcinolone acetonide (0.1–0.2%) was found to be as effective as a combination therapy for the prevention of macular edema associated with laser-assisted cataract surgery . Clinical acuity .Though liposomes are used to decrease the systemic toxicity generated by the loaded agent, they can cause toxicity to normal tissues and, hence, can initiate an immunogenic response. Cationic liposomes are extensively evaluated for gene delivery, are known to trigger toxicity in macrophages and to modify the secretion of prominent immunomodulators . Despite®, Ikervis®, or Cequa®. NLCs are widely probed as carriers for the transport of drugs to the eye due to their improved drug loading and permeation properties besides their sufficient safety profile. Nanoemulsions are a comparatively most effective delivery vehicle to increase the solubility, bioavailability, and functionality of hydrophobic compounds, which further affirm their practical relevance in ocular drug delivery systems. The utilization of nanoemulsions continues to hold challenges of thermodynamic instability that need to be addressed for successful scale-up for the production process, patient safety, and acceptance. Targeting posterior segmental diseases using nanoemulsions having high drug loading capacity and sustained/controlled release profile by non-invasive route would be the future direction of research. After the approval of several liposome-based drug products for clinical use, tremendous progress has been taking place concerning liposome lipid-drug conjugates for enhanced trans-corneal permeation and targeting. The development of biocompatible and biodegradable polymers, delivery of biotechnology, and tissue engineering products are urgently required now for immediate advancement in the field of ophthalmology.SLNs combine the beneficial properties of both liposomes and polymer carriers besides having the ability to encapsulate hydrophilic, as well as lipophilic, drugs. SLNs carry significant potential of targeted and constant drug delivery and the potential capacity to penetrate all ocular tissues, including the posterior chamber, which has recently drawn the attention of many researchers worldwide. Recent investigations revealed that drug-loaded SLNs can penetrate phagocytic cells and cross bacterial membrane barriers, thus, opening new horizons for the treatment of ocular infections and defining a strategy to overcome the challenges of microbial resistance. Several SLN based patents have already been filed and more functionalized SLNs based drug delivery systems would rapidly emerge soon. With the current progression and efforts being undertaken in ocular research, it is anticipated to result in high ocular residence time, to restrict non-specific tissue accumulation and to deliver therapeutic drug concentration into desired ocular tissue sites besides replacing invasive modes of drug administration, such as periocular and intravitreal injection. The application of quality-by-design-based approaches could ease and optimize the formulation development of nanocarrier systems, particularly during the early preformulation stage . Rapid pTargeting drugs to the posterior chamber of the eye through the topical route is still a difficult task because of formulation constraints and the complex anatomical, physiological, and efflux barriers that exist in ocular tissues. Lipid nanocarriers demonstrated excellent ocular permeation characteristics and penetration-enhancing capabilities besides displaying high drug loading and entrapment efficiencies. Lipid nanocarriers will be of considerable interest to researchers who aspire to design and develop ophthalmic drug products with improved efficacy, safety, and acceptability. Drug transporter proteins such as P-gp and multidrug efflux pumps are found in the eye; therefore, lipid nanocarriers could be utilized to target these transporters to promote ocular bioavailability with reduced toxicity. Coordinated efforts between academia, industry, and regulatory authorities are vital to facilitate the potential of these nanoparticles while addressing issues of safety and efficacy.


