Despite the growth of psychotherapy trials in low- and middle-income countries, there have been limited follow-up studies of more than 2 years. This study follows up female sexual violence survivors approximately 6 years after completing a 12-session group cognitive processing therapy (CPT) program in the eastern Democratic Republic of Congo.Baseline trial data were collected in December 2010 from 134 women in 7 study villages randomly allocated to CPT. Study women were over 18 years, reported personally experiencing or witnessing sexual violence, and reported elevated depression, anxiety and/or posttraumatic stress symptoms. Women were followed up (1) post-treatment (6-months after baseline); (2) 6 months later; (3) 12 months later; and (4) in March 2017 (6.3 years after baseline). At the long-term follow-up, 103 women (77%) in 6 of 7 CPT villages were re-assessed; one village was not visited due to ongoing insecurity.We found strong continued intervention effects; nearly all women maintained treatment impacts over the first two years; at long-term follow-up, approximately half continued to maintain low symptom scores. Relapse rates for probable PTSD and probable depression and anxiety were 20%.This study extends prior research to show that treatment impacts can be maintained for several years despite experiences of ongoing trauma. The women described continuing to meet with the women in their therapy group and using the skills they learned in the psychotherapy, providing evidence of the potential for these programs to provide valuable social supports and skills that people use as they continue to face adversity. This study looks at mental health outcome data collected approximately 6 years after intervention completion among women who participated in a 12-session group CPT program in the eastern Democratic Republic of Congo. The research questions are: (1) what proportion of women maintained the symptom reductions observed 1-year posttreatment and (2) what client- or treatment-level variables were associated with continued treatment impact.et al., N = 7 villages) or individual support conditions (n = 8 villages). Baseline assessment was completed in December 2010 and the intervention period was from April through July 2011 with CPT provided by local psychosocial workers trained and supervised by US-based CPT trainers. Primary study outcomes were reduction in symptoms of depression, anxiety and posttraumatic stress and improvement in functional impairment. Treatment impacts at 6-month post treatment were previously reported . Average per-person scores were generated, with scores ranging from 0 to 4; higher scores indicate greater functional impairment.et al., et al., CPT is a protocol-based therapy with strong evidence for reducing the burden of depression, anxiety, and PTSD symptoms in sexual-violence survivors. For this study, CPT was provided in a group format with 6–8 women per group. We used the cognitive-only version of the treatment (i.e. without a trauma narrative) because its efficacy is similar to that of the full version of the therapy  who provided therapy were village-based Congolese women who had been providing general psychosocial care to sexual violence for at least 1 year prior to the trial; none had prior mental health training. The PSAs received 2 weeks of in-person training with CPT trainers from the United States in the use of a manual that was adapted to the local context and educational level of the PSAs (most had completed secondary education) and translated locally. Direct supervision was provided by Congolese psychosocial supervisors who were employees of The International Rescue Committee (IRC), the partner research and implementing organization, through weekly telephone or in-person meetings. A bilingual clinical social worker trained in the U.S. supervised the psychosocial supervisors and communicated with the U.S. trainers weekly to monitor quality and fidelity. Each PSA concurrently led three CPT groups.t tests and Fisher's exact tests due to small cell sizes. We also calculated descriptive statistics for women's responses to questions about the frequency of meeting with CPT groups and the types of activities they engaged in related to use of CPT skills.Descriptive statistics were calculated for demographic and symptom variables at baseline for the full baseline sample from the six targeted villages randomized to CPT and separately for women who completed the long-term follow-up. Characteristics were compared for women in these villages who did and did not participate in the long-term follow-up using student's t tests for continuous mean symptoms scores and McNemar's test for binary probable diagnosis using all available data from women who participated in the long-term follow-up.To assess differences in mental health and functional impairment outcomes at long term follow-up as compared to pre-CPT, baseline mean symptom and function scores were compared to mean scores at each subsequent time point  using paired n = 76) who met our baseline criteria for probable clinically significant symptoms (a score of 1.75 or higher on either measure), we explored remission and recovery over the follow-up period. Remission was defined as dropping below the cut-off score at one of the follow-up assessments but then moving back above the cut-off at a later follow up. Recovery was defined as dropping below the cut-off and remaining below the cut-off at all follow-up assessments.For the subsample of women  to final assessment. We categorized women into those who: (1) maintained reductions: whose symptom score changed by only a small amount (⩽0.5 points); (2) women who partially maintained reductions: whose symptom score increased by more than 0.5 but less than 1.0 points; or (3) women who did not maintain their reductions: whose symptom scores increased by 1.0 points or more. The possible symptom score range for the mental health measures is 0 to 3.0 points ; an increase in 1.0 suggests a full category change.et al., We used multinomial logistic regression to assess whether the following characteristics of women recorded at study baseline were associated with symptom maintenance by comparing women who maintained symptom reductions (group 1) with women who partially maintained reductions (group 2) and women who did not maintain reductions (group 3): age, marital and pregnancy status, whether they had lived in the area for 10 years or more, number of people living in the home, number of children women were responsible for, total number of types of traumas witnessed, total number of types of traumas experienced, and functional impairment, as well as depression, trauma, and total symptoms. These are the same predictors that were explored in the original trial paper  of the Johns Hopkins University and the Kinshasa School of Public Health approved the original trial protocol; the Johns Hopkins University IRB approved the amendment for the current follow-up. The sample of women who participated in the CPT treatment groups in the original trial was contacted by their local psychosocial staff and personally invited to participate in this follow-up interview; consent was obtained by study interviewers for those women who agreed to participate.Of the 134 women from the treatment arm in the 6 follow-up villages of the original trial, 16 (12%) were unable to be located, had passed away, or were no longer living in the study area. In addition, 15 women (11%) declined or were unable to participate. Thus, 103 (77%) participated in this follow-up study; 99 (96%) of these women participated in at least 2 previous follow-up assessments. The trajectory of symptom changes for the 103 women in this follow-up study did not differ across the mental health and function outcomes . Lookingn = 76) of the CPT participating women met criteria for a probable diagnosis of clinically significant depression and anxiety at baseline. 9 (12%) of these women did not move into remission at the post-intervention assessment and of these, 6 remained above the cut-off at the final long-term assessments. Among the 67 women who remitted at the post-intervention assessment, 53 (79%) would be classified as recovered at the long-term follow-up assessment and 14 (21%) as having relapsed. For the posttraumatic stress symptoms, 61% (n = 63) of the CPT participating women met criteria for a probable diagnosis at baseline. Of these, 4 (6%) did not move into remission at the post-intervention assessment and 2 of these women remained above the cut-off at the long-term follow-up. For the 59 women who remitted at the post-intervention assessment, 47 (80%) would be classified as recovered at the long-term follow-up assessment and 12 (20%) as having relapsed.At baseline, 74%  women reported that they had continued to meet with the other women from their CPT groups, with 16 (18%) indicating they had met once or twice in the past 6 months, 17 (19%) indicating they had met about once a month, 29 (32%) reported meeting once per week and 24 (27%) reported meeting more than once per week. The activities they reported engaging in with the women from their group ranged from discussing their problems and feelings, socializing, and doing microfinance or other income generation activities. When asked directly, nearly all of them (97%) reported they used CPT skills to help one another.When asked about their ongoing use of specific skills learned during the CPT program, more than 75% of them women reported sometimes or often using each of the 5 CPT-specific skills.et al., et al., We found strong continued intervention effects in this long-term follow-up of 103 women who participated in a group psychological treatment program in rural DRC. Nearly all of the women maintained the initial treatment impacts during the first 18 months after treatment was complete, and after more than 5 years from the end of treatment, approximately half continue to maintain their relatively low symptom scores. This despite ongoing regional instability and women's continued exposure to traumatic events including sexual violence. Nearly 1 in 5 women reported experiencing sexual violence in the 6-months prior to this final assessment; this is approximately twice the number of women who reported sexual violence in the long-term follow up of rape survivors participating in CPT in the U.S.  that differentiated which women were able to maintain the treatment impacts. Some potentially important factors, including cumulative trauma exposure and other life events that may have occurred in the intervening period were not captured in our assessments. Consistent with past research on predictors of poorer long-term outcomes in CPT, it is possible that the economic hardships faced by women in these communities may also be a factor in the higher rate of relapse in this sample, as compared to US samples (Wachen et al., et al., There are several limitations that must be noted, including the lack of an available comparison group as the women in the original control villages received CPT after the trial was complete. In addition, there are unmeasured predictors that may be important that were not collected as part of this study including cumulative trauma exposure, change in trauma-related cognitions (Iverson The original trial was the first to implement CPT with lay providers for sexual violence survivors in a context of ongoing conflict and sexual violence; the results indicated strong impacts of CPT participation on reducing the burden of depression, anxiety and PTSD symptoms and improving daily functioning. This long-term follow-up study shows that after 5 years from the final post-treatment follow-up, most CPT participants continue to meet with their original CPT groups, continue to use the cognitive skills that are fundamental to the CPT model, and continue to live with relatively low symptom burdens despite the presence of ongoing violence and conflict. This despite the CPT providers not having continued formal support to deliver this intervention, which is a challenge of many programs that begin as studies but are not embedded within existing systems of care. This study provides evidence that psychotherapies can act not only as treatments for reducing the burden of mental illness but can also act as prevention programs that can provide valuable skills that people can use as they continue to face adversity. This study also provides an argument for considering the long-term feasibility of implementing these types of programs from the initiation of the studies, including identifying systems to maintain the employment of trained providers.


