Prostaglandin analogue topical medications are one of the most effective therapeutic approaches for the chronic management of glaucoma and ocular hypertension, through the reduction of elevated intra ocular pressure (IOP). While many of the first generations of anti-glaucoma eye drops were preserved with benzalkonium chloride, their repeated use may induce chronic ocular surface toxicity that leads to ocular surface disease (OSD) signs and symptoms. As a result, soft-preservatives and preservative-free formulations have been developed with the goal to avoid the long-term iatrogenic toxicity of the preservative agents. In addition, it has been suggested that OSD and its associated inflammation may negatively impact the efficacy of the IOP-lowering medications, including treatment adherence and compliance. Hence, it may be particularly interesting that glaucoma medications can concomitantly protect and “heal” the ocular surface and its environment while lowering elevated IOP, for the greater benefit of glaucoma patients. The objective of the present review is to briefly present the preclinical data of the cationic oil-in-water emulsion of latanoprost (latanoprost-CE) to shed some light on its mechanisms of action. It overall supports the following hypothesis: the restoration of a healthy ocular surface environment and treatment of the OSD signs and symptoms will allow for an improved elevated IOP reduction and glaucoma management. This would be achieved with a once daily dosing regimen to preserve glaucoma patients' vision, ocular surface, and quality-of-life and wellness. It is estimated that 3.5% of the global population aged 40–80 years have any glaucoma, with the primary open-angle glaucoma (POAG) and the primary angle-closure glaucoma being the most prevalent forms.2 Glaucoma affects more than 70 million people worldwide with ∼10% being bilaterally blind, making it the leading cause of irreversible blindness in the world.Glaucoma is a worldwide leading cause of irreversible vision loss.The pathogenesis of glaucoma is not fully understood. The level of intraocular pressure (IOP) is related to retinal ganglion cell death; generally, the higher the IOP the more the cell death, and reduction of IOP is the only proven method to treat the disease.3–5Increased IOP results from a decreased elimination rate of aqueous humor constantly produced by the ciliary body. Hence, either decreasing the production of aqueous humor or increasing the drainage of the aqueous humor will lead to a decreased IOP. The vast majority of glaucoma patients worldwide use anti-glaucoma eye drops formulated with β-adrenergic antagonists, carbonic anhydrase inhibitors, α-adrenergic agonists, prostaglandin analogues (PGA), or a combination thereof.6–9 PGAs are currently the standard of care and first-line treatment for POAG. They are among the most potent and efficacious medical therapies for lowering elevated IOP.These eye drops are either preserved  or preservative-free. PGAs primarily increase the flow of aqueous humor through the uveoscleral pathway and may also act on the trabecular meshwork and relax the ciliary muscle, hence decreasing IOP.10 The presence of preservative agents, such as BAK, induces iatrogenic toxicity with signs and symptoms of ocular surface disease (OSD) such as dry eye disease (DED).11 They lead to decreases in tear film (TF) stability and TF break-up time, Schirmer test values, and worsening of ocular surface inflammation and in DED symptom scores, as evaluated with the OSD index scale, and staining scores.The poor compliance associated to the use of IOP-lowering eye drops is often due to the ocular complications associated with their chronic use.13 Indeed, there is a long history of evidence that a large population of glaucoma patients also presents OSD signs and symptoms, that is, close to 60% of medically treated glaucoma patients also have DED.14–16It is hypothesized that the inflammation starting at the ocular surface and diffusing toward deeper ocular tissues such as the trabecular meshwork following the long-term use of preserved IOP-lowering medications induces a vicious cycle of ocular alterations worsening the glaucoma condition and further jeopardizing the long-term outcome of the disease management.17–19 However, it is noteworthy that although the preservative-free formulations of PGA are considered non-inferior when compared with their preserved reference, none formally demonstrated benefits on OSD and showed numerically lower levels of IOP reduction.17As a consequence, preservative-free anti-glaucoma eye drops were developed that appear to be better tolerated than the preserved formulations, and provide a significantly higher patient satisfaction.21 Removing the preservative agents in preservative-free anti-glaucoma eye drops halts the worsening of the underlying ocular surface inflammation. However, these formulations do not possess “healing” properties per se.There is also evidence that OSD and ocular surface inflammation negatively affect the efficacy of the anti-glaucoma treatment, and that DED signs management may result in improved IOP control.It may be reflected by their less than ideal “equivalent” IOP performance and the need for concomitant use of an effective OSD treatment option for the management of OSD signs and symptoms, such as artificial tears. Multiplying number of treatments, that is, adding repeated instillations of artificial tears eye drops on top of the anti-glaucoma eye drops, might also have some deleterious outcomes in term of patient compliance and adherence.22 The TF has a vital role in providing lubrication and protection to the ocular surface, as well as maintaining a smooth, refractive surface for optimal visual performance.23 A stable state of equilibrium  of the ocular surface with a stable TF and TF lipid layer (TFLL) is expected to create a healthy ocular surface environment that can contribute to the management of the underlying ocular surface inflammation existing in glaucoma patients with OSD and consequently to a better IOP control in these patients.24A stable preocular TF is a hallmark of ocular health.® technology of cationic oil-in-water emulsions that have demonstrated their excellent performance for the management of mild/moderate and severe DED signs and symptoms, and their capacity to effectively deliver lipophilic drugs to the ocular surface tissues seem to be the ideal drug delivery system (DDS) platform for the lipophilic prodrug latanoprost.25–27The NovasorbThe objective of the present paper is to review the preclinical data of a well-tolerated cationic emulsion of latanoprost (latanoprost-CE) to shed some light on its mechanisms of action and the following hypothesis: the concomitant restoration of a healthy ocular surface environment with less conjunctival and trabecular inflammation may allow for a better IOP control and glaucoma management in glaucoma patients with OSD; and this with a convenient once-daily dosing regimen to protect glaucoma patients vision, and their ocular surface.26The Novasorb technology is a DDS designed to mimicry the functional composition of a healthy TF with the aim to be the best vehicle for the effective delivery of lipophilic drugs to the anterior segment tissues of the eye . This DDS is a cationic oil-in-water emulsion that contains non-polar  and polar  lipids, non-ionic surfactants , and the osmoprotectant glycerol.28 Based on the good performances of the cationic oil-in-water emulsions DDS at successfully delivering a lipophilic drug ,29 and at restoring a healthy precorneal ocular surface environment ,31 it was decided to use this DDS for the delivery of the lipophilic latanoprost (0.005%) prodrug. The The presence of the polar lipid CKC, which is positively charged, brings the cationic feature to the oil nanodroplets of the oil-in-water emulsion. This positive charge contributes to the stabilization of both the drug product  and the TF once it is instilled on the eye. The ®).32 The ocular tissues distribution was demonstrated to be similar between the 2 latanoprost formulations. A better ocular penetration of the prodrug after latanoprost-0.02%BAK instillation, especially in the cornea and aqueous humor at the shortest time points  study in the rabbit and compared with the reference product latanoprost-0.02%BAK  white rabbits over a period of 28 days, and it was compared with latanoprost-0.02%BAK.34 The systemic exposure of latanoprost-free acid was negligible in both groups after the repeated instillations. Only one animal (out of 8) in each group had a quantifiable level of latanoprost-free acid above the lowest limit of quantification (30 pg/mL): 32.1 and 33.9 pg/mL for latanoprost-CE and latanoprost-0.02%BAK group, respectively.This is of particular interest, since it has been largely described in the literature that the use of latanoprost-0.02%BAK is hampered by a relatively high incidence of hyperemia/red eye, a side effect that has a detrimental consequence on patients' treatment compliance and quality-of-life.35 it will be of particular interest to evaluate the impact  of latanoprost-CE and latanoprost-0.02%BAK on diseased cornea presenting epithelial defects.In conclusion, these safety data demonstrate that both latanoprost formulations were overall well tolerated by the healthy ocular surface of NZ white rabbit, with latanoprost-CE inducing less conjunctiva irritation than latanoprost-0.02%BAK. However, since glaucoma patients also present an ocular surface with a damaged corneal epithelium, with 45%–60% of patients with glaucoma using IOP-lowering drugs have some degree of OSD,It is expected that only a safe formulation will be able to contribute positively to the restoration of a diseased corneal epithelium  immunohistology staining demonstrated that a higher number of proliferating cells were present in the scrapped area on treatment with latanoprost-CE, when compared with latanoprost-0.02%BAK, or other PGAs with different BAK concentrations. These data by Liang et al. (2022) confirmed that BAK was, in a dose-dependent manner, deleterious for the wound-healing process.In an They also demonstrate that the latanoprost-CE can maintain a normal healing process comparable to the one observed on treatment with control PBS. To further analyze the impact of latanoprost-CE instillations on a healthy ocular surface, an established severe rabbit toxicological model  was used. This experiment confirmed that repeated instillations of latanoprost-CE were well tolerated with no signs of ocular surface irritation or lesions as it was observed on treatment with latanoprost-0.02%BAK.40 In this severe toxicity model, latanoprost-0.02%BAK was not tolerated by the healthy rabbit ocular surface.Indeed, the repeated use of BAK-preserved anti-glaucoma medications have demonstrated their long-term toxic and immunoinflammatory effects in the ocular surface's conjunctiva and the inner structure of the trabecular meshwork.in vivo confocal microscopy, or CD45-positive cells by impression cytology) in the conjunctiva-associated lymphoid tissue (CALT) were observed on latanoprost-CE treatment. In contrast, latanoprost-0.02%BAK treatment resulted in a drastic increase of inflammatory cells count in the CALT, as seen in 41Indeed, no infiltrating inflammatory cells  were able to promote corneal wound healing in DED patients with ocular signs of OSD and vernal keratoconjunctivitis patients.Previous preclinical as well as clinical data indicate that the cationic oil-in-water emulsion , and cyclosporine-loaded oil-in-water emulsion  suffer from OSD.43 Moreover, as the treatment proceeds, the corneal surface is wounded by the repeated applications of eye drops containing 0.02% BAK.In this case, pre-existing OSD conditions are generally worsened and exacerbated by the repeated use of the BAK-preserved antiglaucoma drugs. The ocular surface of these patients generally displayed subclinical to clinical signs of inflammation leading to the loss of goblet cells and to a dramatic reduction of the production of protective mucins .Apoptosis of corneal epithelial cells develops, leading to the formation of scars accompanied by irritation and itching sensations that led the patients to end their treatment and have a poor quality-of-life and treatment experience, and are at risk of becoming blind.in vitro, as well as in a rat model of corneal debridement, it appeared that latanoprost-CE was able to improve the wound-healing pace, when compared with the control.44 Treatments with latanoprost-CE or its vehicle (CE) allowed for a complete and almost scar-free re-epithelization of the cornea. It is of particular notice that the remaining scar after treatment with latanoprost-CE was approximately one-third of the one observed after PBS treatment.In an in vitro, and resulted in the formation of scar tissue within the reconstructed rat corneal epithelium  produced by many epithelial tissues. They bind to pathogens as part of the immune system and can retain water and help maintain a wet environment that protects the ocular surface. Collectively, these data demonstrate that Latanoprost-CE possesses potential beneficial properties, that is, it improves corneal wound healing and protects conjunctival goblet cells. On the contrary, latanoprost-0.02% BAK treatment on de-epithelialized cornea worsens the condition of the ocular surface.47These benefits on the corneal wound-healing process and the protection of the goblet cells by latanoprost-CE may find their explanation in the fact that the cationic oil-in-water emulsion are able to protect the ocular surface by: (1) mechanically creating a healthy environment for the ocular surface via the stabilization of the TF and TFLL, and (2) by controlling, via the ancillary pharmacological action of CKC, a known protein kinase Cα (PKCα) inhibitor, the level of ocular surface inflammation.48Indeed, Chen et al. (2010) have demonstrated with a PKCα-knockout mouse exposed to a dry environment, to mimic DED with its corneal epithelium lesions, that blocking PKCα resulted in a lower number of inflammatory cells on the ocular surface and a better wound-healing process of corneal epithelium lesions.50 Mice with a scopolamine transdermal patch placed in a controlled environment chamber were treated for 7 days with various PGA eye drops once dry eye signs peaked at day 3.The efficacy at reducing signs of corneal damage  of the cationic oil-in-water emulsion loaded with various PGAs was evaluated in a well-described mouse model of dry eye.27 These data are corroborated by previous similar studies using cationic oil-in-water emulsion vehicles loaded with cyclosporine and developed for the management of severe DED signs.51The mean CFS change at day 10  demonstrated that the PGAs formulated with the cationic oil-in-water emulsion performed better at reducing CFS scores than BAK-preserved and preservative-free PGA formulations . The catThe preclinical study results described so far demonstrate that latanoprost-CE possesses interesting features that define it as an effective drug formulation for reducing elevated IOP in glaucoma patients suffering from OSD: (1) latanoprost-CE is able to delivers an effective dose of latanoprost-free acid to the target tissue, (2) latanoprost-CE is as effective as the PGA gold standard antiglaucoma eye drop at reducing elevated IOP, (3) latanoprost-CE appears to have an improved local tolerance profile compared with latanoprost-0.02%BAK, (4) latanoprost-CE is able to mechanically improve the TF and TFLL stability and film properties and restore a healthy ocular surface environment, (5) latanoprost-CE is able to contribute effectively in the management of ocular surface inflammation, (6) latanoprost-CE improves the healing process of damaged cornea, and (7) latanoprost-CE is able to improve CFS scores in the context of dry eye.However, the remaining question is the following: do all the aforementioned properties translate to the clinic? And is latanoprost-CE able to treat concomitantly elevated IOP and OSD signs and symptoms in glaucoma patients while preserving their compliance and well-being and improving their treatment satisfaction and experience?®Z , it appeared that IOP change from baseline for latanoprost-CE was non-inferior to TravatanZ, with a trend toward a superior IOP reduction at each time point at month 3: −7.2 ± 2.9 versus −6.0 ± 3.0, −6.7 ± 3.0 versus −5.9 ± 3.5, −6.0 ± 3.3 versus −5.4 ± 3.7 at 8:00 AM, 10:00 AM, 4:00 PM for latanoprost-CE and TravatanZ, respectively.27In a phase II clinical study comparing latanoprost-CE with the soft-preserved Travatan21 A similar trend toward better CFS scores  and TF break-up times with latanoprost-CE was also observed when compared with TravatanZ .This trend for a better IOP management is not unexpected, considering that it has been described that reducing ocular surface inflammation in glaucoma patients is accompanied by better IOP outcomes.However, these promising results will need to be confirmed in a larger clinical trial. The results of the comparison of latanoprost-CE to latanoprost-0.02%BAK  in a phase III clinical trial on both IOP management and on OSD signs and symptoms tend to support our hypothesis (data on file).42 The negative impact preservative agents have on the ocular surface, extensively described for BAK, due to its long history of use to preserve eye drops, is largely accepted worldwide, and the recent development of preservative-free eye drop formulations is just the clear confirmation that such molecules should be avoided.52OSD is a very common comorbidity associated to glaucoma, as close to 60% of glaucoma patients suffer from OSD signs and symptoms of various severity.in vitro studies suggest pro-inflammatory response and cytotoxicity in corneal cells with these excipients .53However, this should not prevent the ophthalmic community from maintaining its watch on the excipients that made their apparition in these preservative-free formulations, as their ocular impact after long-term use is not yet well established and since some recent Preservative-free anti-glaucoma eye drops have greatly benefited glaucoma patients, and not only those presenting signs and symptoms of OSD. They contributed to their elevated IOP management, while reducing the discomfort associated to the repeated instillations. However, for a better long-term vision protection outcome, it is important that the IOP reduction is as important as possible, and unfortunately, most of the preservative-free “generic-like” PGA formulations consistently show a lower IOP reduction performance, while remaining within the 1.5 mmHg margin at each time point.This might be explained by the fact that preservative agents, such as BAK, are also effective permeation enhancers. This is nicely demonstrated by the backward reformulation of 0.03% bimatoprost/0.005% BAK to 0.01% bimatoprost/0.02% BAK while the IOP lowering efficacy was maintained within the non-inferiority margin. It is also possible that while being preservative-free, these PGA formulations are unable to address the underlying inflammation of the ocular surface that is known to negatively impact elevated IOP management outcomes; in the context of anti-glaucoma eye drop treatment, with possible trabecular meshwork inflammation and damage, or filtering surgery.Removing preservatives such as BAK from PGA formulations may not be enough to improve OSD and may still require the concomitant use of artificial tears to effectively alleviate both signs and symptoms. This polymedication, while certainly effective, requires the repeated instillation of various eye drops during the day, which can negatively impact glaucoma patients' quality-of-life and treatment experience, if not their compliance.28The latanoprost-CE described in these pages takes advantage of the Novasorb technology of the cationic oil-in-water emulsion, an eye drop formulation that mimics a healthy TF that helps mechanically restore a safe TF and ocular surface environment and which is at the same time an effective DDS for the delivery of the lipophilic prodrug latanoprost.54The latanoprost-CE was demonstrated to protect the ocular surface through: (1) the restoration of a thick and stable TFLL via oil supplementation, (2) the stabilization of the oil/water interface between the TFLL and the aqueous phase of the TF by bringing optimized amounts of the cationic polar lipid agent  and a non-ionic surfactant (polysorbate 80). The resulting restored TFLL protects the TF from evaporation, thus restoring a healthy ocular surface environment that contributes to the homeostasis of the ocular surface.47And in addition to these mechanical beneficial effects on the TFLL and TF stabilization, the cationic oil-in-water emulsion is also able to promote the healing process of the corneal epithelium via, up to a certain extent, the management of the ocular surface inflammation .This wealth of preclinical evidence explains the excellent clinical outcomes observed in the phase II clinical trial, and it corroborates the hypothesis postulated by Batra et al., (2014) and Dubrulle et al., (2018). The improvement of the ocular surface environment and its inflammation concomitant with an IOP-lowering drug translate into a better IOP lowering efficacy, possibly through decreased inflammation of both conjunctiva and trabecular meshwork among other eye tissues leading to improved outflow facility of the eye.27 Based on cost-utility analyses over a 5-year time horizon, the latanoprost-CE resulted in an incremental 0.35 Quality-Adjusted Life Years compared with Latanoprost.55Hence, the simultaneous treatment of the ocular surface while treating elevated IOP with the same eye drop formulation may represent the next generation of glaucoma eye drop treatment with possibly a better quality-of-life and treatment experience for glaucoma patients.56 These results should be confirmed by future economic evaluations carried out alongside empirical trials.Similarly, it was concluded as potentially highly cost-effective versus standard latanoprost formulation for glaucoma-OSD patients from the Italian National Health Service perspective.