Methylacidiphilales are known as extremely acidophilic, thermophilic or mesophilic bacteria that inhabit acidic geothermal ecosystems. The occurrence of verrucomicrobial methanotrophs in other types of acidic environments remains an open question. Notably, Methylacidiphilales-affiliated 16S rRNA gene sequences are commonly retrieved from acidic (pH 3.5–5.5) peatlands. In this study, we compared the patterns of verrucomicrobial diversity in four acidic raised bogs and six neutral fens located in European North Russia. Methylacidiphilales-like 16S rRNA gene reads displaying 83–86% similarity to 16S rRNA gene sequences of currently described verrucomicrobial methanotrophs were recovered exclusively from raised bogs. Laboratory incubation of peat samples with 10% methane for 3 weeks resulted in the pronounced increase of a relative abundance of alphaproteobacterial methanotrophs, while no response was detected for Methylacidiphilales-affiliated bacteria. Three metagenome-assembled genomes (MAGs) of peat-inhabiting Methylacidiphilales bacteria were reconstructed and examined for the presence of genes encoding methane monooxygenase enzymes and autotrophic carbon fixation pathways. None of these genomic determinants were detected in assembled MAGs. Metabolic reconstructions predicted a heterotrophic metabolism, with a potential to hydrolyze several plant-derived polysaccharides. As suggested by our analysis, peat-inhabiting representatives of the Methylacidiphilales are acidophilic aerobic heterotrophs, which comprise a sister family of the methanotrophic Methylacidiphilaceae.Methanotrophic verrucomicrobia of the order  Verrucomicrobia was discovered fifteen years ago in three independent studies that focused on exploring methanotrophic microbial communities of three geographically remote acidic geothermal sites located in New Zealand, Italy and Kamchatka  group 3 hydrogenase and formate dehydrogenase indicates that hydrogen and formate also could be generated as fermentation products. Pathways for anaerobic respiration were not found. Two other MAGs obtained in this study, SRB-18 and SRB-116, also encoded an aerobic respiratory chain indicating that these bacteria have similar metabolic properties.Sphagnum-dominated peatlands, such as Fimbriiglobus ruber [The search for potential hydrolytic enzymes revealed that SRB-291 bacterium has a rather limited capacity to degrade complex organic substrates compared to specialized polysaccharide-degrading bacteria from us ruber . NeverthThe finding of beta-N-acetylglucosaminidase, an enzyme that cleaves N-acetyl-D-glucosamine (GlcNAc) from the non-reducing end of chitin oligomers , indicatPlanctopirus limnophila [Humisphaera borealis [An interesting feature of SRB-291 genome is the presence of the pathway for L-rhamnose metabolism . The genmnophila  and Humiborealis .Methylacidiphilales are common inhabitants of acidic (pH 3.5–5.5) Sphagnum-dominated raised bogs. These bacteria, however, are absent from neutral (pH 6.5–7.6) eutrophic fens even though these two types of wetlands are located in a close proximity to each other. This suggests an obligate character of acidophily in peat-inhabiting representatives of the Methylacidiphilales. Analysis of the three MAGs reconstructed in our study allowed making an insight into the metabolic capabilities of these as-yet-uncultivated bacteria. As suggested by our analysis, peat-inhabiting Methylacidiphilales are aerobic heterotrophs, which have the potential to degrade xylan and to utilize a number of plant cell wall sugars, such as xylose and L-rhamnose. This range of potential growth substrates and the predicted presence of catabolic BMCs in cells suggest a notable lifestyle similarity of these verrucomicrobia to the recently described Phycisphaerae planctomycetes from wetlands [Methylacidiphilales. None of the genomic determinants characteristic for methanotrophic metabolism, such as the genes encoding methane oxidation enzymes and C1 assimilation pathways were detected in MAGs assembled for Methylacidiphilales from acidic wetlands.As shown in our study, representatives of the order wetlands . Both grMethylacidiphilales from raised peat bogs together with related bacteria from Alpine bogs, forest soils and some freshwater habitats comprise a sister family of the methanotrophic Methylacidiphilaceae. The only cultivated representative of this potential novel family is an epiphytic verrucomicrobium, strain LW23, which was characterized as being associated with the surface of rice roots [Astrimicrobium roseum’ was proposed for this bacterium but its formal taxonomic description remains to be published. Strain LW23 was characterized as aerobic, neutrophilic to acidotolerant, mesophilic heterotrophic bacterium, which possesses a number of features potentially involved in plant-associated lifestyle. Notably, most MAGs of the candidate family UBA1321 were assembled from the samples composed of live and dead plant remains, such as the vegetation of Alpine bogs or poorly decomposed peat from upper peat bog layers. It may well be that many members of this verrucomicrobial family tend to associate with plants.As suggested by the phylogenomic analysis , Methylace roots . A tentaMethylacidiphilaceae within the order Methylacidiphilales appears to host bacteria with methanotrophic capabilities, which apparently were acquired via the horizontal transfer of methane monooxygenase genes from an ancestral methanotroph [Methylacidiphilales, most likely, are acidophilic or acid-tolerant heterotrophic bacteria with various substrate utilization preferences and environmental adaptations. Extending characterized diversity of these bacteria by using the information retrieved in metagenomic studies is one of the most promising strategies for further cultivation efforts.In summary, only the family anotroph  and furt