In all cases, the cause of the SIRVA was from an incorrect COVID-19 vaccine administration technique. Two patients developed shoulder pain immediately following the injection, one patient about 3 h after the injection, and the other four patients within the next few days. Two of the seven patients visited the orthopedic clinic after the persistent shoulder pain for 3 and 4 days and the other five patients 1–9 weeks following their injections. One of the seven patients was treated with combined intravenous antibiotic and oral non-steroidal anti-inflammatory drug (NSAID) because septic arthritis of the shoulder could not initially be ruled out, and recovered within 2 weeks. The other six patients had shoulder pain without acute fever, and five of them were treated with only oral prednisolone 30 mg/day for 5–10 days, following which the pain improved and they all could return to normal activities within 14 days, with no side effects from the prednisolone such as stomachache, nausea, vomiting, headache, or dizziness. Discussion and conclusion: In our series, the most common cause of SIRVA was an incorrect vaccination technique. Most patients responded well to oral NSAIDs or oral prednisolone. Clinical relevance: All SIRVAs were from an incorrect injection technique and not actually the vaccination, so our series highlights the importance of ensuring all vaccinators understand the importance of taking proper care with the injection technique. Additionally, most of our patients with SIRVA from a COVID-19 injection responded well to oral prednisolone (30 mg/day). If there are no contraindications, we suggest this as the first line treatment for COVID-19-related SIRVA.Background: A shoulder injury related to vaccine administration (SIRVA) is a vaccination complication that can affect daily life activities. To date, there have been no case series of patients diagnosed as SIRVA following a COVID-19 vaccination. We offer a series of seven SIRVA cases including clinical presentations, investigations and treatment outcomes. Methods: A retrospective chart review was performed for seven patients who developed SIRVA following a COVID-19 vaccination between April 2021 and October 2021. All patients had no prior shoulder pain before their vaccination and then developed shoulder pain within a few days following the vaccination, which did not spontaneously improve within 1 week. Results: Four of the seven patients were male, and the average age was 62.29 ± 7.76 years. The average body mass index was 25.1 ± 2.2 kg/m The COVID-19 pandemic has had a great effect on normal life activities worldwide. To aid the return to normal life activities, one of the important key factors has been the COVID-19 vaccines, which have been widely applied for immunization since early 2021. However, there have been some adverse effects after the COVID-19 vaccinations, which can be divided into local and systemic adverse effects. Most of these effects usually spontaneously resolve within a few days after the vaccination. If the clinical symptoms persist for more than two days, the doctor should be concerned about more serious complications. A shoulder injury related to vaccine administration (SIRVA) is one complication that begins within 48 h following a vaccination, with the main symptoms of shoulder pain with limited range of motion. The pathogenesis of SIRVA is still uncertain, but a widely accepted theory is that injecting the vaccine into the subdeltoid bursa produces a prolonged inflammatory response . This coTo date, there have been no case series published in the literature on patients diagnosed as SIRVA following a COVID-19 vaccination. In this case series, we present seven cases of SIRVA including clinical presentations, physical examinations, investigations, medications, and treatment outcomes after treatment with some combination of oral prednisolone, oral non-steroidal anti-inflammatory drugs (NSAIDs), intraarticular steroid injection, and/or intravenous antibiotic.In our institution we had seven patients between April 2021 and October 2021 who were diagnosed as SIRVA according to the definition of the National Vaccine Injury Compensation Program (VICP) by one of two sports medicine orthopedists. The VICP definition is “shoulder pain with limited range of motion within 48 h after vaccination with no prior history of pain, inflammation, or dysfunction of the affected shoulder before vaccine administration” [2. In all cases, our investigations found that the cause of the SIRVA was from an incorrect COVID-19 vaccine administration technique. The cause of the SIRVA in three of the seven patients was use of an incorrect landmark (thus resulting in the injection being given too high on the arm) for the injection, and the cause in the other four cases was from an incorrect direction of the needle causing the vaccine to be injected into the bursa rather than the deltoid muscle. Two patients developed their shoulder pain immediately following the injection, one patient about 3 h after the vaccination, and the other four patients within the next few days. Two of the seven patients visited the orthopedic clinic after persistent shoulder pain for 3 and 4 days and the other five patients 1–9 weeks after their injections. One of the seven patients was treated with combined intravenous antibiotic and oral NSAID because septic arthritis of the shoulder could not initially be ruled out and recovered within 2 weeks. The other six patients had shoulder pain without acute fever, and five of them were treated with only oral prednisolone 30 mg/day for 5–10 days following which the pain improved and they all could return to normal activities within 14 days, with no side effects from the prednisolone such as stomachache, nausea, vomiting, headache, or dizziness. The 7th patient received combined oral prednisolone and an intraarticular steroid injection and recovered in 7 days.During the study period, seven patients visiting the orthopedic clinic of our hospital with clinical symptoms of shoulder pain were diagnosed as SIRVA, as they had had no prior shoulder pain before their vaccination and the shoulder pain developed quickly or within a few days after the vaccination and did not spontaneously improve within 1 week. The patients’ characteristics are shown in 3, monocytes 1%, polymorphonuclear neutrophils 99%, red blood cell count 23,400 cells/mm3, and no crystals or organisms. The fluid was sent for culture which showed no organisms. These findings were indeterminate, so he was admitted and treated with combined intravenous cefazolin 1 gm every 6 h for 3 days and oral celecoxib (400 md/day) for 7 days, when the intravenous cefazolin was changed to oral cephalexin (1000 mg/day) for a further 7 days. One day after admission for the intravenous antibiotic, his fever resolved, and he was discharged on day 4 after switching to an oral antibiotic. Over the next few days his symptoms gradually improved, and he could move his shoulder with a full range of motion in all directions 2 weeks after the initial diagnosis and treatment.The first case was a 52-year-old Thai male without prior shoulder pain who developed right shoulder pain on day 2 after his Sinovac vaccination. In this case, the injecting nurse used a 1.5-inch 27-gauge needle, with an injection site 3 fingerbreadths below the mid-lateral edge of the acromial process. The direction of the needle was oblique to the skin cephalad A. He begThe second case was a 51-year-old Thai female with no history of shoulder pain. She received an Oxford-AstraZeneca COVID-19 vaccine with a 1.5-inch 25-gauge needle, based on the landmark of 1 fingerbreadth below the midlateral edge of the acromial process B. The diThe third case was a 66-year-old Thai male who began to have right shoulder pain immediately after a second dose of AstraZeneca. The injection was given with a 1.5-inch 25-gauge needle. The injection landmark was 3 fingerbreadths below the mid-lateral edge of the acromial process. The direction of the needle was 30◦ cephalad to the skin A. The paThe fourth case was a 71-year-old Austrian male who began to feel left shoulder pain one day after a second dose of AstraZeneca vaccine. The injection was given with a 1.5-inch 25-gauge needle and the landmark was 1 fingerbreadth below the mid-lateral edge of the acromial process B. The neThe fifth case was a 68-year-old Thai female who began to feel right shoulder pain with limited range of motion 24 h after a second dose of AstraZeneca vaccine. She had had no shoulder pain before the vaccination. The vaccination landmark was 3 fingerbreadths below the acromial process and the needle direction was 45 degrees cephalad to the skin. Her symptoms had persisted for 14 days without improving. They were worse at night, and she could not lay on her right shoulder. She decided to see an orthopedist and the clinical examination showed tenderness at the deltoid muscle. She had a full range of shoulder motion, but with pain at some points. She was sent for ultrasonography which showed a thin layer of subdeltoid bursal fluid and a partial thickness tear of the subscapularis tendon A,B. She The sixth case was a 64-year-old Thai male who had received his first dose of AstraZeneca vaccine. The vaccination landmark was 3 fingerbreadths below the acromial process with a needle direction of 30 degrees cephalad to the skin. He had developed right shoulder pain with limited range of motion in all directions within 48 h of receiving the vaccine. He did not take any medications, and when the symptoms had not improved in a month he decided to see an orthopedist. A physical examination showed tenderness at the deltoid muscle with limited range of motion in all directions. An ultrasonography showed tenosynovitis at the long head of the biceps and a low-grade partial tear of the subscapularis tendon A,B. He wThe seventh case was a 64-year-old Thai female who presented with right shoulder pain 9 weeks after a first dose of the AstraZeneca vaccine. The symptoms had begun immediately after the vaccination and gradually gotten worse, to the point that it was painful to complete daily activities and she could not sleep on her right side. In her case, the injection landmark was 1 fingerbreadth below the mid-lateral edge of the acromial