Clarity about the role of delamanid in longer regimens for multidrug-resistant TB is needed after discordant Phase IIb and Phase III randomized controlled trial results. The Phase IIb trial found that the addition of delamanid to a background regimen hastened culture conversion; the results of the Phase III trial were equivocal. We evaluated the effect of adding delamanid for 24 weeks to three-drug MDR/RR-TB regimens on two- and six-month culture conversion in the endTB observational study. We used pooled logistic regression to estimate the observational analogue of the intention-to-treat effect (aITT) adjusting for baseline confounders and to estimate the observational analogue of the per-protocol effect (aPP) using inverse probability of censoring weighting to control for time-varying confounding. At treatment initiation, 362 patients received three likely effective drugs (delamanid-free) or three likely effective drugs plus delamanid (delamanid-containing). Over 80% of patients received two to three Group A drugs  in their regimen. We found no evidence the addition of delamanid to a three-drug regimen increased two-month (aITT relative risk: 0.90 (95% CI: 0.73–1.11), aPP relative risk: 0.89 (95% CI: 0.66–1.21)) or six-month culture conversion , aPP relative risk: 0.93 ). In regimens containing combinations of three likely effective, highly active anti-TB drugs the addition of delamanid had no discernible effect on culture conversion at two or six months. As the standard of care for MDR/RR-TB treatment becomes more potent, it may become increasingly difficult to detect the benefit of adding a single agent to standard of care MDR/RR-TB regimens. Novel approaches like those implemented may help account for background regimens and establish effectiveness of new chemical entities. An estimated 10 million people globally developed tuberculosis (TB) disease in 2021 –5.M. tuberculosis in nearly 50 years—offers the potential to improve treatment for RR/MDR-TB. In 2014 the European Medicines Agency (EMA) conditionally approved delamanid for TB [The introduction of delamanid —one of the first drugs with a novel mechanism of action against One major difference between the trials was the composition of the background regimen. Phase II trial participants received four to five drugs on average, as compared to a mean of 6.5 drugs in Phase III trial. And, Phase III trial regimens tended to be more potent, which could have potentially masked delamanid’s contribution to treatment outcomes. The conflicting findings and different treatment regimens used across these trials beg for further investigation of delamanid’s role in MDR-TB regimens.Based on the Phase III trial results, delamanid was categorized as a lower priority drug, to be used if a regimen with more effective drugs cannot be composed  , 10. TheHere, using a robust analytic design and methods grounded in causal inference, we evaluated the comparative effect on two- and six-month culture conversion of adding versus not adding delamanid for 24 weeks to MDR-TB regimens comprising only three likely effective drugs instead of the four recommended by WHO at the time of the study.We used data from the endTB observational cohort , a prospective research cohort across 17 countries, and included participants with a positive culture and documented MDR/RR-TB at enrollment. Full details on the study protocol have been published previously . In summWe designed our analysis using target trial emulation to answer the causal question of interest: –28 what Culture conversion is used as an interim microbiological indicator and surrogate endpoint in both observational studies and RCTs , 30. We M.tb strain was not resistant to the drug, or (2) no resistance testing had been conducted and the participant had not previously received the drug for one month or more, according to the medical record. All drugs in the WHO hierarchy were considered. Baseline regimens  were categorized as follows, based on the number of likely effective drugs and irrespective of WHO drug group hierarchy:[We included participants with a positive baseline sputum culture, defined as any culture on a sputum specimen collected ≤90 days before treatment initiation in the endTB cohort , 32. We ierarchy: (1) receWe estimated the observational analogue of the intention-to-treat (aITT) effect and the observational analogue of the per-protocol effect (aPP). The analysis estimating the aITT effect includes all participants, classified by their baseline treatment, and adjusted for baseline confounders. This analysis estimates the effect of initiating delamanid plus a background regimen of three drugs versus initiating a background regimen of three drugs, not including delamanid. Treatment could change during follow-up. For some participants in the delamanid-containing group, delamanid was discontinued; for some participants in the delamanid-free group, delamanid was started; and in both groups, some patients experienced changes in the number of background drugs. The aPP effect estimates the effect of adding and remaining on delamanid among participants who received a regimen of three likely effective drugs for the duration of follow-up (up to 24 weeks). Because MDR-TB treatment can vary over time, aPP analyses may be biased by time-dependent confounding –35.We fitted a pooled logistic regression model and its predicted probabilities to estimate the risk of conversion for delamanid-containing versus delamanid-free regimens. The model was adjusted for the following baseline confounders chosen a priori using content knowledge and directed acyclic graphs: age, sex, hospitalization at treatment initiation, the number of Group A drugs , whether the patient was receiving imipenem-cilastatin, body mass index <18.5, HIV infection, and hepatitis C antibody positivity. Confidence intervals were estimated using nonparametric bootstrapping with 500 samples . MissingTo estimate the aPP effect, we artificially censored participants when their treatment deviated from that administered at baseline. To adjust for selection bias due to this artificial censoring, we applied inverse probability of censoring weights .To simulate the per-protocol population, we censored observations in the delamanid-containing group when delamanid had been discontinued for >2 consecutive weeks prior to the end of treatment and there was no evidence that delamanid discontinuation was in response to an adverse event. Therefore, estimated effects reflect continuation of delamanid for up to 24 weeks, unless contraindicated due to an adverse event. In the delamanid-free group, we censored observations when delamanid was added for >2 consecutive weeks. In both groups, observations were censored if drugs were added or removed from the regimen such that it contained either less than three or more than three drugs likely to be effective for >2 consecutive weeks. To control for selection bias due to artificial censoring, for each individual and for each week, we estimated time-varying inverse probability of censoring weights equal to the inverse of the probability of being uncensored, i.e. maintaining a regimen consistent with the baseline treatment group.To estimate time-varying weights, we fitted a pooled logistic regression model to estimate the probability each participant remained on their baseline treatment  conditional on time-varying predictors of changing treatment and time since baseline. These predictors included time-varying number of Group A drugs according to WHO classification, sputum smear result, number of adverse events, hospitalization, time, and a quadratic function of time , the MSF Ethics Review Board , IRD Institutional Review Board  and in all 17 implementing countries by appropriate government authorities or local ethics committees . Participants provided written informed consent.Between April 1, 2015 and September 30, 2018, 2757 patients were initiated on a first MDR/RR regimen containing bedaquiline and/or delamanid and consented to participate in the endTB observational study . We exclMost patients were treated in Kazakhstan (30.1%), Georgia (16.9%), Peru (14.1%), or Pakistan (12.2%) . TreatmeAlthough participants in both groups received a background regimen of three drugs likely to be effective, there was substantial heterogeneity in companion drugs   participants in the delamanid-containing group and 7 (2.9%)  participants in the delamanid-free group were not retained in care. In aITT analyses, the RR of culture conversion for delamanid-containing versus delamanid-free regimens was 0.90  and RD was -0.05 . In the aPP analysis the RR of culture conversion for delamanid-containing versus delamanid-free regimens was 0.89  and the RD was -0.06 ) .By six months, 81.3% of participants in the delamanid-containing and 88.7% of participants in the delamanid-free group experienced sputum culture conversion. Fifteen (12.2%)  participants in the delamanid-containing group died or were LTFU before conversion versus 14 (5.9%)  in the delamanid-free group. In aITT analyses, the RR of culture conversion for delamanid-containing versus delamanid-free regimens was 0.94  and the RD was -0.06 . In the aPP analysis the RR of culture conversion for delamanid-containing versus delamanid-free regimens was 0.93  and RD was -0.06  .When we restricted analyses to participants receiving bedaquiline in their baseline regimen, the two-month aPP RR of culture conversion suggested comparable frequencies, with an RR of 0.94  and RD of -0.04  . EstimatIn the context of regimens containing exactly three likely effective drugs, many of which comprised at least two Group A drugs and clofazimine, we found no evidence of an effect of adding delamanid on culture conversion within two and six months. This finding is consistent with the previously reported Phase III delamanid trial but different from that of the Phase IIb trial , 8. A maquantity of drugs in a regimen. However, the efficacy of drugs in the regimen cannot be ignored. The advent of bedaquiline, repurposed drugs, such as linezolid and clofazimine, and late generation quinolones, such as levofloxacin/moxifloxacin, have transformed the TB treatment landscape. Regimens comprised of drugs with lesser efficacy, like those used in the Phase IIb trial that showed a significant effect of delamanid, [Our treatment groups were defined by the lamanid,  are not lamanid, , 39 whicFuture MDR-TB treatment research aimed at comparative effectiveness should examine both the number and efficacy of drugs in a regimen. Delamanid-free regimens comprised of only three, primarily Group A drugs rather than the recommended four drugs performed exceedingly well, with >90% culture conversion at six months. These findings point to the possibility that three potent drugs may be sufficient in many patients. In addition, that these findings corroborate those of the Phase III trial, and are at odds with the Phase II, raises red flags about the strategy used for evaluating both delamanid and bedaquiline: each was added as a single drug to a background regimen. The relative efficacy of bedaquiline compared to placebo in the Phase IIB trial was even more pronounced than that of delamanid . It is cThis study highlights important considerations for investigators analyzing MDR-TB treatment cohorts. Analyses of baseline regimen compositions might not answer the most relevant clinical question when regimen composition commonly changes throughout the course of treatment .aITT and aPP estimates, when the objective is to estimate the effect of starting and remaining on a treatment, the approach applied here is one strategy that also resolves the potential for time-dependent confounding [We did not identify a clinically meaningful difference between results of standard baseline-adjusted models—which estimates the aITT—and the inverse probability censoring weighted analysis estimating the aPP. This tells us that, in this cohort, the effect of adding delamanid to a three-drug regimen at baseline is similar to that of adding and maintaining delamanid in a three-drug regimen for the first six months of treatment. The likely reason for not identifying differences between these analyses is that among patients who changed their regimen, changes to the primary drug of interest, delamanid, were rare . Only one patient discontinued delamanid due to an adverse event, reinforcing the safety of delamanid and potential use of delamanid in regimens compromised by toxicity. Changes in the number of likely effective background drugs in the background regimen were more common; however, meaningful differences between estimates will be observed if censoring  is highly (many-fold) associated with both the treatment group  and the outcome. This was not the case in this analysis  and three-drug regimen plus delamanid (58% had 2 or 3 Group A drugs). Few efforts have been made to meaningfully capture the heterogeneity of individualized MDR-TB regimens in comparative effectiveness studies, likely because hundreds or even thousands of distinct regimens can be represented in any one cohort. For example, the 2012 individual patient data meta-analysis comprises over 9000 patients on 1626 different baseline regimens . FurtherIn conclusion, although, we did not identify a benefit for 2- or 6- month culture conversion of adding delamanid to an MDR/RR-TB regimen with only three likely effective, and often highly-potent drugs, the rarity of delamanid suspension reinforces existing evidence about its safety. Important questions remain about how to optimize the use of delamanid, including whether delamanid can improve the effectiveness of regimens comprised of drugs with suboptimal efficacy or improve the safety (or efficacy) of treatment through substitution for more toxic (less potent) agents. Our findings also highlight the risk of equivocal results if the drug-development approach used for bedaquiline and delamanid is applied to new chemical entities in the context of the current, improved background regimen. Finally, the analytic methods used here can facilitate articulation of precise research questions and should be considered as a strategy for reducing bias in analysis of MDR/RR-TB regimens, when treatment changes are frequent.S1 Text(DOCX)Click here for additional data file.