Recently, a new gene regulation mechanism called competing endogenous RNA (ceRNA) interactions has been discovered. Certain genes targeted by common microRNAs (miRNAs) “compete” for these miRNAs, thereby regulate each other by making others free from miRNA regulation. Several computational tools have been published to infer ceRNA networks. In most existing tools, however, expression abundance sufficiency, collective regulation, and groupwise effect of ceRNAs are not considered. In this study, we developed a computational tool named Crinet to infer genome-wide ceRNA networks addressing critical drawbacks. Crinet considers all mRNAs, lncRNAs, and pseudogenes as potential ceRNAs and incorporates a network deconvolution method to exclude the spurious ceRNA pairs. We tested Crinet on breast cancer data in TCGA. Crinet inferred reproducible ceRNA interactions and groups, which were significantly enriched in the cancer-related genes and processes. We validated the selected miRNA-target interactions with the protein expression-based benchmarks and also evaluated the inferred ceRNA interactions predicting gene expression change in knockdown assays. The hub genes in the inferred ceRNA network included known suppressor/oncogene lncRNAs in breast cancer showing the importance of non-coding RNA’s inclusion for ceRNA inference. Crinet-inferred ceRNA groups that were consistently involved in the immune system related processes could be important assets in the light of the studies confirming the relation between immunotherapy and cancer. The source code of Crinet is in R and available at  MicroRNAs (miRNAs) are small RNA types that bind to other RNAs such as mRNA, long non-coding RNA (lncRNA), and circular RNA to regulate their expression post-transcriptionally. Recently, a new regulatory layer related to miRNAs has been discovered : certainDespite existing computational tools –9, thereIn this study, we developed a computational tool named Crinet (CeRna Interaction NETwork) to infer genome-wide ceRNA interactions and groups to address the aforementioned drawbacks. To build our ceRNA network on a proper miRNA-target interaction set, we integrated expression datasets and binding scores for miRNA-target pairs considering expression abundance sufficiency. We computed ceRNA pairs considering strong regulation jointly. To cope with the spurious interactions, we excluded ceRNA pairs which were potentially inferred because they had significant overlapping of common ceRNA partners. We inferred ceRNA groups and integrated these groups into the ceRNA network. Getting the benefit of multiple biological datasets and including non-coding RNAs, this approach facilitates a better understanding of ceRNA regulatory mechanisms addressing important drawbacks, which would shed light on the underlying complex regulatory circuitry in disease conditions.Crinet was applied to breast cancer dataset to infer ceRNA interactions and groups. We evaluated Crinet-selected miRNA-target interactions with protein expression-based benchmarks having increasing performance following filtering steps. Expression change in the inferred ceRNAs was highly affected following knockdown of their ceRNA partners. Inferred ceRNAs, hub ceRNAs, and some ceRNA groups were significantly associated with the cancer-related genes and processes, and consistently involved in the immune system related processes, thus Crinet-inferred ceRNAs could be important virtues in the view of the studies validating the relation between immunotherapy and cancer.Crinet is a computational tool to infer genome-wide ceRNA interactions and groups . BrieflyCrinet incorporates miRNA-target interactions with binding scores, gene-centric copy number aberration (CNA), and expression datasets. If binding scores are not available, the same score for all interactions could be used.https://www.cancer.gov/tcga). We preprocessed each datatype separately obtaining normalized expression values (gene expression as FPKM and miRNA expression as RPM) and filtered lowly expressed genes (if FPKM <1 and RPM = 0 for at least 15% samples). To get gene-centric CNA from a segmented dataset, we ran CNTools R package [To collect datasets for Crinet, we used TCGAbiolinks R package  and obta package .We obtained all conserved and nonconserved miRNA-target interactions with weighted context++ scores from TargetScan . To compSince there is a corresponding weighted context++ score in TargetScan results even for weak miRNA-target