process. At the OPD, the physical examination showed limited range of right shoulder motion and pain on the forward motion elevation resistance test and external rotation. Ultrasonography showed calcific tendinopathy of the supraspinatous tendon without bursitis A,B. She We report a series of seven patients who developed a SIRVA after a COVID-19 vaccination, all of which were the result of an incorrect COVID-19 vaccination technique. Following their examinations, four of the five patients who received only oral prednisolone had rapid improvement, and one patient who received combined oral prednisolone and TA injection also had rapid improvement. One patient received an antibiotic and NSAID due to fever with the shoulder pain, following which he gradually improved over the next 14 days and the 7th patient, who received only oral prednisolone also improved over 14 days. In cases of SIRVA, if the patient has no contraindications such as fever, we recommend giving oral prednisolone (30 mg/day) for at least 5 days.2. The other factor which has been reported as associated with SIRVA is sex. Two large series reported that SIRVA was predominant in females with incidences of 82.6% and 82.8% [SIRVA is a rare complication that can occur following a vaccination. The risk factors for this complication are BMI, sex, age and type of vaccine. One study reported that overweight patients had an increased risk of SIRVA , while ond 82.8% ,6. In ound 82.8%  and AtanHowever, the most common cause of SIRVA, as with most other vaccines, remains an incorrect injection technique. The recommended vaccine injection technique for the COVID-19 vaccines involves three things. First, the landmark should be 2 or 3 fingerbreadths, depending on the size of the patient, below the acromial process C. If theThe main symptom of SIRVA is shoulder pain beginning a few minutes to a few days after the vaccination which does not resolve by a week or so. This is different from the quite common local effect of a vaccination which is usually not severe and clears up in 2–3 days. The key factor in considering SIRVA is the absence of prior shoulder pain before the vaccination. All patients in our study met these conditions, and all complained of difficulty in performing their normal daily activities. There have been reports of neurological symptoms such as weakness or numbness, but these were not found in our study [The most common physical examination finding of SIRVA is pain while moving the shoulder with incidences of 55.9% and 85% in reported studies ,3. The dNot all patients with a SIRVA are sent for further investigations such as radiography, ultrasonography and/or MRI. Atanasoff et al.  reportedThere have been several modalities for treating SIRVA reported in case series, including nonsteroidal anti-inflammatory drugs, lidocaine patches, opioid medications, intra-articular steroid injections, oral steroids, acupuncture and even surgery ,3,6. A SAll SIRVAs following a COVID-19 vaccination in this series were the result of an incorrect COVID-19 vaccination technique. Most patients responded well to a short course of oral prednisolone.


Lipid droplets (LDs) are organelles formed in the endoplasmic reticulum (ER) to store triacylglycerol (TG) and sterol esters. The ER protein seipin is key for LD biogenesis. Seipin forms a cage-like structure, with each seipin monomer containing a conserved hydrophobic helix and two transmembrane (TM) segments. How the different parts of seipin function in TG nucleation and LD budding is poorly understood. Here, we utilized molecular dynamics simulations of human seipin, along with cell-based experiments, to study seipin’s functions in protein–lipid interactions, lipid diffusion, and LD maturation. An all-atom simulation indicates that seipin TM segment residues and hydrophobic helices residues located in the phospholipid tail region of the bilayer attract TG. Simulating larger, growing LDs with coarse-grained models, we find that the seipin TM segments form a constricted neck structure to facilitate conversion of a flat oil lens into a budding LD. Using cell experiments and simulations, we also show that conserved, positively charged residues at the end of seipin’s TM segments affect LD maturation. We propose a model in which seipin TM segments critically function in TG nucleation and LD growth. The lipid droplet (LD) is a fat-storing organelle, surrounded by numerous coat proteins and a phospholipid (PL) monolayer . LDs stoCurrent models of LD biogenesis posit that lipid droplet assembly complexes (LDACs) in the endoplasmic reticulum (ER) bilayer determine LD formation sites and facilitate LD growth . LDACs, Human seipin is an undecamer, forming a ring structure in the ER. Each subunit contains a lumenal domain, flanked by two transmembrane (TM) segments and short cytoplasmic tails . The lumIn this study, we capitalized on new information on seipin TM segments to investigate their roles in TG nucleation and LD maturation using all-atom (AA) and coarse-grained (CG) molecular dynamics (MD) simulations. We discover a cage-like geometry of seipin TM segments facilitates a conversion of a planar oil lens into a unique ER-LD neck structure. Using cell experiments and CG simulations, we provide evidence that conserved, positively charged residues at the end of seipin’s TM segments are critical for LD growth.Seipin TM segments are thought to be critical for seipin functions . The resD-glycero-1-phosphatidylcholine (POPC) bilayer containing 6% triolein for 3 µs. To study protein–lipid interactions, we reduced the resolution of the AA simulation by molecularly grouping each lipid or protein residue as illustrated in How each part of seipin functions in LD biogenesis is not clearly known. To analyze the interactions between protein residues and lipids, we performed the AA simulation of human seipin in a 3-palmitoyl-2-oleoyl-We further compared protein residues’ attractions to TG glycerol groups or TG tail groups by normalizing the coordination number by the number of grouped atoms. While the coordination number per molecule  and the end of the C-terminal TM segment  are essential for the seipin function. Those charged residues located at the borders of the TM segments are conserved , and in r) between the residues at the membrane interface and PL interfacial and head groups . The resulting CG simulations demonstrated that those residues no longer interacted with the interface of the cytosolic leaflet . The normalized coordination number by molecule or the coordination number per molecule  were used for MARTINI CG simulations , run by kBT for PL was used in this study. A CG model for seipin was constructed by linearly mapping four amino acids into each CG bead. Such a resolution was chosen to match the resolution of the CG lipids, preventing hydrophobic mismatch. We also placed 24 PL molecules inside the HH ring with their orientations consistent with the other PL molecules in the lumenal leaflet. Three models were constructed with different elastic networks with bond potentials k is the spring constant and r0 is the equilibrium bond length. The first two models utilized a standard ENM with a spring constant of 0.2 or 2 kcal/mol Å2 and with a distance cutoff of 15 Å. The third model utilized the hENM that more correctly represented the fluctuations of seipin in the underlying AA simulation  were obtained from the laboratory of Dr. Tomas Kirchhausen  and were maintained in DMEM/F-12 GlutaMAX (Life Technologies) supplemented with 5 μg/ml insulin (Cell Applications), 1 μg/ml hydrocortisone (Sigma), 5% FBS , 50 μg/ml streptomycin, and 50 U/ml penicillin. Cell lines were tested monthly for mycoplasma contamination using the PCR Mycoplasma Test Kit (ABM Cat# G238) and always came back negative. The cell lines were authenticated via STR profiling.SUM159 breast cancer cells  and restriction enzymes were from New England Biolabs. The fragment DNA of tipA mutant construct was synthesized .Cells were plated on 35-mm glass-bottom dishes (MatTek Corp). Imaging was carried out at 37°C approximately 24 hr after transection. Before imaging, cells were transferred to prewarmed FluoroBrite DMEM supplemented with 2 mM GlutaMAX , 5 mg/ml insulin (Cell Applications), 1 mg/ml hydrocortisone (Sigma Aldrich), 5% fetal bovine serum , 50 mg/ml streptomycin, and 50 U/ml penicillin.2 levels, a stage top chamber was used (Okolab). Images were acquired through a 603 Plan Apo 1.40 NA objective or 1,003 Plan Apo 1.40 NA objective (Nikon). Image pixel size was 0.065 mm. Green or red fluorescence were excited by 488 or 560 nm  lasers. All laser lines shared a quad-pass dichroic beamsplitter . Green and red emission was selected with FF03-525/50 or FF01-607/36 filters (Semrock), respectively, mounted in an external filter wheel. Multicolor images were acquired sequentially.Spinning-disk confocal microscopy was performed using a Nikon Eclipse Ti inverted microscope equipped with Perfect Focus, a CSU-X1 spinning-disk confocal head (Yokogawa), Zyla 4.2 Plus scientific complementary metal–oxide semiconductor cameras , and controlled by NIS-Elements software (Nikon). To maintain 85% humidity, 37°C and 5% CO2) at room temperature. 