Prodromal characteristics of psychosis have been described for more than a century. Over the last three decades, a variety of studies have proposed methods to prospectively identify individuals (and youth in particular) who are at high risk of developing a psychotic disorder. These studies have validated various screening instruments and made them available in several languages. Here, we describe the translation into Brazilian Portuguese and cross-cultural adaptation of two such screening tools – the Prodromal Questionnaire-16 (PQ-16) and the Prevention through Risk Identification, Management, and Education (PRIME)-Screen. Two bilingual native speakers of Brazilian Portuguese translated the questionnaires from English. A native English speaker then performed back-translations into English. These back-translated versions were submitted to the original authors. They provided feedback and later approved the final versions. After translation and cross-cultural adaptation, no items needed to be changed in the adapted PQ-16 and four items were revised in the PRIME-Screen. After the peer-review process, we included two suggestions in the PQ-16 to facilitate use of the tool in our cultural and social contexts. The PRIME-Screen did not need further changes. These new instruments can help screen Brazilian Portuguese-speaking patients who are at risk of psychosis in primary care. Prospective assessment of subsyndromal symptoms enabled researchers to identify patients who were at higher risk of developing psychosis, which led to definition of the at-risk mental states (ARMS). Previous studies have developed brief questionnaires to screen for ARMS, but to date few have been available for low- and middle-income countries.Psychotic disorders are the ninth-leading cause of global health problems.6 A few years later, an American group called Prevention through Risk Identification, Management, and Education (PRIME) developed their Structured Interview for Psychosis-Risk Syndrome (SIPS). This tool aimed to evaluate the same phenotype.7 These tools have been proven to reliably identify people who are in ARMS and have opened the door for a new field of study called early intervention in psychiatry.8 Comparative studies have found that both instruments perform similarly and both were able to identify people who are likely to transition from ARMS to full psychosis after two years.9Some commonly reported subthreshold psychotic symptoms that characterize the ARMS syndrome include thought disorders, altered beliefs, and changes in perception and speech. In the early 1990s, an Australian group led by McGorry and Yung developed a semi-structured clinical interview to prospectively assess young people who presented such symptoms. They called their interview the Comprehensive Assessment of At-Risk Mental States (CAARMS).10Early intervention has already become government policy in several developed countries . However, implementing early intervention in clinical practice has proven to be a challenge. For instance, it is difficult to identify ARMS subjects correctly. To conduct CAARMS and SIPS interviews, clinicians require extensive training, specialized staff, and considerable time to administer the interviews. Consequently, using these interviews to identify people who are at risk of developing psychosis in daily clinical practice is unfeasible, especially in low-resource settings. Over time, these challenges led to development of self-reported screening tools such as the Prodromal Questionnaire (PQ) and the PRIME-Screen.12 It has also been translated into Portuguese.13 Unfortunately, this 92-item questionnaire is very long and therefore not appropriate as a screening tool for use in non-specialized settings. A Dutch group recently tested a condensed version of the tool – the PQ-16 – and demonstrated that it is a reliable and much more time-effective tool.14 Similarly, the North-American group developed the PRIME-Screen, a brief questionnaire based on the SIPS.15 The PRIME-Screen is more specific and sensitive than most other screening tools.16 Consequently, the PRIME-Screen has been widely adopted in studies of early detection of psychosis that use a two-stage screening process, comprising an initial brief screening procedure and a subsequent clinical interview.17The long version of the PQ has been proven to have adequate psychometric properties and a fair degree of reliability.To date, there are no brief screening instruments for ARMS available in Brazilian Portuguese. Therefore, this study performs and describes the translation into Brazilian Portuguese and cross-cultural adaptation of the PQ-16 and the PRIME-Screen.18 for cross-cultural adaptation of psychometric instruments.Prior to beginning the translation process, the authors of the present study obtained consent from the authors of the original screening tools to translate their work. We followed the guidelines proposed by Guillemin et al.19 Respondents mark their response to each item as true or false and also provide an answer on a Likert-type scale that ranks their level of associated distress on a scale from “0” (no distress) to “3” (severe distress).The PQ-16 has 16 items – nine items related to perceptual abnormalities, five items related to changes in thought content, and two items related to negative symptoms.20 Respondents indicate how much they agree or disagree with the propositions in each item regarding their experiences in the past year using a Likert-type scale ranging from “0” (definitely disagree) to “6” (definitely agree).The PRIME-Screen has 12 items. It is based on the positive symptom domain of the SIPS, which tracks presence of unusual mental activity such as ideas of grandeur and persecution, hallucinatory experiences, and lack of insight.Our translation process included the following steps: 1) we obtained consent from the authors to translate their work; 2) two bilingual native speakers of Brazilian Portuguese independently translated the instruments into Brazilian Portuguese; 3) two psychiatrists with experience in ARMS clinical practice revised these translations; 4) a native English speaker performed back-translations into English; 5) these back-translated versions were submitted to the authors of the original scales for feedback; and 6) feedback was incorporated and a final Brazilian Portuguese version was completed.These steps are illustrated in As mentioned above, we sent the screening tools to the original authors after back-translating them into English. The original authors did not suggest any changes to the PQ-16 . The ori16 Early identification of ARMS facilitates early intervention and might potentially mitigate the psychosocial impact of this condition. Here, we present translations of the PQ-16 and PRIME-Screen into Brazilian Portuguese, which may constitute simple and time-effective tools to identify ARMS in low-resource settings.Previous studies have shown that the PQ and PRIME-Screen are among the best instruments for screening people who are at risk of developing psychosis.21 and the PQ-16.14 Although both have been shown to be effective and reliable instruments, a recent study found that the PQ-16 outperformed the PQ-B.22 A cutoff of six or more symptoms in the PQ-16 exhibited a high true positive rate (87%) and high specificity (87%) when differentiating AMRS and FEP from help-seeking youth who were not at high clinical risk, according to the CAARMS.14 A recent review,19 based on four studies evaluating the diagnostic accuracy of the PQ-16, found that a total symptom score of ≥ 6 and a distress score of ≥ 9 were valid cut-offs for community-based samples. In help-seeking populations, lower scores were suggested for distress (cut-off of ≥ 8) and symptoms (cut-off of ≥ 5). For the PRIME-screen, as proposed by the original authors, any item endorsed with a score of 6 or three items endorsed with a score of 5 are considered positive for ARMS.15Previous studies have proposed two short versions of the PQ; the PQ-B, with 21 items,Finally, it is important to address some limitations of the present work. First, although previous studies have validated these instruments across several languages and cultures, we suggest that the psychometric properties of these Brazilian Portuguese versions should be investigated. Second, we also suggest that the norms and cut-offs provided in previous research should be adopted with caution, since they were not investigated in Brazilian samples. Third, the original studies typically investigated samples of young people aged 17-18 years old; therefore, utilization of these instruments in other age groups, particularly younger individuals, still needs further validation. Finally, the average educational level of Brazilian youth may differ from youth in high-income countries, which may possibly impact their ability to read and understand the items and, therefore, affect the instruments’ accuracy and applicability.The Brazilian Portuguese versions of the PQ-16 and PRIME-Screen have been successfully translated and are now available for clinicians and researchers. Future studies should test their accuracy and set norms for the Brazilian population.