interactions, we kept the top 40% of ranked interactions with respect to weighted context++ scores. To fit different distributions of scores for mRNAs, lncRNAs, and pseudogenes, we combined z-normalized scores from each and applied min-max normalization after having all the scores in the range of -1 and 1. We used these normalized scores as the weight for each miRNA-target interaction assuming that these scores show the binding strength between miRNA and its gene target.In this step, we computed final miRNA-target interactions leveraging expression datasets and considering expression abundance sufficiency.Since miRNAs are known to repress their target genes, we kept only miRNA-target pairs having negative correlation between their expressions. By default, we used the correlation coefficient threshold of -0.1, which was the median correlation coefficients of all miRNA-target pairs. We also applied random sampling with replacement to compute correlation coefficient of each miRNA-target pair for 1000 times and required that the threshold was satisfied for ≥99% of the samplings.Interaction Regulation (IR) formulated as:IR is the IR of the regulator r and the target t across samples, Exp(.) is the expression vector across samples, scorert is the normalized binding score for the interaction between the regulator r and the target t. Using this formula, we kept the final miRNA-target interactions having high IRs  > −4.89, which was third quartile of all IRs through samples).In the miRNA-target interaction sets, a miRNA could be assigned to mediate thousands of RNAs, and similarly an RNA could be assigned to many miRNAs as a potential target. To quantify the expression sufficiency for our putative interactions, we introduced Effective Regulation (ER) formulated as:To exclude genes from analysis if they were not under strong miRNA regulation based on the final miRNA-target interactions, we introduced To keep genes with proper effective regulation by miRNAs, we filtered out genes without strong negative correlation (< −0.01) between its expression and ER, assuming that they did not have strong miRNA regulation for our specific dataset. We also applied random sampling with replacement to compute correlation for 1000 times and required that the threshold was satisfied for ≥99% of the samplings. We used the remained genes  for further analysis.To infer ceRNA interactions, we generated all possible gene-gene combinations using candidate genes and filtered them based on the following criteria:Since ceRNAs should have common miRNAs to compete for, we kept gene pairs having a significant number of common miRNA regulators .Since the ceRNA pairs indirectly positively regulate each other and CNA considerably affects the expression values, we kept the gene pairs having significant partial correlation when excluding the CNA effect from the gene expression. By default, we used the correlation coefficient threshold of 0.55 , which was the third quartile of the positive correlation  formulated as:S is a set of genes having ceRNA interactions and corr is the Pearson correlation function. We kept the ceRNA pairs if they had a CR < −0.01.If there exists ceRNA regulation between two genes then both genes compete for common miRNAs and those miRNAs affect both genes simultaneously. We called this regulation We applied random sampling with replacement for both partial correlation and CR measurements (last two steps) 100 times separately and kept the interactions when the threshold was satisfied for ≥99% of the samplings.Having common ceRNA partners between any two genes will increase the correlation between their expression. If a gene pair has too many common ceRNA partners, then some of the ceRNA interactions could be superior due to the high number of common ceRNA partners. To exclude such spurious interactions from our network, we employed a network deconvolution algorithm  and keptIn ceRNA regulation, each ceRNA pair compete for common miRNA(s) and act as a decoy to make the other RNA free from miRNA regulation. However, this competition could occur among more than two RNAs, or between two groups of RNA. Based on this premise, Crinet inferred ceRNA groups in addition to ceRNA interactions.To obtain ceRNA groups, we utilized one of the popular community detection algorithms named Walktrap  on the wTo be able to compete for, all the group members were required to have at least one common miRNA regulator.corr(Exp(g), ER(g)) for each gene g in the group was >0.CeRNAs in a group were expected to have a stronger miRNA regulation effect as a group than as individual ceRNAs. Thus, we required that CR of a group must be stronger (i.e. reduced) than the correlation between expression and ER of ≥90% of the group members. Moreover, to ensure that most of the group members would be under strong collective regulation effect, we required that an average difference between CR of the group and neighbor and expected the group to satisfy any two of the three conditions for at least 90% of its neighbors. The conditions are i) at least one common miRNA regulator between the group and the neighbor, ii) a strong Pearson correlation