40 μM digitonin and 100 μg/ml Proteinase K were added to the media during imaging acquisition to lyse plasma membrane and kill fluorescence proteins, respectively.The membrane topology of seipin constructs were determined by fluorescence protease protection assay as described in LD size quantification was done with the fiji software. Individual images were first converted to a binary mask using ‘Threshold’ plugin in fiji with Otsu method. Then, LD sizes were measured by the fiji plugin ‘Particle Analysis’.2 and 80% humidity in Multitron-Pro shaker at 125 rpm. When cell density reached 2.5 × 106 cells per ml, the pCAG-LNK plasmids were transfected into the cells. For 2 l of cell culture, 2 mg of plasmids were premixed with 6 mg of 25 kDa linear polyethyleneimine (Polysciences) in 200 ml of Opti-MEM medium for 30 min at room temperature before transfection. At 16 hr after transfection, 10 mM sodium butyrate were added to boost protein expression. To enrich a status of the LDAF1–seipin complex that does not contain neutral lipids inside the complex, cells were treated with 50 μ DAGT1 and DGAT2 inhibitors for 48 hr and with 5 μM Triacsin C for 12 hr prior to cell harvest. At 48 hr after transfection, cells were collected, and cell pellets were snap frozen by liquid nitrogen and stored at −80°C.The complex of LDAF1-FLAG and seipin (1–310) were expressed in suspension cultures of Expi293F cells (Life Technologies) which were cultured in Expi293 Expression Medium  at 37°C under 8% CO2, 10% vol/vol glycerol) supplemented with cOmplete Protease Inhibitor Cocktail tablet, ethylenediaminetetraacetic acid (EDTA)-Free (Roche). Cells were lysed by sonication. The cell debris were removed by centrifugation at 5000 × g for 15 min. To get membrane fractions, the supernatant was subjected to centrifugation in a Ti45 rotor (Beckman) at 43,000 rpm for 1 hr. The membrane pellet was collected and homogenized with a Dounce homogenizer in Buffer A supplemented with 1% Lauryl Maltose Neopentyl Glycol (LMNG), and cOmplete Protease Inhibitor Cocktail tablet, EDTA-Free, and membranes were solubilized with gentle rocking for 1.5 hr. Insoluble material was then removed by centrifugation at 43,000 rpm for 35 min. The supernatant was incubated with 1.2 ml of anti-FLAG M2 resin (Sigma) for 1.5 hr. The resins were then collected and washed with 12 ml of Buffer A with 0.05% digitonin and the proteins were eluted with 3 ml of washing buffer containing 0.2 mg/ml of 3xFLAG peptide (Sigma). The eluted protein was concentrated and PMAL C8 was added at a mass ratio of 3–1 protein. Detergent was removed by biobeads SM-2 overnight. The sample was further purified by size-exclusion chromatography on a Superose 6 3.2/300 Increase column, equilibrated with buffer containing 25 mM HEPES, pH 7.4, 150 mM NaCl. Peak fractions were pooled and concentrated to 2 mg/ml for cryo-EM analysis.All purification procedures were performed at 4°C. Cell pellet was thawed and resuspended in the Buffer A 12 methyl-PEG-NHS-ester (Thermo Fisher) at a 1:10 molar ratio for 2 hr on ice to reduce aggregation of particles on the grids. PEGylated sample (3 μl) was applied to a glow-discharged quantifoil grid . The grids were blotted for 2.5 s at ~90% humidity and plunge-frozen in liquid ethane using a Cryoplunge 3 System (Gatan).2 fractionated into 49 frames. The defocus range for the sample was between 0.8 and 2.5 μm.Cryo-EM data were collected on a Krios operated at 300 kV and equipped with a K3 Summit direct electron detector (Gatan) at Harvard Medical School. All cryo-EM movies were recorded in counting mode using SerialEM. The nominal magnification of ×81,000 corresponds to a calibrated physical pixel size of 1.06 Å. The dose rate was 22.8 electrons/Å2 s. The total exposure time was 2.2 s, resulting a total dose of 50.6 electrons/Åhttp://www.mrc-lmb.cam.ac.uk/kzhang/). After manual inspection and sorting to discard poor images, classifications were done in Relion 3.0  based on previously determined structure of lumenal domain and our cryo-electron microscopy data . The sei Kim et al., investigate interactions between Seipin transmembrane domains and triacylglycerol using molecular dynamics simulations. They identify the leading steps in droplet formation and provide a physical basis for understanding the initial phases of this process, highlighting the importance of transmembrane helices in the function of seipin protein. This paper will be of interest to cell biologists and biophysicists aiming to unveil and understand how lipid droplets are formed inside cells. The topic is important given that lipid droplets are key organelles used for energy storage, and that the failure in their formation can result in various metabolic diseases. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Lucie Delemotte as Reviewing Editor and José Faraldo-Gómez as Senior Editor. All reviewers have opted to remain anonymous.Thank you for submitting your article “Seipin transmembrane segments critically function in triglyceride nucleation and lipid droplet budding from the membrane” for consideration by eLife. However, editors and reviewers recognize the interest and originality of the work; therefore, rather than declining this submission, we would like to invite you to revise the manuscript in a manner that convincingly resolves the critical issues raised by the reviewers. To enable you to do so, we will reconsider your revised manuscript irrespective of the timeframe required for these improvements. Please also note it is probable that your revised manuscript will be sent to the same reviewers for a re-evaluation.The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this letter to help you prepare a revised submission. We appreciate that the revisions required might entail a substantial effort, which might also exceed the typical scope and timeframe permitted for revisions at Essential revisions:1) A more thorough investigation of the quality of the models of the transmembrane regions of Seipin.2) Improved sampling of the conformational space in atomistic simulations (introducing replicas).3) A thorough characterization of the role of the charged residues and the residues in transmembrane position on protein expression, membrane insertion and topology.4) A list of concrete predictions from the coarse-grained model that can be tested experimentally or by atomistic MD simulations.5) Release of data and models to be openly available to the community.Reviewer #1 (Recommendations for the authors):Whilst a well-structured project, and representing a considerable amount of data, I have a couple of concerns on how well the experiments support the findings.Chiefly, I am uncertain of how confident we can be of the modelled TM regions. Whilst fine to include, I feel that the bulk of the figures which interpret these regions are at risk of over-interpreting what might be low-accuracy input models. Seeing as even a small change in TM helix angle or register can cause dramatic artefacts, the model here seems potentially insufficient for use.At the very least the following points should be addressed:– These models should be made available for download, for verification and use by other researchers.– The packing of side chains in the TM domains should be shown, as the image in Figure 1a has very limited use.– How do the helices compare to the recent structure of the seipin from yeast ? Are there any conserved geometries or side chain packing that could be used to refine or support the model used here?– It is stated that the structural density in S1 was used to guide the modelling, but from the figure it is impossible to see how this was done. If there is reasonable density for the TM helices that can be shown, it should be. Otherwise, this comparison is misleading.– The principal component analysis in Figure 3 suggests huge movements of the TM helices. This might be due to naturally high dynamics, however might also reflect a low quality input model. Ideally, some other data could corroborate these dynamics, but failing that at the very least the progression of each pair of TM helices (out of 11) should individually be projected on PC1 and PC2. This would show if the dramatic dynamics seen in the figure is happening in each subunit (which would support the author's claims) or if each subunit is doing something different. This would both show that the simulation is not ergodic, and also suggest that subunits are "collapsing" into different conformations, so possibly a result of an issue in the input model.The second part, which focuses on the lens formation, is a neat piece of work, and directly addresses the biological question of LD formation. The outcomes from this section seem plausible, however I have a couple of points which should be addressed:– The simulations are very coarse-grained, and it's open for debate how well a molecule with 3 tails like TAG can be represented by a 4-bead model. Ideally, some "bridging" simulations using something like Martini could be used on a smaller system to demonstrate that the same observations can be made.– Are the dynamics of the CG system the same as the AT system, as in, are the same extremes in TM angles observed?Combining these last two points – I wonder if random modelling of the TMs would not produce the same results, as the effect seems to be mostly due to the length of the TM helices and the position of the basic residues. If so, I'd argue that the first part of the study is not really necessary for the study, and overall makes the work weaker.Reviewer #2 (Recommendations for the authors):– The conclusions of the study rely heavily on a structural model that is described only superficially. How was the density map/model presented in Figure S1 determined? The transmembrane residues were modelled based on the structure of the yeast protein that is not publicly available . How does this model compare with the predicted Alphafold structure? Information on the transmembrane segment residues and the transmembrane segment conformational changes would be particularly relevant.– It is stated that the switch region leads to lower lipid permeability on the luminal leaflet. This is potentially interesting but could the presence of the bulky luminal domain explain this behaviour (and not the Switch region per se)? Please comment.– It would be interesting to show the analysis on the evolutionary conservation of the transmembrane residues. Additional mutagenesis of conserved residues to demonstrate their functional relevance would also be helpful.– In Figure 6 the conclusion is "Therefore, high protein density at the seipin site provides for collective and cooperative interactions with TG, catalyzing TG nucleation". This appears unjustified since Seipin monomers were not used as control. Instead, it seems that a single TM protein lacking the TAG-binding hydrophobic helix was used.– The identification of the charged residues flanking transmembrane segments is potentially interesting. However, given the role of charged residues in protein topogenesis the authors should exclude that the phenotype is not indirect due to poor expression or changes in membrane topology.