The study included 158 patients (mean age was 61.63 ± 10.14 years) with angina and low to intermediate clinical likelihood of CAD, with 75 having a previous COVID-19 infection and 83 without infection. The results showed that patients who had a previous COVID-19 infection had higher levels of pericoronary inflammation than those who did not have a COVID-19 infection, suggesting that COVID-19 may increase the risk of coronary plaque destabilization. This study highlights the potential long-term impact of COVID-19 on cardiovascular health, and the importance of monitoring and managing cardiovascular risk factors in patients recovering from COVID-19 infection. The AI-powered CaRi-Heart® technology may offer a non-invasive way to detect coronary artery inflammation and plaque instability in patients with COVID-19.Inflammation is a key factor in the development of atherosclerosis, a disease characterized by the buildup of plaque in the arteries. COVID-19 infection is known to cause systemic inflammation, but its impact on local plaque vulnerability is unclear. Our study aimed to investigate the impact of COVID-19 infection on coronary artery disease (CAD) in patients who underwent computed tomography angiography (CCTA) for chest pain in the early stages after infection, using an AI-powered solution called CaRi-Heart It is well-established that vascular inflammation is a key factor in the development and progression of atherosclerosis. Plaques that are stable have a chronic inflammatory response, while those that are vulnerable or have ruptured have an “active” inflammation that can compromise the protective fibrous cap and raises the risk of rupture, which is the main cause of acute vascular events (AVE) .Atherosclerosis is a prevalent condition that affects the blood vessels and increases the risk of serious AVE, such as acute coronary syndrome (ACS) and cerebrovascular events. These events occur when the plaques rupture and form clots, which can lead to severe complications and even death. The concept of “vulnerable plaque” (VP) was introduced to describe the unpredictable nature of the progression of atherosclerosis , a VP isIn recent years, epicardial adipose tissue (EAT) and perivascular adipose tissue (PVAT) have been extensively researched for their roles as markers and promoters of local coronary inflammation ,7. PVAT The COVID-19 pandemic has had a considerable impact on cardiovascular health, due to its effects on inflammation and destabilization of the immune system, both during the acute phase of the infection and over the course of the disease, as well as in long-COVID syndrome ,13. SARSThe optimal method to detect inflammation in the PVAT using coronary computed tomography angiography (CCTA) is through the use of the fat attenuation index (FAI). The FAI is a measure of the CT attenuation gradients in the perivascular space and is calculated by comparing the attenuation values of the PVAT and the adjacent vessel. In cases of coronary inflammation, the composition of the PVAT undergoes a change towards higher aqueous and lower lipophilic content, which can be observed in the FAI due to a decrease in the CT attenuation of the PVAT. Chronic inflammation can also lead to unfavorable fibrotic and other remodeling of the PVAT, which can be identified using progressed radiomic texture analysis of the perivascular adipose tissue ,17. The CCTA is commonly used in COVID-19 patients with diagnosed or suspected CVD, especially when the results are likely to significantly affect the patient’s treatment plan or save their life ,20. As pn = 75) had a history of COVID-19 infection a few months prior to the CCTA examination, while the remaining 52.53% did not have any prior infection. The participants had an average age of 61.63 ± 10.14 years, and 67.08% (n = 106) of them were male. There were no statistically significant differences between the two groups in terms of gender, age, and comorbidities such as hypertension, diabetes, and obesity. In contrast, it was observed that group 1 exhibited a lower incidence of hypercholesterolemia in comparison to group 2 , alongside having a higher body mass index  and significantly reduced levels of triglycerides . The groups did not show any significant differences in terms of PCI after CCTA, multi-vessel PCI, heart failure, left ventricular ejection fraction (LVEF), serum creatinine, and total cholesterol. The average time from COVID-19 infection to CCTA examination for group 2 was 138.1 ± 103.2 days and there were no significant differences between the groups in terms of vaccination against COVID-19. The baseline characteristics, comorbidities, and risk factors of the two study groups are presented in This study recruited 158 patients who reported chest pain and had a low to intermediate clinical likelihood of CAD. In total, 47.46% of the participants (p = 0.05), LCX , FAI-score TOTAL  (According to the standard adipose tissue Hounsfield unit range (−190 to −30 HU), there were no significant differences in the coronary FAI index values between the two groups as revealed by the conventional CT scan. The FAI-score was consistently higher in the non COVID-19 group; more precisely: LAD  .The FAI-score of the LAD, LCX, and RCA was used to create percentile curves across various age and sex groups. These curves were evaluated for their prognostic significance in Cox models that were adjusted for hypertension, diabetes mellitus, smoking, hyperlipidemia, high-risk plaque characteristics, and the modified Duke prognostic CAD index .p = 0.05), FAI-Score Centile LCX , FAI-Score Centile RCA . FAI-Score Centile values of the two study groups are presented in For the FAI-score Centile, the overall pattern shifts significantly, as the values for all three coronary arteries are higher for the subjects in the COVID-19 positive group, as follows: FAI-Score Centile LAD  included patients who had a previous COVID-19 infection and group 2 (n = 83) included patients matched for age and gender who did not have a COVID-19 infection prior to the CCTA examination. We ensured the accuracy of patient selection in the first group by only including individuals who had laboratory-confirmed COVID-19, exhibited mild to moderate symptoms, and had not received any treatment in healthcare facilities. Demographic and laboratory characteristics, cardiovascular risk factors, and symptom development were all monitored and assessed for each patient before the CCTA examination.Our cross-sectional observational study, conducted at the Center of Advanced Research in Multimodality Cardiac Imaging, Cardio-Med Medical Center  included 158 patients presenting with pain in the chest area and a low to intermediate likelihood of CAD. The patients underwent 128-slice CCTA to assess coronary anatomy and atherosclerosis, determine FAI score, and perform plaque analysis. The study participants were categorized into two main groups based on their COVID-19 status: group 1  were also excluded.All participants underwent a CCTA scan using a 128-slice Siemens Somatom Definition AS  from the Centre of Cardio-Med . The scan was retrospectively gated with a heart rate below 65 beats per minute and the parameters included a tube voltage of 120 kV, gantry rotation time of 0.33 s, and a collimation of 128 × 0.6. Beta-blockers were given to patients with a resting heart rate above 65 beats per minute, and blood pressure was monitored during the administration of intravenous or oral beta blockers. The acquisition process began with a native scan for coronary calcium assessment, followed by the administration of 80–100 mL of iodine-based contrast material based on body weight with a 50-mL saline chase at a flow rate of 5.5–6 mL/s during inspiratory breath-hold. All obtained scans were saved in a devoted electronic imaging database for offline image post-processing and for cloud delivery.All the acquired images were converted to DICOM format, then anonymized and transferred to the partner center  via the cloud for post-processing. The inclusion criteria for the analysis included the presence of plaques with a stenosis effect of at least 50% and with at least one vulnerability marker present . The FAI and AI-based FAI scores were determined for each of the major coronary arteries in all patients.®, CARISTO Diagnostics, Oxford, UK) that deliver precise and consistent attenuation measurements in concentric 1-mm 3D layers of perivascular adipose tissue surrounding the human arterial wall [The calculation of the PVAT-FAI employs AI-enhanced algorithms . Statistical analysis was performed using GraphPad Prism 9.5 software . The study included a PCAT-FAI analysis of 474 coronary arteries: 158 on the left anterior descending artery (LAD), 158 on the circumflex artery (LCX), and 158 on the right coronary artery (RCA). In addition, the CaRi-Heartdividual .2) and its variables. Numeric data were expressed as mean ± standard deviation, and a Mann–Whitney or unpaired Student’s t-test was used. Pearson correlation analysis was conducted to determine correlations between the PVAT-FAI and other variables as appropriate. Results were considered statistically significant if the two-sided p-value was 0.05.The data were analyzed comparatively between patients with and without previous SARS-CoV-2 infection. Nominal  variables were reported as integer values (percentages) and compared between groups using the Chi-square test . Although these limitations exist, additional research is required to validate the clinical significance of the FAI-Score in managing coronary inflammation.In order to expand on the results of this proof-of-concept study, future research plans include conducting a follow-up period to evaluate the outcome prediction capability of the FAI mapping technique and its correlation with systemic inflammation as measured by biomarker analysis. Additionally, to address the limitations of this study, our research team intends to explore the correlation between local vascular inflammation and hemodynamic differences in vessels by computing shear stress using CCTA imaging. In an effort to gain a comprehensive understanding of overall coronary vulnerability, future studies will incorporate computerized plaque analysis, which provides insight into intrinsic vulnerability, in conjunction with FAI.Lesions with higher pericoronary FAI-Score Centile values were more commonly found in patients who had previously been infected with COVID-19. The pericoronary FAI-Score percentile curves illustrate the level of coronary inflammation and provide a uniform interpretation of perivascular FAI mapping on CCTA. Patients in the upper percentile curves have an increased risk of cardiovascular disease, regardless of the presence of traditional risk factors or established atherosclerotic changes.An association has been observed between COVID-19 infection and an elevated risk of destabilizing coronary plaque, as indicated by higher levels of inflammation in the pericoronary adipose tissue. The use of pericoronary FAI as a marker for identifying vulnerable patients at high risk for cardiovascular events could have significant implications in the deployment of targeted prevention strategies. However, further validation of these findings through larger studies with more consistent data is necessary to establish the utility of FAI in clinical practice.