based on expression, and iii) strong collective regulation between the group and the neighbor.To hold inference consistency of the ceRNA network, for a given group, we called each ceRNA partner of group members as Data preparation for details). We computed miRNA-target interactions (We tested Crinet on breast tumor samples from TCGA (Section ractions  and usedractions . Using tR2 = 0.93), indicating that the inferred ceRNA network was scale-free  suggesting that consistently inferred ceRNAs were the hub ceRNAs highly involving in our inferred ceRNA network. Moreover, to examine the effect of each step in Crinet, we disabled major steps in ceRNA inference and evaluated the results. Disabling individual steps made a substantial difference in the inferred results  dataset for MDA-MB-231 breast cancer cell line from The Cancer Proteome Atlas (TCPA) database (accession number: TCPA00000001)  to assesUsing protein expression dataset from TCPA matching with breast tumor samples in our analysis, we analyzed the negative correlation between miRNA expression and protein expression of their targets for each applicable miRNA-target interaction.While the anticorrelation tendency slightly decreases after selecting top 40% miRNA-target interactions, our filtering steps substantially increased the ratios showing the anticorrelation tendency in our selected interactions . Also, wTo evaluate predicted miRNA-target interactions in , the autTo assess the accuracy for ceRNA inference, we used the Library of Integrated Network-based Cellular Signature (LINCS)  L1000 shTo analyze the biological significance of the inferred ceRNA network, we applied enrichment analysis for the inferred ceRNAs. We used ClusterProfiler R package  for all −105) between the known cancer genes and the inferred ceRNAs having 789 out of 3078 known cancer genes in our inferred network. When we repeated the same analysis for non-inferred genes, they did not show significant p-value . We also had 54 breast cancer-related genes from LncRNADisease and Network of Cancer Genes 6.0 databases, 14 out of 54 breast cancer-related genes were inferred in our network. These results indicated that inferred ceRNAs were significantly associated with known cancer genes.To check if the inferred ceRNAs were associated with the cancer-related genes, we collected 3078 known cancer genes obtained from Cancer Gene Census in COSMIC v91 , BushmanMoreover, we analyzed the hub ceRNAs in our network. The degree distribution of our network had a median of three with a maximum of 152 and a minimum of 1. We got the top 81 ceRNAs having a degree of 50 or more in our network as hub genes. The hub genes were significantly enriched in 58 GO terms and eight KEGG pathways. We investigated the GO Slim terms from biological processes ontology for hub genes, and enriched terms included immune system process, cell death, cell differentiation, cell motility, and cell cycle. Also, these hub genes were among the known cancer genes with a hypergeometric p-value of 0.0009. Specifically, 15 out of 81 genes were involved in the known cancer genes, while three of them were breast cancer-related. These suggest that hub ceRNAs in the inferred network were involved in the important biological processes in cancer.Among hub genes were some lncRNAs with known involvement in cancer. For instance, MAGI2-AS3 had a degree of 89 being highly connected for ceRNA regulation in our network, and it is known as suppressor involving in cell growth . MALAT1,To evaluate the inferred ceRNA groups, we performed enrichment for four of 81 inferred ceRNA groups that have more than three members  score, and confirmed that the miRNAs with the highest number of targets and low expression levels were successfully filtered out  genes in order to have a computationally manageable number, we included all lncRNAs and pseudogenes in addition to all coding RNAs (including non-DE mRNAs) to understand comprehensive ceRNA regulation. We had the known lncRNA oncogenes and suppressors as highly connected in the inferred ceRNA network indicating that the lncRNAs could have an important role in ceRNA regulation. When the expression profiling of other data types such as circular RNAs is available, including them into the pipeline could be worthy of further investigation to improve ceRNA inference .We also started with a large set of miRNA-target interactions since we utilized weighted context++ score as a proxy for binding probability. This enabled us to include all possible gene-gene interactions based on common regulators especially for the measurements like our collective and effective regulation. Our carefully designed miRNA-target interaction filtering steps were able to eliminate high likely false-positive interactions confirmed by the protein expression-based benchmarks. As a negative control, we analyzed non-inferred interactions in addition to the inferred ones in our evaluations based on miRNA transfection and protein expression data. Especially for the gene phase, we observed that the the number of down-regulated non-inferred interactions to up-regulated ones were less than 1 Click here for additional data file.