– According to Yan et al., (2019), Seipin luminal domain (without the TMs) expresses well and appears to oligomerize. This contrasts with the what is described on page 14: “Finally, we simulated the seipin model that only contained the luminal domain. Such a complex does not form a mobile focus in cells likely because it fails to form an oligomer, it is not stable in bilayers, or it is degraded”. Please comment on the discrepancy.Reviewer #3 (Recommendations for the authors):The authors have done a very nice piece of work. Overall, the quality is high, the results are very interesting, and the discussion of the results is fresh and insightful.Nonetheless, please let me propose a few ideas to improve the quality of the manuscript further.The main weakness of the study is the limited sampling of the atomistic simulation model. If the reviewer is not mistaken, the authors did only one simulation. While its length was 3 microseconds, it is short given that the formation of a triglyceride (TG)-rich lipid phase is limited by lipid diffusion, which is a slow process, and since the transmembrane helices of seipin block diffusion, hence slowing down the formation of the TG-rich phase. This concern is increased by the fact that the development of the coarse-grained (CG) model is partly based on atomistic simulation data. The authors are encouraged to improve the sampling of atomistic simulations by carrying out a couple of additional simulations that would start from a different initial state / lipid configuration.In Methods, the methodology of cryo-EM imaging spectroscopy is not presented. These descriptions would be useful, since the structure of the transmembrane region in the simulation models is based on cryo-EM imaging data  and educated guesses.The results in Figure 4 are interesting since they present (first) evidence of the rate of lipid diffusion inside a cage-like protein complex. The lipid diffusion data outside but in the vicinity of the seipin oligomer, in turn, is consistent with previous studies (supplementary) and Niemela et al., ))), which could be briefly discussed here.Page 13 ("To study the impact of the cage-like structure of the seipin oligomer and their TM segments in LD biogenesis…"). These simulations were carried out at 6 mol% TG, which is above the critical concentration of TG clustering. What happens if the TG concentration is 2 mol%, as in previous simulations discussed earlier in the paper? These additional simulations would be relevant, since they would clarify the relative importance of different seipin units in the clustering of TG .Main conclusions of the paper are based on the CG model, and the conclusions are consistent with current experimental knowledge. However, since the CG model is phenomenological and its properties can be tuned , the question is, what new insight the present CG simulation data can generate that could be tested in experiments and more detailed atomistic simulations? In other words, are the authors able to present experiments to test the key predictions in this paper? Reviewer #1 (Recommendations for the authors):Whilst a well-structured project, and representing a considerable amount of data, I have a couple of concerns on how well the experiments support the findings.Chiefly, I am uncertain of how confident we can be of the modelled TM regions. Whilst fine to include, I feel that the bulk of the figures which interpret these regions are at risk of over-interpreting what might be low-accuracy input models. Seeing as even a small change in TM helix angle or register can cause dramatic artefacts, the model here seems potentially insufficient for use.We appreciate the reviewer’s input. We understand the reviewer’s concern about the low-resolution of the TM segments. Although the cryo-EM is low-resolution, it provided valuable information such as the orientations and locations of the TM segments of seipin. Therefore, this information based on cryo-EM builds a higher quality of the seipin model than the previous papers, PNAS 2021, 118, 10 and PLoS biology 2021, 19, 1, e3000998, where they used a random guess. In our updated manuscript, reflecting the reviewer’s suggestion, we discussed this in more detail and removed the results and discussion of the TM movements.At the very least the following points should be addressed:– These models should be made available for download, for verification and use by other researchers.We have made the model publicly available.– The packing of side chains in the TM domains should be shown, as the image in Figure 1a has very limited use.We have now included a detailed structural model in Figure 1.– How do the helices compare to the recent structure of the seipin from yeast ? Are there any conserved geometries or side chain packing that could be used to refine or support the model used here?We have compared our model against the recently resolved yeast structure in Figure 1.– It is stated that the structural density in S1 was used to guide the modelling, but from the figure it is impossible to see how this was done. If there is reasonable density for the TM helices that can be shown, it should be. Otherwise, this comparison is misleading.The TM helices are shown in Figure 1 —figure supplement 1, and the orientations of those helices were used in modelling our structure.– The principal component analysis in Figure 3 suggests huge movements of the TM helices. This might be due to naturally high dynamics, however might also reflect a low quality input model. Ideally, some other data could corroborate these dynamics, but failing that at the very least the progression of each pair of TM helices (out of 11) should individually be projected on PC1 and PC2. This would show if the dramatic dynamics seen in the figure is happening in each subunit (which would support the author’s claims) or if each subunit is doing something different. This would both show that the simulation is not ergodic, and also suggest that subunits are “collapsing” into different conformations, so possibly a result of an issue in the input model.The depicted PC1 and PC2 motions were associated with their maximum and minimum values; Therefore, they can look exaggerated. We would not expect all-atom simulations can show complete ergodicity of the TM segment movements even if the input model was very accurately resolved due to the limited time scale and the large size of seipin.The second part, which focuses on the lens formation, is a neat piece of work, and directly addresses the biological question of LD formation. The outcomes from this section seem plausible, however I have a couple of points which should be addressed:– The simulations are very coarse-grained, and it’s open for debate how well a molecule with 3 tails like TAG can be represented by a 4-bead model. Ideally, some “bridging” simulations using something like Martini could be used on a smaller system to demonstrate that the same observations can be made.We have now included additional higher CG resolution MARTINI simulations in Figure 3e; The 4-bead CG models are now published in J. Phys. Chem. B 2022, 126, 2, 453–462.– Are the dynamics of the CG system the same as the AT system, as in, are the same extremes in TM angles observed?Combining these last two points – I wonder if random modelling of the TMs would not produce the same results, as the effect seems to be mostly due to the length of the TM helices and the position of the basic residues. If so, I’d argue that the first part of the study is not really necessary for the study, and overall makes the work weaker.The RMSF from the CG simulations agrees reasonably well with that from the AA simulations; Also, the radius of the TM segments in CG simulations becomes larger due to TG nucleation and LD maturation, which was not possible to see in the more limited AA simulation. We feel the first part of the study is valuable and adds to the paper.Reviewer #2 (Recommendations for the authors):– The conclusions of the study rely heavily on a structural model that is described only superficially. How was the density map/model presented in Figure S1 determined? The transmembrane residues were modelled based on the structure of the yeast protein that is not publicly available . How does this model compare with the predicted Alphafold structure? Information on the transmembrane segment residues and the transmembrane segment conformational changes would be particularly relevant.We have updated our methods section to explain how the density map/model was obtained in detail. Arlt et al., is now published in Nat Struct Mol Biol 2022. The Alphafold-predicted TM structure of human seipin seems to deviate from the expectations in the field. Despite its publicity, Alphafold is not a panacea, especially for membrane proteins. We have publicly made our model available and updated – It is stated that the switch region leads to lower lipid permeability on the luminal leaflet. This is potentially interesting but could the presence of the bulky luminal domain explain this behaviour (and not the Switch region per se)? Please comment.We agree with the reviewer. We have updated our manuscript.– It would be interesting to show the analysis on the evolutionary conservation of the transmembrane residues. Additional mutagenesis of conserved residues to demonstrate their functional relevance would also be helpful.We feel this is interesting but beyond the current scope of the manuscript.– In Figure 6 the conclusion is "Therefore, high protein density at the seipin site provides for collective and cooperative interactions with TG, catalyzing TG nucleation". This appears unjustified since Seipin monomers were not used as control. Instead, it seems that a single TM protein lacking the TAG-binding hydrophobic helix was used.We performed a new simulation that includes only one seipin monomer. TG did not nucleate at 2 mol %. We have updated Figure 3d.– The identification of the charged residues flanking transmembrane segments is potentially interesting. However, given the role of charged residues in protein topogenesis the authors should exclude that the phenotype is not indirect due to poor expression or changes in membrane topology.We have included additional experiments to address this. See Figure 6a.