The TF–TG identification methods fill the gap of lacking a method for genome-wide TG prediction of a TF, and potential for validating ChIP/DAP-seq results, while the two Net-based methods are instrumental for predicting pathway GRNs.Four statistical selection methods for inferring transcription factor (TF)–target gene (TG) pairs were developed by coupling mean squared error (MSE) or Huber loss function, with elastic net (ENET) or least absolute shrinkage and selection operator (Lasso) penalty. Two methods were also developed for inferring pathway gene regulatory networks (GRNs) by combining Huber or MSE loss function with a network (Net)-based penalty. To solve these regressions, we ameliorated an accelerated proximal gradient descent (APGD) algorithm to optimize parameter selection processes, resulting in an equally effective but much faster algorithm than the commonly used convex optimization solver. The synthetic data generated in a general setting was used to test four TF–TG identification methods, ENET-based methods performed better than Lasso-based methods. Synthetic data generated from two network settings was used to test Huber-Net and MSE-Net, which outperformed all other methods. The TF–TG identification methods were also tested with SND1 and  Construction and delineation of transcriptional regulatory networks are essential for a systematic understanding of how various biological processes and complex traits are regulated and how plants grow and develop in response to environmental cues. Although biological experiments can be carried out to obtain gene regulatory relationships, they are labor-intensive and time-consuming, and can therefore only be used to infer a small number of true regulatory relationships. In the last two decades, the advent of high throughput technologies, including microarray and RNA-Seq, has made it easier to generate a terabyte of transcriptome data for inferring gene regulatory relationships and gen regulatory networks (GRNs). As the high-throughput data in public repositories increases exponentially, computational algorithms and tools that utilize high-throughput transcriptome data and ChIP/DAP-seq data provide an alternate approach to infer gene regulatory relationships and GRNs. However, the validity of this approach relies on the accuracy of the methods.Initially, high-throughput transcriptome data sets were primarily generated from the single-celled organisms like bacteria and yeast, or the eukaryotic cell lines. These organisms and cell lines allowed for the generation of time-course microarray data with small time intervals e.g. 5–10 min), which spurred the development of dynamic methods, including differential equations 0 min, wh, dynamicPlant researchers often produce a large number of transgenic lines in which a TF is up-/down-regulated, or use a transient expression system, for instance, CRISPR-CAS9  or dTALEIn this study, four statistical selection methods were developed to infer the potential TGs for a given TF by combining two loss functions, mean squared error (MSE) or Huber loss function (Huber), with two penalty functions, elastic net (ENET) or least absolute shrinkage and selection operator (Lasso). These four methods are referred to as Huber-ENET, Huber-Lassso, MSE-ENET and MSE-Lasso. The MSE and Huber loss functions were used to measure the errors between the predicted values and the observed values. Huber can more readily avoid the sensitivity of heavy-tailed errors or outliers than MSE. The penalty functions, ENET and Lasso contain the ediction .gl3 overexpression studies. In addition, the two Net methods were tested with the real transcriptome data of all metabolic pathway genes from the maize B73 line from public repository. Our study showed that the four TF–TG identification methods had higher efficacy in genome-wide prediction than the three comparison methods, CLR, MRNET and TIGRESS, implying that the methods can be used to validate TGs of a TF resulting from ChIP-seq or DAP-seq experiments, while the two Net-based methods can identify pathway GRNs.To solve the regressions for the methods described above, an accelerated proximal gradient descent (APGD) algorithm was developed for the parameter optimization in all six methods. Our simulations showed that the APGD was equally effective but much faster than a commonly used method called convex optimization solver (CVX). To obtain stable selection results, we applied a stability selection method, the half-sample approach, which does not need to choose the optimal tuning parameters in selection methods. All the methods were tested using simulated data, and the four TF–TG identification methods were also tested with the real transcriptomic data from SND1 and The simulated data were generated in three settings: (i) a general setting; (ii) two network settings: a hierarchical network setting and a Barabasi-Albert (BA) network setting. In the general setting, escribed  , a scaling method contained in the edgeR package. The normalized data was used for real data simulation to validate the methods we developed in this study.The poplar data used for simulation was from our previous study . The datgl3 promoter for overexpression were constructed. The two regions targeted are located 5 and 48 bp upstream of the gl3’s transcription start site. The 14-day-old seedlings were used to test the dTALes-mediated induction of gl3. Three samples and three controls, upon being infected with Xv1601 bacteria carrying dTALes, dT1 or dT2, were harvested in a time-series with four time points: 18 and 24 h. Sequencing data were trimmed by Trimmomatic (version 0.38)  that target two non-overlapping 16-bp regions of the on 0.38)  and trimon 0.38) . Fragmenon 0.38) , and DEGon 0.38) . FPKM dagl3 data as described above. 2539 unique pathway genes were extracted from the Plant Metabolic Network (PMN)  repository. The accession numbers are shown in Table S1. The sequence reads were preprocessed as described for rk (PMN)  and 23 lrk (PMN)  were useConsider that the expression levels of a TF where Based on the above statistical model, we developed four statistical selection methods to infer the potential TGs for a given TF and two methods to infer pathway regulatory networks based on the penalized regression model. The general objective function of the penalized regression model was defined aswhere In the above general objective function of the penalized regression model, we considered the following two loss functions, mean squared error (MSE) and Huber. The MSE loss function is defined as ng et al.. For anyl1 norm and l2 norm. The Lasso penalty is the special case of ENET ; more detailed information on how to install and run the tool was enclosed in the packages.Six statistical selection methods based on the penalized regression models and the APGD algorithm for solving these six statistical methods had been implemented in both Python3 and R and then packed into TGPred. Both of them used commonly used libraries for scientific computing. For Python3 version of TGPred, we used numpy, scipy and sklearn to support efficient mathematical and dataframe computing, cvxpy to compare the runtime and estimated results of APGD with commonly used CVX, and networkx to generate synthetic data based on the BA network setting. For R version of TGPred, we used Matrix and MASS to support the efficient mathematical computing, and mvtnorm and igraph to generate synthetic data. TGPred can be directly used within Python and R. Both regulation effect Simulation studies were used to evaluate the performance of the six statistical selection methods we developed based on the penalized regression models. We considered three simulation settings, the general setting and two network settings, and we used In the general setting, TGs were independent with each other. Therefore, we only compared the performances of Huber-Lasso, MSE-Lasso, Huber-ENET and MSE-ENET with the three comparison methods, CLR, MRNET and TIGRESS. Figure For the network settings, we considered two network structures, the hierarchical network  and the entclass1pt{minima10-scale from 10-scale from We also compared the computational efficiency and the regression coefficients estimated by APGD and CVX, a commonly used package for convex optimization, for several pairs of tuning parameters  setting . All ana setting . A disad setting .We compared the regression coefficients estimated by APGD and CVX for several pairs of tuning parameters  of APGD . However setting  and \doc setting . We obseSND1 overexpression were identified in our early publication  system. Two TALes, dT1 and dT2, were constructed to target two non-overlapping 16-bp regions (4 and 48 bp upstream of the transcription start site) in the gl3’s promoter to activate it. Analysis of RNA-seq data yielded at 24 h revealed 144 DEGs (93 upregulated and 51 downregulated genes), that were activated by both dT1 and dT2  and pathway genes (PWGs). A total of 2539 PWGs belonging to 446 pathways were obtained after the PWGs with 90% or more expression values being zero were removed. The Laplacian matrix  species . We spec species  as ligniWe applied Huber-Net and MSE-Net to two input subsets of transcriptomic data: one contains Huber-Net identified the unique pathway genes that were not identified by any other methods including the three comparison methods. For example, C4H regulated by MYB103-2, and HCT-1 by KNAT7. MSE-Net uniquely identified CCoAOMT1-2 regulated by MYB69, 4CL3 by MYB42, HCT-1/2/3 by MYB42. Huber-Net and MSE-Net together uniquely identified FAH1-1, FAH1-2 by MYB63. To show the overlaps of the regulatory relationships identified by different methods, we generated a Venn diagram Figure  based onThe loss functions and the penalty functions we used in this study are all convex functions ,39. AlthWith the improved new APGD algorithm, we set out to develop novel methods to predict the TGs of a TF of interest using omics data, an important issue that has not been resolved in current bioinformatics. Using two loss functions, Huber and MSE, and three penalty functions, Lasso, ENET and Net, we developed four statistical selection methods, MSE-ENET, Huber-ENET, MSE-Lasso and Huber-Lasso for identifying TF–TG relationships, and two additional methods, MSE-Net and Huber-Net, for building pathway GRNs. The Huber loss function, which is a hybrid of squared errors for relatively small errors and absolute errors for relatively large errors (see Formula ), has beaccuracy ,49. Howeaccuracy . Fortunaaccuracy  that useP-values or q-values) to validate their functions; (ii) using a correlation method  un method ,17,53,54Instead of identifying TGs of a TF independently, we sometimes need to investigate if a TF regulates a pathway or a biological process. In this case, we can determine if a TF’s TGs contain multiple genes belonging to a pathway or a gene ontology that represents a biological process. Toward this goal, we developed Huber-Net and MSE-Net methods based on a network-based penalty. In our extensive simulation studies based on the network setting, we showed that Huber-Net and MSE-Net performed better than the other four methods in terms of the true positive discovery rates. We then applied these two methods to identify true TGs of 23 TFs, which are known to regulate lignin pathway , from alIn this study, we combined statistics , machine learning (regularization in unsupervised learning) and convex optimization (solving regularization with APGD) to identify TGs of a TF of interest, the flowchart depicting the methods is illustrated in Figure In our methods, we utilized two loss functions. The Huber loss function is a combination of linear and quadratic loss functions, and the MSE loss function, which measures the average of the squared errors, ensures that our trained model has no outlier predictions with huge errors. MSE puts more weights on these errors due to the squared portion of the function. The mathematical benefit of MSE is particularly evident in its use at analyzing the performance of linear regression, as it allows one to partition the variation in a dataset into variation explained by the model and variation explained by randomness. Huber loss is more robust to outliers than MSE loss and least absolute deviation (LAD) loss, and has higher statistical efficiency than the LAD loss function in the absence of outliers .In addition, we utilized three different penalty functions: Lasso, ENET and Net. Lasso penalty adds a penalty for non-zero coefficients to penalize the sum of their absolute values (We demonstrated that the improved APGD had less computational complexity for solving the convex optimization problem with both differentiable and undifferentiable functions. Traditional regularization methods need to choose optimal tuning parameters. One limitation of traditional regularization methods with cross-validation is that they depend on the saturation of the data; different data sets may obtain different tuning parameter sets, leading to different or unstable results. APGD is a highly efficient approach to solve our proposed methods as well as the other penalized regressions.The incorporation of half-sample-based selection probability allows for more stable results and avoids the choice of optimal tuning parameters. Therefore, integration of statistics, machine learning and optimization enables us to take the advantage of all methods and combines them to generate a powerful approach to identify true TGs of a TF with high efficacy.Due to the disadvantage of the feature selection procedure, we cannot check if the selected genes have strong evidence related to the outcome. For future studies, we plan to integrate statistical inference in the selection procedure and further investigate the selection performance by integrating both selection and statistical inference.Four new statistical selection methods, referred to as Huber-ENET, MSE-ENET, Huber-Lasso and MSE-Lasso for identifying TGs of a TF of interest and two new methods, Huber-Net and MSE-Net, for inferring pathway GRNs have been developed by integration of statistics, machine leaning and convex optimization approaches. An APGD algorithm was developed to solve convex optimization with significantly reduced computation times. Comprehensive simulations and analyses of the four TF–TG identification methods using synthetic data under a general setting indicated that Huber-ENET, MSE-ENET, Huber-Lasso and MSE-Lasso could be used to identify true TGs of a TF with high efficacy, especially in genome-wide predictions. In simulations using the data from two network settings, Huber-Net and MSE-Net outperformed any other non-Net methods for identifying true TGs involved in a pathway or biological process. For real data, the Huber optimization has a noticeable contribution to the identification of true TGs of a given TF by increasing the selection probabilities as compared to MSE, and Huber-Net and MSE-Net could identify many unique regulatory relationships as compared to CLR, MRNET and TIGRESS. The ENET penalty function contributed greatly to enhancement of the method efficacy as compared to Lasso. AuROC plotting showed that all six methods could rank more positive known TGs to top of TG lists. The TF–TG identification methods developed will fill a methodological gap for genome-wide TF–TG prediction and have a great potential for being used to validate ChIP/DAP-seq results, while the Net-based methods will be instrumental for inferring pathway GRN.lqad083_Supplemental_FilesClick here for additional data file.