Stomach cancer is a common malignancy and one of the leading causes of cancer death in Taiwan. Although tumor characteristics are the main determinants of oncological outcomes, modulation of the immune system may also play some role in cancer progression. Despite the hypothesis that perioperative blood transfusion may suppress the immune reactivity and promote tumor recurrence, the role of perioperative transfusion in the prognosis of stomach cancer remains controversial. To fill this gap, we designed this retrospective study using sound analytical approaches to investigate the impact of perioperative transfusion on oncologic outcomes after curative resection for stomach cancer. We demonstrated that perioperative transfusion was associated with inferior disease-free and overall survival after stomach cancer surgery and a dose-response relationship was also noted between the amount of transfusion and risk of cancer recurrence or mortality.p < 0.001) and overall survival . A non-linear dose-response relationship was noted between the amount of transfusions and worse disease-free or overall survival. Conclusions: Perioperative pRBC transfusion was associated with worse disease-free and overall survival after stomach cancer surgery, and strategies aiming to minimize perioperative transfusion exposure should be further considered to reduce the potential risk.Background: Whether perioperative packed red blood cell (pRBC) transfusion is associated with inferior long-term outcomes after stomach cancer surgery remains controversial. Methods: This research used a retrospective cohort study. Patients with stage I~III stomach cancer undergoing tumor resection were collected at a tertiary medical center. Patient characteristics, surgical features and pathologic findings were gathered from an electronic medical chart review. The associations of perioperative pRBC transfusion with postoperative disease-free and overall survivals were evaluated using Cox regression analysis with an inverse probability of treatment weighting (IPTW). Restricted cubic spline functions were employed to characterize dose-response relationships between the amount of transfusion and cancer outcomes after surgery. Results: Among the 569 patients, 160 (28.1%) received perioperative pRBC transfusion. Perioperative transfusion was associated with worse disease-free survival (IPTW adjusted HR: 1.42, 95% CI: 1.18–1.71,  Stomach cancer is a common malignancy and one of the leading causes of cancer death in Taiwan . SurgicaIn order to elucidate the relationships between perioperative pRBC transfusion and oncologic outcomes after stomach cancer surgery, we conducted this retrospective cohort study and applied robust statistics to a detailed list of clinicopathologic features associated with oncologic outcomes to isolate the effects of perioperative transfusion on recurrence and survival and determine if dose influences outcomes. We used inverse probability of treatment weighting (IPTW) based on a propensity scoring model to eliminate imbalances in patient characteristics that might affect outcomes and preserve sample size for better precision. In light of the positive findings observed in previous studies, we hypothesize that perioperative pRBC transfusion increases the risk of stomach cancer recurrence and mortality after curative surgery. We also suggest that the amount of blood given could modify the relative risk further. The purpose of our analysis is to provide health care providers with detailed information about the transfusion risk to guide clinical decision making in the perioperative period.After the approval of Institutional Review Board of Taipei Veterans General Hospital (IRB-TPEVGH No.: 2017-01-021AC), we reviewed the electronic medical database of all patients who received surgery for stomach cancer at a medical center from January 2009 to December 2014 and followed up them until December 2020. Those with repeated surgery, palliative surgery or metastasis, resection for benign lesion, missing demographic data, surgical and anesthetic features, and pathologic records were excluded . The remThe specialist anesthesiologist who reviewed the electronic medical records was not involved in data analysis. We collected the following data: age, sex, height, weight, body mass index (BMI), underlying disease based on Charlson comorbidity index  ; preoperThe primary outcome was disease-free survival (DFS), which was defined as the time from the date of surgery to the date of tumor recurrence. Patient death without recurrence was treated as a censored case. The secondary outcome was overall survival (OS), defined as the time from the date of surgery to the date of death. For those without cancer recurrence or death, the survival time was regarded as the censored observations.The frequency of transfusion is presented in the Among the 569 patients included in the analysis, 160 (28.1%) received perioperative pRBC transfusion for stomach cancer resection. The median follow-up interval for all patients was 59.8 months (interquartile range: 22.8~90.7). Note that in the original dataset, more patients in the perioperative pRBC transfusion group tended to have lower preoperative hemoglobin and albumin levels, less laparoscopic surgery, longer anesthesia time and more advanced cancer . Supplemp < 0.001, p < 0.001) in the weighted Cox regression analysis. With respect to the dose-response relationship, the IPTW-adjusted method using restricted cubic spline transformation revealed a significantly non-linear association between perioperative pRBC transfusion and cancer recurrence after surgery (p = 0.003) as well as after the adjustment for the other significant predictors. The propensity score-based quintile method also identified a significant association between perioperative pRBC transfusion and DFS after stomach cancer surgery In the univariate analysis, perioperative pRBC transfusion was