– According to Yan et al., (2019), Seipin luminal domain (without the TMs) expresses well and appears to oligomerize. This contrasts with the what is described on page 14: "Finally, we simulated the seipin model that only contained the luminal domain. Such a complex does not form a mobile focus in cells likely because it fails to form an oligomer, it is not stable in bilayers, or it is degraded". Please comment on the discrepancy.It was hard to find an article only with the last name (Yan) of the first author and the publication year (2019). Given the context, we assumed the reviewer tried to refer to Dev. Cell. 2018, 47, 2, 248-256. We have carefully reviewed this paper again. However, this article does not include any in-vivo experiments with the seipin luminal domain (without the TMs).Reviewer #3 (Recommendations for the authors):The authors have done a very nice piece of work. Overall, the quality is high, the results are very interesting, and the discussion of the results is fresh and insightful.Nonetheless, please let me propose a few ideas to improve the quality of the manuscript further.The main weakness of the study is the limited sampling of the atomistic simulation model. If the reviewer is not mistaken, the authors did only one simulation. While its length was 3 microseconds, it is short given that the formation of a triglyceride (TG)-rich lipid phase is limited by lipid diffusion, which is a slow process, and since the transmembrane helices of seipin block diffusion, hence slowing down the formation of the TG-rich phase. This concern is increased by the fact that the development of the coarse-grained (CG) model is partly based on atomistic simulation data. The authors are encouraged to improve the sampling of atomistic simulations by carrying out a couple of additional simulations that would start from a different initial state / lipid configuration.We appreciate the reviewer’s positive feedback. We agree with the reviewer that the sampling of atomistic simulations was limited as this is a fairly large system and TG does not nucleate within the all-atom time scale. To address the reviewer’s concerns, we have argued the followings:First, many lipids moved more than a dimension of the periodic box.Second, this is the longest all-atom seipin simulation to date as a single trajectory.Third, we have complemented our all-atom simulations with the MARTINI CG model simulations, which are now included in Figure 3e.Finally, we have extended our all-atom simulations by 500 ns, and the results were consistent with the previous 3 microsecond simulation.In Methods, the methodology of cryo-EM imaging spectroscopy is not presented. These descriptions would be useful, since the structure of the transmembrane region in the simulation models is based on cryo-EM imaging data  and educated guesses.We have updated our Methods section.The results in Figure 4 are interesting since they present (first) evidence of the rate of lipid diffusion inside a cage-like protein complex. The lipid diffusion data outside but in the vicinity of the seipin oligomer, in turn, is consistent with previous studies (supplementary) and Niemela et al., ))), which could be briefly discussed here.We appreciate the reviewer’s input on this. We have discussed those aspects in our updated manuscript.Page 13 ("To study the impact of the cage-like structure of the seipin oligomer and their TM segments in LD biogenesis…"). These simulations were carried out at 6 mol% TG, which is above the critical concentration of TG clustering. What happens if the TG concentration is 2 mol%, as in previous simulations discussed earlier in the paper? These additional simulations would be relevant, since they would clarify the relative importance of different seipin units in the clustering of TG .Main conclusions of the paper are based on the CG model, and the conclusions are consistent with current experimental knowledge. However, since the CG model is phenomenological and its properties can be tuned , the question is, what new insight the present CG simulation data can generate that could be tested in experiments and more detailed atomistic simulations? In other words, are the authors able to present experiments to test the key predictions in this paper?Although we did not indicate this in the manuscript, the idea of the mutant construct (seipin-∆TERM-tipA) was actually proposed to the experimentalists from the simulators based on the coarse-grained simulations. While we analyzed our coarse-grained simulations, we found that the charged residues at the borders seem to be important, and therefore we proposed the mutant construct (tipA construct) to our experimental collaborators. Consistent with our initial expectations, the mutant construct could not produce mature lipid droplets properly. To support this finding more, we added additional experiments during the revisions .


This cross-sectional study assesses whether state-specific implementation of opioid prescribing is associated with a shorter duration of opioid prescriptions from dentists for children and adult dental patients. Are state opioid prescribing limits associated with reductions in the duration of opioid prescriptions from dentists?In this cross-sectional study of nearly 90 million dental opioid prescriptions dispensed to adults and children in the US from January 2014 through February 2020, the median duration of these prescriptions was 3.0 days. Implementation of state opioid prescribing limits, most of which allowed up to a 7-day supply of opioids, was not associated with changes in the duration of these prescriptions.The findings of this study suggest the need for future research evaluating the potential role of alternative interventions in reducing dental opioid prescribing. In part to prevent the harms associated with dental opioid prescriptions, most states have enacted policies limiting the duration of opioid prescriptions for acute pain. Whether these limits are associated with changes in the duration of opioid prescriptions written by dentists is unclear.To evaluate the association between state opioid prescribing limits and the duration of opioid prescriptions from dentists.This difference-in-differences cross-sectional study used data from the IQVIA Longitudinal Prescription Database, an all-payer database reporting prescription dispensing from 92% of retail pharmacies in the US. The sample included opioid prescriptions from dentists dispensed to children aged 0 to 17 years and adults 18 years or older from January 2014 through February 2020. Treatment states were those that implemented limits between January 2016 and December 2018. Control states were those that did not implement limits during the study period. Data on opioid prescribing limits were derived from the Prescription Drug Abuse Policy System. Data were analyzed from January 1 to September 30, 2022.State opioid prescribing limits.The outcome was opioid prescription duration, as measured by days’ supply. The association between limits and duration was evaluated using a linear model with a 2-way fixed-effects specification. Covariates included patient characteristics, prescription characteristics, and indicators of implementation of prescription drug monitoring program use mandates. Separate analyses of data from adults and children were conducted owing to differences in the number of treatment states and restrictiveness of limits by age.The adult analysis included 56 607 314 opioid prescriptions for 34 364 775 patients  in 22 treatment states and 12 control states. The child analysis included 3 720 837 opioid prescriptions for 3 165 880 patients  in 23 treatment states and 12 control states. In both analyses, the median (25th-75th percentile) duration of opioid prescriptions was 3.0 (2-5) days. Implementation of limits, most of which allowed up to a 7-day supply of opioids, was not associated with changes in the duration of opioid prescriptions for adults  or children .In this study of national pharmacy dispensing data, opioid prescribing limits were not associated with changes in the duration of opioid prescriptions from dentists. Future research should investigate the potential role of alternative interventions in reducing opioid prescribing by dentists. On the other hand, these limits may be associated with minimal changes if the duration was already below the maximum level allowed.10 To date, few national studies have evaluated these possibilities. In an analysis of Medicare claims, implementation of state opioid prescribing limits was associated with a 0.9-day decrease in the duration of opioid prescriptions from dentists and surgeons.11 However, that study did not separately analyze opioid prescriptions from dentists and did not include patients without Medicare coverage, who received 85% of opioid prescriptions from US dentists in 2019.1In part to achieve this goal, at least 39 states have enacted restrictions on the duration of opioid prescriptions for patients with acute pain, patients who are new to opioids, or both.12The objective of this cross-sectional study was to evaluate the association between state opioid prescribing limits and the duration of opioid prescriptions from dentists. We hypothesized that limits would not be associated with reduced duration, as most limits allow for a 7-day supply of opioids and most dental opioid prescriptions are written for a 3-day supply.STROBE) reporting guideline.13We analyzed data (2014 to 2020) from the IQVIA Longitudinal Prescription Database. This database captures 92% of prescriptions dispensed in US retail pharmacies (including chain and independent) and the majority of prescriptions dispensed in mail-order and long-term care pharmacies. Because the data used were deidentified, the University of Michigan Medical School Institutional Review Board deemed this difference-in-differences cross-sectional study exempt from review and waived the informed consent requirement. We followed the Strengthening the Reporting of Observational Studies in Epidemiology  database before prescribing controlled substances , a potential confounding policy.During the study period, the degree of pharmacy coverage in the database remained stable, according to Allen Campbell, BS, at IQVIA . Data elements included patient demographic characteristics , prescription method of payment, prescriber specialty, and days’ supply. We obtained details on state limits from the Prescription Drug Abuse Policy System (PDAPS), an organization that conducts professional legal