The association between tobacco smoking and the risk of COVID-19 and its adverse outcomes is controversial, as studies reported contrasting findings. Bias due to misclassification of the exposure in the analyses of current versus non-current smoking could be a possible explanation because former smokers may have higher background risks of the disease due to co-morbidity. The aim of the study was to investigate the extent of this potential bias by separating non-, former, and current smokers when assessing the risk or prognosis of diseases.We analysed data from 43,400 participants in the Stockholm Public Health Cohort, Sweden, with information on smoking obtained prior to the pandemic. We estimated the risk of COVID-19, hospital admissions and death for (a) former and current smokers relative to non-smokers, (b) current smokers relative to non-current smokers, that is, including former smokers; adjusting for potential confounders (aRR).The aRR of a COVID-19 diagnosis was elevated for former smokers compared with non-smokers  =1.00–1.15); including hospital admission with any COVID-19 diagnosis ; or with COVID-19 as the main diagnosis ; and death within 30 days with COVID-19 as the main or a contributory cause . Current smoking was negatively associated with risk of COVID-19 .Separating non-smokers from former smokers when assessing the disease risk or prognosis is essential to avoid bias. However, the negative association between current smoking and the risk of COVID-19 could not be entirely explained by misclassification. The coronavirus disease COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused more than 605 million confirmed cases of COVID-19 and about 6.5 million deaths around the world by 12 September 2022 9 pandemi,4, yet oIt has been suggested that the observations of a negative association between tobacco smoking and COVID-19 may result from biases, including confounding, selection or bias due to misclassification of the exposure. This latter bias may be present, for instance, when the exposure is defined as current versus non-current smokers (therefore including former and never smoker in the same reference category). Smokers who develop smoking-related chronic diseases are more likely to quit smoking than smokers without these conditions . For insThe presence of such morbidity may, in turn, increase the risk of a SARS-CoV-2 infection and/or its adverse prognosis. The risk of infection among smokers may be enhanced by several biological or behavioural factors. First, smoking-related co-morbidity may imply accelerated lung function decline , and thisnus’) has been very common among men quitting smoking [snus users also have a high risk of infections of the airways due to frequent contact between the hands and the oral mucosa. Also, unlike smokers, snus users are not subjected to restrictions on tobacco use in proximity to others, which can have unfavourable consequences on scoial distancing during the pandemic.In Sweden, the transition to smokeless tobacco use . For this purpose, we explored the relative risk of COVID-19 and its adverse outcomes comparing (a) current and former smoking contrasted to non-smokers; (b) current smokers contrasted to non-current smokers, where the reference group includes both never and former smokers.The study was based on longitudinal data collected within the framework of the SPHC. The SPHC is a population-based cohort that had been established within the region of Stockholm, with participants recruited in subsequent surveys conducted in 2002, 2006, 2010 and 2014. For the purpose of this study, we used information collected during the 2010 and 2014 surveys. Data were collected using postal or web-based questionnaires covering health-related and lifestyle information, including tobacco use. Self-reported information has been complemented by information from healthcare and socio-demographic registries.The data collection was managed by Statistics Sweden in collaboration with the Department of Public Health Sciences at Karolinska Institute . The cohThe derivation of the study sample is shown in Participants were initially grouped according to their self-reported tobacco smoking status in 2014, whereby they were required to answer the following questions: A. Have you ever smoked tobacco daily, or almost daily, for at least six months? ; B. Do you currently smoke tobacco daily or almost daily? . For the purpose of this analysis, we first grouped the answers into three categories: non-smoker (if A=2); current smoker ; former smoker . Smoking status in 2010 was then used to refine this latter category to also include former smokers, those who reported not smoking in 2014 but reported former or current daily smoking in 2010. To assess the potential bias induced by the inclusion of former smokers in the category of non-current smokers, we then re-categorised the answers into a dichotomous variable ‘Current non-smoker’ ; and ‘Current smoker’ .Diagnoses were included between 1 March 2020 and 31 August 2021 (date of last update of record linkage). The rollout of COVID-19 vaccinations started in Sweden in January 2021. Five binary outcomes (Yes/No) were included in the analysis:Any diagnosis of COVID-19, whether in a hospital or outside, consisting of at least a positive polymerase chain reaction (PCR) test reported by the laboratories to Sweden’s national electronic surveillance system for communicable diseases, SmiNet.Hospital admission with a diagnosis of COVID-19 (ICD-10 codes U071 and U072) registered either as main or as a concomitant diagnosis.Admission to an intensive care unit (ICU) with a diagnosis of COVID-19 (ICD-10 codes as above).Death by COVID-19, established using the Swedish Cause of Death Registry, which is based on the death certificate filled in by physicians. All deaths occurring during the follow-up period with COVID-19 registered as the main cause were included. The restriction to the main cause of death was done to maximise the specificity of the diagnosis [Death within 30 days from a diagnosis of COVID-19, listed as either the main or a contributory cause. This latter analysis was conducted for sensitivity purposes.Supplemental material Figure S1 online). Socio-demographic information  was extracted through record-linkage with the register of the total population of the region of Stockholm held by Statistics Sweden. These covariates were categorised as follows: Sex , Age in years (continuous), Education ; Disposable yearly income in Swedish crowns (continuous); Cohabitation with others (Yes/No); Country of birth . Occupational risk for infection with SARS-CoV-2 was categorised as ‘high’, ‘moderate’ or ‘low’ based on a priori knowledge of exposure to the transmission of the virus.Adjustments were made for potential confounders, selected using a directed acyclic graph  and their corresponding 95% confidence intervals (CIs) for COVID-19 infection, hospitalisation, ICU or death due to COVID-19 were estimated through generalised linear models. We specified the Poisson family for the binary health outcomes with robust standard errors estimated using maximum likelihood . We ran snus use, existence of sexually transmitted or chronic diseases as covariates in the study. Missing data in any co-variate (less than 10%) were considered ignorable . All analyses were conducted using STATA®, version 17 .Adjustments were made for the putative confounders listed above: sex, age, cohabitation, education, income, occupational risk for infection, country of birth, A total of 43,400 participants  were included in the study. The derivation of the study sample is shown in a flowchart . Table Isnus use was more frequent among former smokers (12.2%) compared with non-smokers and current smokers . The cumulative incidence of COVID-19 diagnoses was 9.1%, and that of death was 0.43%. The proportion of current smokers in this sample was 7.5%, and that of former smokers was 38.9%. Former smokers were older compared with both non-smokers and current smokers. The proportion of chronic diseases self-reported by current and former smokers (35.8%) was similar, and it was higher than among non-smokers (22.0%). The proportion reporting sexually transmitted diseases in the past 12 months was similar in all groups of smoking behaviour. Current The risk of being diagnosed with COVID-19 between 1 March 2020 and 31 August 2021 was higher among former smokers than among non-smokers  . The risIn this population-based cohort, we examined the prospective association between tobacco smoking and SARS-CoV-2 infection risk and its adverse outcomes six years later, looking for evidence of potential bias introduced by the comparison between current and non-current smoking. The data allowed a refined definition of non-, former and current daily smoking, thus enabling the comparison between results potentially biased because they were obtained with the use of a misclassified reference group and those obtained with a correctly specified reference group. Former smoking was associated with a higher risk of COVID-19 infection, hospitalisation, and death compared with non-smoking, even after considering the presence of chronic diseases. On the other hand, current tobacco smoking was associated with a lower risk of COVID-19 infection after controlling for potential confounders. Our study showed that the results based on the misclassified comparison (categorising former smokers as non-smokers) were similar, but the negative association between current smoking and infection appeared to be stronger, thus speaking for a moderate amount of downward bias (about 4%).The results from this study are in line with previous reports of a higher risk of COVID-19 infection and adverse outcomes among former smokers when compared with non-smokers ,24. AlsoPrevious studies indicate that former smokers are more likely to be older and have smoked for a longer time than current smokers or suffer more comorbidities when compared with non- or current smokers –28. Thissnus are at a higher risk of infection compared with non-users [snus delivers high amounts of nicotine, these results did not support a protective role of this substance. It is also possible that our study may not have accounted for all confounding factors that could influence the association between smoking and SARS-CoV-2 infection risk. Smokers and non-smokers may differ in their health-seeking behaviour, including their likelihood of seeking medical care and testing for SARS-CoV-2. This could lead to a spurious negative association between smoking and the risk of infection. It is crucial to emphasise that the findings of this study should not be taken as an encouragement to start or continue smoking. Smoking is a well-established risk factor for numerous health issues, including lung cancer, heart disease and COPD. The low SARS-CoV-2 infection risk observed in this and other studies, even if causal, does not outweigh the negative health effects of smoking. Additional studies are needed to better understand the relationship between smoking and SARS-CoV-2 infection.Current smokers were at lower risk of infection with SARS-CoV-2 in this study, irrespective of smoking categorisation, while the prognostic outcomes of a diagnosis of COVID-19 among smokers in this study were not different from those of non-smokers. The negative association between current smoking and the risk of COVID-19 in our study could not be entirely explained by the misclassification of the exposure. Other studies also reported a lower risk of infection among smokers . A convion-users ,33. BecaOur study has several strengths. First, the longitudinal study design allowed the study of the association of interest in a truly prospective fashion, avoiding the risk of reverse causality. Second, this study is population-based, hence minimising the risk of selection bias  present Third, cohort participants, and particularly those retained between 2010 and 2014, may be a selected group with different behavioural and risk profile from those originally sampled. Caution should therefore be employed in extrapolating the results to the underlying source population. Also, we did not have information on occasional smoking; therefore occasional smokers are included in the ‘non-smoking’ group. Depending on assumptions on the risk profile of these smokers, the estimates of the relative risks of COVID-19 for former and current smokers may have been biased in either direction.Former smokers in this study had a higher risk of COVID-19 infection and adverse disease outcomes. These findings indirectly support the public health efforts to curb smoking, given the effect of tobacco-related comorbidities. On methodologic grounds, separating non-, former, and current smokers when assessing the risk or the prognosis of diseases with an impact on the respiratory system emerges as an important suggestion.Click here for additional data file.Supplemental material, sj-docx-1-sjp-10.1177_14034948231174279 for Does misclassification of former tobacco smokers explain the ‘smoker’s paradox’ in the risk of COVID-19? Insights from the Stockholm Public Health Cohort by Ahmed N. Shaaban, Filip Andersson, Cecilia Magnusson, Nicola Orsini, Ida H. Caspersen, Sebastian Peña, Sakari Karvonen, Per Magnus and Maria R. Galanti in Scandinavian Journal of Public Health