associated with inferior DFS (crude hazard ratio (HR) = 2.86,  surgery . Note th surgery a. For se surgery . Note thp < 0.001, p = 0.014) after IPTW. A significantly non-linear dose-response relationship was identified between the amount of transfusions and OS, and the peak effect was noted around 4 U (p < 0.001). A significant association between perioperative pRBC transfusion and OS after stomach cancer surgery was also demonstrated using the quintile method based on the propensity score .In the univariate analysis, perioperative pRBC transfusion was associated with worse OS after surgery for stomach cancer (crude HR = 2.79, ound 4 U b. For seound 4 U . The effFour independent predictors of perioperative pRBC transfusion were identified from the multivariable logistic regression analysis, including hemoglobin level, surgical blood loss, Charlson comorbidity index and BMI .Our study revealed that perioperative pRBC transfusion was an independent predictor of disease-free and overall survivals in patients receiving stomach cancer surgery, and a non-linear dose-response relationship was noted in contrast to some previous studies that proposed that more transfusion would lead to more cancer recurrence and mortality . In thisThe mechanism of perioperative pRBC transfusion influencing cancer recurrence may be attributed to the modulation of the immune system . PeriopeBMI is a protective factor in our study. The previous literature revealed the U-shaped association between BMI and the oncological outcomes of stomach tumor . PatientThe choice of total or subtotal gastrectomy is based on the location, size and extent of the tumor . In patiThere are several limitations in our study. First, potential selection bias and confounding factors cannot be totally excluded due to the retrospective design. Second, outcomes of patients lost to follow-up were difficult to obtain and the corresponding censoring time was included in the analysis instead. Third, we only evaluated the effects of pRBC transfusion on cancer outcomes but did not further investigate the influences of other blood products that may also induce a different immune response. Finally, the immune status of collected patients was not further assessed due to data unavailability.Perioperative pRBC transfusion was associated with worse disease-free and overall survival in patients receiving curative surgery for stage I to III stomach cancer. In order to improve patient outcome, a systematic method should be considered to weight the relative risks of anemia compared to blood transfusion in order to guide treatment. Strategic measures to reduce surgical blood loss and perioperative transfusion will also be beneficial to long-term outcomes after stomach cancer surgery. Well-designed prospective studies are still necessary to verify the causal relationships between perioperative transfusion and oncological outcomes after stomach cancer surgery and to elucidate the underlying mechanisms in order to identify more potential interventions.


Background: The exact focus of computed tomography (CT)-based artificial intelligence techniques when staging liver fibrosis is still not exactly known. This study aimed to determine both the added value of splenic information to hepatic information, and the correlation between important radiomic features and information exploited by deep learning models for liver fibrosis staging by CT-based radiomics. Methods: The study design is retrospective. Radiomic features were extracted from both liver and spleen on portal venous phase CT images of 252 consecutive patients with histologically proven liver fibrosis stages between 2006 and 2018. The radiomics analyses for liver fibrosis staging were done by hepatic and hepatic–splenic features, respectively. The most predictive radiomic features were automatically selected by machine learning models. Results: When using splenic–hepatic features in the CT-based radiomics analysis, the average accuracy rates for significant fibrosis, advanced fibrosis, and cirrhosis were 88%, 82%, and 86%, and area under the receiver operating characteristic curves (AUCs) were 0.92, 0.81, and 0.85. The AUC of hepatic–splenic-based radiomics analysis with the ensemble classifier was 7% larger than that of hepatic-based analysis (p < 0.05). The most important features selected by machine learning models included both hepatic and splenic features, and they were consistent with the location maps indicating the focus of deep learning when predicting liver fibrosis stage. Conclusions: Adding CT-based splenic radiomic features to hepatic radiomic features increases radiomics analysis performance for liver fibrosis staging. The most important features of the radiomics analysis were consistent with the information exploited by deep learning. Chronic liver disease prevalence is increasing globally . RepetitHistopathology is currently considered the gold standard for liver fibrosis diagnosis and staging. To enable histopathological examination of liver parenchyma, a percutaneous image-guided biopsy can be performed. However, up to 6% of patients experience postprocedural complications, with a reported mortality rate of 1.6% .To overcome these disadvantages, various radiology-based liver fibrosis staging methods have been explored to replace biopsy, with among them, radiomics analysis ,7,8 and Therefore, the primary aim of the current study was to determine whether adding splenic information to hepatic information can improve the performance of liver fibrosis staging by CT-based radiomics analysis and to identify the most important radiomic features in this setting. The secondary aim was to explore the correlation between important radiomic features and location maps generated by deep learning models.All consecutive patients who were diagnosed with liver fibrosis at our tertiary referral center between 2006 and 2018, aged >16 years, and of whom both a