epidemiological research on substance use policies.We initially included all opioid analgesic prescriptions from general dentists, dental subspecialists, and oral and maxillofacial surgeons that were dispensed from January 2014 through February 2020 to children aged 0 to 17 years and adults 18 years or older residing in 1 of the 50 US states or the District of Columbia. Prescriptions that were dispensed after February 2020 were not included to avoid potential confounding related to the COVID-19 pandemic. Among prescriptions meeting inclusion criteria, we excluded those with missing data for covariates or potentially invalid dosing information .We identified opioid analgesics using IQVIA’s market definition. This definition included liquid and transdermal opioid products but did not include opioid cough-and-cold medications or buprenorphine formulations used for opioid use disorder. The eAppendix 1 in 15We used a difference-in-differences approach. In this approach, the difference in mean outcomes between the preintervention and postintervention periods in the control group is subtracted from the corresponding difference in the treatment group. The resulting difference-in-differences estimate is only unbiased if the outcome in the treatment group would have trended in the same direction as the control group in the absence of the intervention. Confidence in this assumption is increased if the preintervention trends among the treatment and control groups are parallel.We conducted separate analyses for adults and children, as some states imposed opioid prescribing limits for only 1 age group and other states imposed limits that were more stringent for children than for adults. In both the adult and child analyses, the control states included 11 states  that did not enact an opioid prescribing limit from January 2014 through February 2020, as well as the District of Columbia, which also did not enact a limit during this period. To mitigate bias from staggered treatment timing and to capture sufficient preintervention and postintervention data, we allowed only those states that enacted limits between January 2016 and December 2018 to be the treatment states.We excluded states that enacted limits during this period in the same month that they implemented or amended a PDMP use mandate for dentists because disentangling the implications of the limits from these other policy changes would be challenging. We also excluded states that only enacted limits for opioid prescriptions written under certain circumstances . After these exclusions, there were 22 treatment states in the adult analysis and 23 treatment states in the child analysis; the notes for The outcome was the duration of opioid prescriptions, as measured by days’ supply. This outcome is directly reported in the IQVIA database and represents the number of days of therapy that is fully covered by a prescription if the maximum daily number of doses allowed is taken. For example, 24 opioid pills correspond to a 4-day supply if a prescription is written for 1 pill every 4 hours as needed for pain .16Covariates included an indicator that equaled 1 if prescriptions were dispensed on or after the enactment date of a PDMP use mandate for dentists or 0 if otherwise . We began the postintervention period 6 months before the limit implementation because the event study analysis suggested differential declines in days’ supply in the treatment states 5 months prior to the limit implementation in the adult analysis and 6 months prior to the limit implementation in the child analysis. These patterns suggested that dentists may have changed their prescribing behavior in anticipation of the limit enactment.To assess whether the association between limits and duration varied by limit restrictiveness, we conducted 2 difference-in-differences analyses. First, to compare the association among states that restricted dental opioid prescriptions to a 5-day supply or less vs greater than a 5-day supply, we fitted the same model used in the main analysis but included an indicator that equaled 1 if the prescription was dispensed on or after the limit implementation date, minus 6 months, in a state that restricted prescriptions to a 5-day supply or less. For all other prescriptions, the indicator equaled 0. Second, we used a similar approach to assess whether the association varied among states that restricted opioid prescriptions to a 3-day supply vs greater than a 3-day supply.To assess for variation in association by dental specialty, we fitted the same model used in the main analysis. We included in the model an interaction between the primary variable of interest and an indicator for whether the prescription was written by an oral and maxillofacial surgeon or by another type of dentist.To assess the parallel trends assumption, we conducted an event study analysis. We fitted linear regression models with fixed effects for state, fixed effects for year-month, variables for each covariate, and dummy variables for the number of months to the intervention in treatment states. The reference period was the month before limit implementation minus 6 months. The number of leads and lags was capped to ensure that all treatment states contributed equivalent amounts of data, thus avoiding bias from compositional changes in the states contributing to each data point. The time caps were −16 months for leads and +16 months for lags, as 1 treatment state (Oklahoma) implemented a limit in November 2018, 16 months before the end of the study period in February 2020.All models used robust SEs clustered at the state level. This approach accounted for the association between the limits and the duration of dental opioid prescriptions among patients in the same state. Analyses were performed with R, version 4.3.1 , and 2-sided hypothesis tests with α = .05 were used to determine statistical significance. Data were analyzed from January 1 to September 30, 2022.18 Finally, to explore whether the conclusions would change if alternative measures of dosing were used, we repeated the analyses when modeling total morphine milligram equivalents (MMEs), a standardized measure of opioid prescription size,19 and when modeling the proportion of opioid prescriptions exceeding a 5-day or 7-day supply.In sensitivity analyses, we repeated the main analyses when assuming a 3-month anticipation period and no anticipation period. Additionally, we repeated the analyses when using doubly robust difference-in-differences estimation, which combines outcome regression adjustment with inverse probability weighting to reduce the bias that occurs in 2-way fixed-effects estimates when the magnitude of the association between the exposure and outcome changes over time.The IQVIA database included 85 632 541 dental opioid prescriptions dispensed to adults between January 2014 and February 2020. Of these prescriptions, 1 924 812 (2.2%) were excluded from this study owing to missing covariate data or invalid or missing data on days’ supply, and 27 100 415 (31.6%) were excluded because they were not for adults in the treatment states or control states. The characteristics of the remaining 56 607 314 prescriptions are displayed in eAppendix 3 in Among all prescriptions in the sample, the mean (SD) duration was 3.4 (2.2) days and median (25th-75th percentile) duration was 3.0 (2-5) days. During the study period, monthly mean days’ supply decreased from 3.4 to 3.0 days in the treatment states compared with 3.6 to 3.3 days in the control states , A. LimiThe IQVIA database included 5 475 704 opioid prescriptions from dentists dispensed to US children from January 2014 through February 2020. Of these prescriptions, 147 128 (2.7%) were excluded due to missing covariate data or invalid or missing data on days’ supply, and 1 607 739 (29.3%) were excluded because they were not for children in the treatment states or control states. The characteristics of the remaining 3 720 837 prescriptions are displayed in eAppendix 4 in Among all prescriptions in the sample, the mean (SD) duration was 3.6 (2.2) days and median (25th-75th percentile) duration was 3.0 (2-5) days. During the study period, monthly mean days’ supply decreased from 3.6 to 2.9 days in the treatment states compared with 3.9 to 3.2 days in the control states , B. As iFor both the adult and child analyses, conclusions were unchanged when assuming a 3-month anticipation period, when assuming no anticipation period, or when using doubly robust difference-in-differences estimation. Limit implementation was associated with a decrease in the size of opioid prescriptions of −3.7  MMEs for adults and −3.7  MMEs for children, corresponding to a decrease of less than 1 pill containing 5 mg hydrocodone. However, event study plots suggested that the parallel trends assumption was not met, suggesting that the findings should be interpreted with caution  and a policy database created by epidemiologists (PDAPS).9 suggesting that their implementation likely was not associated with changes in the duration of dental opioid prescriptions.9 Second, the IQVIA database does not capture dispensing from pharmacies in facilities affiliated with the US Department of Veterans Affairs or from health system–specific pharmacies, such as those affiliated with Kaiser Permanente. Finally, because the IQVIA database lacked claims data for dental procedures, we could not assess changes in the probability of opioid prescription dispensing after such procedures. However, there is little reason to suspect these changes would occur, as opioid prescribing limits mostly target the duration of opioid prescriptions and not the decision to prescribe opioids.The study also has several limitations. First, the study did not include states that enacted limits in 2019 and thereafter. However, each of the 4 states that implemented limits in 2019 restricted opioid prescriptions to a 7-day supply or greater,In this difference-in-differences cross-sectional study of national pharmacy dispensing data, state opioid prescribing limits were not associated with changes in the duration of dental opioid prescriptions from dentists for either adults or children. Future research is needed to investigate whether other interventions play a role in reducing dental opioid prescribing.