The majority of the United States population is overweight or obese, and obesity bias is frequently reported by patients. Obesity bias is associated with adverse health outcomes, even independent of body weight. Primary care residents are often sources of obesity bias towards patients with weight, yet education regarding obesity bias is significantly lacking in most family medicine residency teaching curricula. The aim of this study is to describe an innovative web-based module on obesity bias and discuss its impact in family medicine residents.The e-module was developed by an interprofessional team of health care students and faculty. It consisted of a 15-minute video containing five clinical vignettes that depicted instances of explicit and implicit obesity bias in a patient-centered medical home (PCMH) model. Family medicine residents viewed the e-module as part of a dedicated one-hour didactic on obesity bias. Surveys were administered prior to and following the viewing of the e-module. They assessed previous education on obesity care, comfort in working with patients with obesity, residents’ understanding of their own biases in working with this population, and the anticipated impact of the module on future patient care.A total of 83 residents from three family medicine residency programs viewed the e-module and 56 completed both the pre and post survey. There was a significant improvement in residents’ comfort in working with patients with obesity as well as their understanding of their own biases.This teaching e-module is a short, interactive, web-based educational intervention that is free and open-sourced. The first-person patient perspective allows learners to better understand the patient’s point of view and its PCMH setting illustrates interactions with a variety of healthcare professionals. It was engaging and well received by family medicine residents. This module can begin the conversation around obesity bias, leading to improved patient care.The online version contains supplementary material available at 10.1186/s12909-023-04310-4. Since the 1990s, obesity rates have rapidly increased from under 10% to a current prevalence of 41.9% in the United States (US) . For womThe most recent studies estimate that 42–61% of US adults have experienced weight bias with effects that may be independent of body weight , 6. ManyPatients experiencing weight bias may avoid preventative and other medical care and a vicious cycle may be initiated, resulting in an increased risk of new medical conditions or aggravation of existing diagnoses . Mental Since 2007, the Association of American Medical Colleges (AAMC) has recommended that the medical education of future physicians incorporate respectful and effective care of patients with overweight and obesity , 17. In 7.Employ strategies to minimize bias towards and discrimination against people with obesity, including weight, body habitus, and the causes of obesity.7.1Describe the ways in which weight bias and stigma impact health andwellbeing7.2Recognize and mitigate personal biases.7.3Recognize and mitigate the weight biases of others.These guidelines reinforce the need for clinically focused education to mitigate effects of obesity bias in the primary care setting.Recommendations for teaching obesity bias, include educating learners about the complexities and biological processes involved in weight issues, as well as removing personal blame for weight gain , 20. It There is a paucity of validated teaching material for obesity bias, especially regarding implicit biases, and few programs have implemented any instruction around the subject of obesity bias , 23. EffE-Learning and distance learning are well established and extensively documented . HoweverAn interprofessional team of health care students  developed a 15-minute interactive web-based e-module on obesity bias under the oversight of obesity medicine experts and a clinical psychologist. Content was based on the Provider Competency guidelines mentioned above, personal experiences of the creators, and the UConn Rudd center , 24. TheThe interactive module depicts five clinical vignettes depicting problematic patient/physician contact in a non-judgmental setting. The vignettes are presented from a first-person patient perspective, intentionally refraining from providing any direct visualization of the patient. This more easily allows learners to experience the patient’s point of view as the visit unfolds.The vignettes occur in a Patient-Centered Medical Home (PCMH) setting and include video clips depicting the patient’s clinical interactions with a medical assistant, physician, dietitian, pharmacist, and social worker Table . They inhttps://www.unmc.edu/elearning/egallery/obesity-bias/).Since lack of time is a well-known limiting factor in medical education, this educational resource was produced and edited with a focus on brevity, with a total length of 15 min, including the self-assessment summary. This is a free, closed-captioned, and open-sourced module that anyone can access to enhance their patient-centered communication skills and is available on the university e-gallery website  approval was obtained (574-16-EX).Descriptive statistics were used to summarize the quantitative data, and paired sample t-tests examined the change from pre to post on their comfort and understanding of their biases. Quantitative data analysis was done using SPSS v27.0. Qualitative responses were analyzed separately by each member of the research team using an immersion/crystallization process. The team members then collectively agreed on common overarching themes.A total of 83 residents completed the e-module. Of these, 73 (88%) completed the pre-survey, and 66 (80%) completed the post-survey; 56 (67%) participants completed both. The educational initiative was mandatory for all residents. However, the completion of the pre/post surveys was voluntary. The ten knowledge assessment questions that were part of the e-module were for the learners benefit only and responses could not be retained. In the pre-survey, 64% of residents reported no prior focused education about the biopsychosocial management of obesity in their medical school curriculum. Results of a paired sample t-test, including only those 56 participants who completed both the pre- and post-surveys, indicate that comfort level increased significantly after completing the module (p = 0.001). The mean level of comfort in working with patients with obesity increased from a mean of 3.0  (SD = 0.7) to a mean of 3.30  Table .Similarly, when respondents were asked how well they understood their own biases in working with patients with obesity, scores increased from a mean of 2.77  (SD = 0.7) to a mean of 3.48 . Residents overwhelmingly endorsed the module; 95.5% percent of residents said that what they learned would moderately or mostly impact their approach to working with patients with obesity.Qualitative analysis revealed that residents found the e-module engaging and enabled them to reflect on the comprehensive nature of caring for patients with obesity Table .Responses indicated that residents enjoyed seeing an interaction from a patient’s perspective and that they appreciated the authenticity of an interprofessional PCMH model of caring for patients with obesity. Residents suggested that the module could be improved by including more strategies to mitigate obesity bias in patient encounters and making some of the examples more complex, thereby increasing the difficulty.We describe an innovative video module that illustrates how obesity bias might be perceived from the point of view of a patient with obesity in a clinical setting. This e-module incorporates Provider Competencies specifically addressing obesity bias, enabling providers and trainees to better recognize and mitigate obesity bias. Family medicine residents using this interactive e-module reported significantly increased awareness of personal biases regarding obesity, increased comfort treating patients with obesity, and favorable ratings of the clinical applicability of the e-module.To date, there are very few studies on techniques for addressing obesity bias . RecentlThese different methods primarily take a third-person perspective in each clinical scenario. In contrast, the e-module described in the present study utilizes first-person patient perspective. Although potential gender bias from voice recordings of the patient could not be eliminated, use of the first-person perspective eliminated concomitant race or age bias by avoiding direct visualization of the patient. This perspective more easily allows learners to experience the patient’s point of view as the visit unfolds and was repeatedly referenced as effective in resident surveys in this study.The UConn Rudd Center for Food Policy and Health produced a 17-minute video depicting a single visit between a patient with obesity and their provider . Our vidThis e-module can be fully incorporated into a completely remote delivery as a stand-alone educational tool or in conjunction with a more robust obesity education curriculum. Because it is open-sourced, it can be accessed by anyone wishing to strengthen their communication skills, particularly when interacting with patients of size.Limitations of our study consist of lack of precedence of similar methodology and exceedingly limited validated resources and published studies , 20. AnoGraduate and undergraduate teaching programs have limited resources and have a low priority in teaching obesity and obesity bias. Our study indicates that a brief web-based interactive video e-module can serve as an engaging and stimulating methodology to begin the conversation around obesity bias using patient-centric clinical vignettes. Given the projected obesity rates in the US, we advocate for medical educators to be aware of the critical need for development and dissemination of patient-centered educational material to optimize the care of our patient population.Below is the link to the electronic supplementary material.Supplementary Material 1