contrast-enhanced portal venous phase CT scan of the upper abdomen  and histopathological proof of liver fibrosis stage were available, with less than 6 months between them, were included in the study . PatientTo improve the balance between patients with and without fibrosis , patients who underwent a contrast-enhanced abdominal CT scan in the portal venous phase (similar image acquisition parameters as mentioned earlier) at the emergency department to rule out traumatic injuries during the same inclusion period were added to our study population. Patients were only included if no focal liver lesions, injuries, or other abnormalities of liver morphology were present, as well as no history of liver disease or associated risk factors.Patients were identified in our institutional database and retrospectively analyzed. The study was approved by the local institutional review board, and the need for obtaining informed consent was waived.The reference standard consisted of histopathological examination of liver parenchyma, either through liver biopsy, liver resection, or after liver transplantation. Liver fibrosis stage was determined by a specialized liver pathologist according to the METAVIR staging system . This syThe algorithm scheme is shown in The radiomic features were then extracted from the original 3D images of the segmented liver and spleen parenchyma, respectively. The images were resampled with multiple spacing parameters by sitkBSpline interpolator provided by the pyradiomics library , includip < 0.05) were included in the selected feature subset.To remove the features that did not show differences among liver fibrosis stages, we performed univariate feature selection by the Kruskal–Wallis test. Features statistically related to the liver fibrosis stage . To visualize data distribution, principal component analysis and t-distributed stochastic neighbor embedding (t-SNE) were combined to reduce the radiomic features to two dimensions, represented by the axes in the plot of After extracting the radiomic features from both the liver and spleen, 77 features from the liver and 65 features from the spleen were selected by the univariate feature selection , while the microaveraged AUCs of logistic regression were not (p = 0.243 and p = 0.277). In addition, we used full feature sets without univariate feature selection, which showed a similar result; the AUCs of hepatic–splenic classifiers improved by 5% at most.The top 5 weighted features of logistic regression classifiers are shown in The top five weighted features of tree-based ensemble classifiers for liver fibrosis staging are shown in This study aimed to determine the value of adding splenic information to liver information in liver fibrosis staging by CT-based radiomics analysis, as well as investigate the correlation between important features of radiomic analysis and the CT-based information exploited by the deep learning model for liver fibrosis staging.p < 0.01). Interestingly, the logistic regression classifiers demonstrated that hepatic radiomic features, especially shape-based hepatic features that can be extracted from the liver surface, were most important for liver fibrosis stage F0. The combination of hepatic and splenic texture features was much more important when it concerned liver fibrosis stage F4. This is in accordance with a recent study in which a deep learning model was trained for liver fibrosis staging and location maps were generated by Grad-cam [In the current study, the microaveraged AUC of best-performing radiomics analysis based on hepatic–splenic features was significantly larger than based on merely hepatic features, namely the XGBoosting classifier -based radiomic features and random forest classifier were used to predict the severity of liver cirrhosis, with clinical decompensation and model for end-stage liver disease (MELD) scores as reference standard. Nitsch et al. concluded that adding splenic MRI-based radiomic features can increase the AUC for predicting a higher median MELD score or clinical decompensation . HoweverOur study found that the machine learning models based on splenic–hepatic features could outperform the models only based on hepatic features. To generalize further, it might be helpful to include information of multiple organs when developing computer-aided diagnostic tools for systemic diseases such as liver fibrosis. In addition, as mentioned earlier, the most important radiomic features selected by machine learning models in the current study were consistent with the location maps generated by a recently reported liver fibrosis staging network . The resSome limitations of our study have to be mentioned. First, our study is a retrospective and single-center study, which might limit the utility of our results in broader clinical practices. A prospective and multicenter study using the same research question could be a possible improvement in the future. Second, the relatively small number of patients with F1, F2, and F3 liver fibrosis in the current study can be considered a limitation. This renders the performance of machine learning models sensitive to the bias-variance tradeoff. Unfortunately, because histopathological proof in patients with less severe liver fibrosis stages is generally not available, it is difficult to prevent imbalances in the dataset. In addition, the stability of texture features could be influenced by the parameters used during CT image acquisition and reconstruction techniques could also influence the robustness of radiomic features ,26, whicAdding CT-based splenic radiomic features to hepatic radiomic features increases radiomic analysis performance for liver fibrosis staging. In addition, the most important features of the radiomic analysis were consistent with location maps generated by deep learning, which could improve physicians’ confidence when using deep learning techniques more regularly in daily practice.