The remote device management (RM) is recommended for patients with cardiac implantable electronic devices (CIEDs). RM underutilization is frequently driven by the lack of correct system activation. The MyLATITUDE Patient App (Boston Scientific) has been developed to encourage patient compliance with RM by providing information on communicator setup, troubleshooting, and connection status of the communicator.ad hoc questionnaire to evaluate their experience.At 14 centers, patients with CIEDs were invited to download and install the App on a mobile device. After 3 months, patients were asked to complete an p=0.033). The use of the app made 84% of patients feel reassured.The App was proposed to 242 consecutive patients: 81 before RM activation, and 161 during follow-up. The App was successfully installed by 177 (73%) patients. The time required for activation of the communicator and the need for additional support were similar between patients who followed the indications provided by the App and those who underwent standard in-clinic training. During follow-up, notifications of lack of connection were received by 20 (11%) patients and missed transmission by 22 (12%). The median time from notification to resolution was 2 days. After 3 months, 175 (99%) communicators of the 177 patients who installed the App were in “Monitored” status versus 113 (94%) of 120 patients without the App installed (The App was well accepted by CIED patients and offered support for communicator management and installation. Its use enabled patients to remain connected with greater continuity during follow-up. According to current guidelines, the use of remote management (RM) is recommended for patients with cardiac implantable electronic devices (CIEDs) , 2. SeveThe MyLATITUDE™ Patient App (Boston Scientific) has been developed to encourage patients’ compliance with RM by providing them with information on communicator setup and troubleshooting, connection status of the communicator, scheduled transmissions, and status of the battery of the implanted device.In the present study, we evaluated the first experience of the adoption of the MyLATITUDE™ Patient App in clinical practice.From May to July 2021, at 14 Italian arrhythmia centers, patients with a compatible Boston Scientific CIED with RM capabilities were invited to download and install the App on a mobile device.The use of the App was proposed to all consecutive patients after CIED implantation or, during a scheduled follow-up visit, to consecutive patients already enrolled and monitored at home through the LATITUDE™ platform. The study design was approved by the Institutional Committee on Human Research of each center and informed consent was obtained from all patients. According to individual patient preference, patients were assigned to the App group or the control group.ad hoc questionnaire to collect anonymous data and evaluate their experience. All data were collected under local standard-of-care conditions of use. The operators at the centers were also asked to complete a questionnaire to provide their feedback on the adoption of the App.In the App group, patients received instructions on how to download the App from the App Store of their own mobile device. The LATITUDE™ communicator was given to all patients before discharge or directly delivered to their homes, according to the standard practice of the center. Patients were invited to follow the indications provided by the App in order to activate the communicator and perform the first transmission. If they had any difficulties, patients could contact the center or the Boston Scientific customer support service. After activation, patients were instructed to follow the indications provided by the App in order to solve possible connection problems in the event of notification of “Not Monitored” status or missed scheduled remote transmission. The group of patients already monitored at home through the LATITUDE™ platform and invited to use the App were included in the analysis of the compliance with remote monitoring. An additional group of 60 consecutive patients served as control group and received standard in-clinic training on the activation and use of the communicator, as well as the patients who did not install the App. In November, the monitoring status of all patients with a LATITUDE™ communicator was checked. Patients in “Monitored” status were considered compliant with remote monitoring at medium-term follow-up. All patients were asked to reply to an The App enables patients to independently obtain instructions and information on their communicator and CIED. It works with all Boston Scientific CIEDs that are monitored on the LATITUDE™ NXT platform and can be used by patients and/or their caregivers Fig. . The Appt-test for Gaussian variables and Mann-Whitney non-parametric test for non-Gaussian variables. Differences in proportions were compared by means of a chi-square analysis. A p-value <0.05 was considered significant for all tests. All statistical analyses were performed by means of R: a language and environment for statistical computing .Descriptive statistics are reported as means ± standard deviation for normally distributed continuous variables or medians with 25th to 75th percentiles in the case of skewed distribution. Categorical data were expressed as percentages. Differences between mean data were compared by means of a The MyLATITUDE™ Patient App was proposed to patients with a compatible CIED at 14 Italian arrhythmia centers. At 11 (79%) centers, the professionals routinely involved in remote device management were nurses and physicians, in accordance with a “Primary Nursing” model. In the remaining 3 (21%) centers, they were physicians alone. The communicator was delivered by nurses at 11 (79%) centers and by physicians at 3 (21%) centers. At 6 (43%) centers, home delivery of the communicator was allowed if preferred by the patient. The professionals involved in training patients in the activation and use of the communicator were nurses at 9 (64%) centers and physicians at 5 (36%) centers.The App was proposed to 242 patients: 81 consecutive patients immediately after CIED implantation and 161 patients who were already being monitored at home through the LATITUDE™ platform and who were consecutively enrolled at the time of an in-clinic visit. The App was successfully installed by 177 (73%) patients; 49 (20%) declined, 12 (5%) did not have a suitable mobile phone, and 4 (2%) experienced technical problems that precluded LATITUDE™ communicator activation (lack of landline and mobile telephone coverage). Baseline parameters of the study population are reported in Table The App was installed by 127 (72%) patients, by 39 (22%) caregivers, and by patient and caregiver in 11 (6%) cases. Installation problems that did not preclude final activation were reported by 15 (8%) patients. In 12 cases, troubleshooting required telephone support.Forty-eight patients successfully followed the indications provided by the App on how to activate the communicator and perform the first transmission. Standard post-implantation in-clinic training was received by the 33 new RM activation patients who did not install the App and by another 60 consecutive patients. Of these latter, one 2%) patient experienced technical problems that precluded communicator activation. The duration of in-clinic training was < 30 min for all patients. The App-guided installation group and the control groups (standard in-clinic training group and App-decliners) were similar in terms of the time required for activation and the need for additional support (Table % patientp=0.033). In particular, 48 (100%) of 48 patients who installed the App immediately after CIED implantation and 54 (92%) of 59 patients of the standard in-clinic training group (excluding App-decliners) were in “Monitored” status at the end of the observation (p=0.063).Of the 177 patients who installed the App, 20 (11%) received notifications of lack of connection during follow-up and 22 (12%) information of missed scheduled transmissions. The median time between notification of “Not monitored” status and resolution was 2 days (<1 week in 90%). After 3 months, 175 (99%) of these 177 communicators were in “Monitored” status. Of the 120 patients without the App installed, 113 (94%) were in “Monitored” status at the end of the observation . This finding was obtained in the overall group of patients who installed the App and confirmed, although with no statistical significance , comparing patients who installed the App immediately after CIED implantation and patients of the standard in-clinic training group. This finding may have clinical implications. Indeed, previous analyses revealed not only better survival of patients enrolled in RM programs [HR 0.80, 95% CI 0.76–0.84) [In patients who enabled the App, we recorded notifications of missed transmissions or lack of connection during follow-up. After notification, problems of connection were promptly solved by the patients. Consequently, these patients were able to remain connected with greater continuity than a similar group of patients who did not use the App, with a very high proportion of communicators regularly transmitting data at 3 months (99% versus 94%, programs , but alsBarriers to the widespread use of RM can arise at the time of program implementation in the center and also owing to difficulties in activating RM and keeping patients monitored. While the implementation depends largely on the local practice environment and the institution, RM activation and transmission are more dependent on patient factors . The adoThe remote management of CIEDs has been proposed as a strategy to improve the efficiency of device follow-up by replacing in-office device follow-up visits with remote transmissions –14. MoreWe recently implemented direct home delivery of the communicator and remote training of patients during the COVID-19 lockdown period in Italy . This stOur findings have potential limitations. This was an observational non-randomized study. Some analyses were performed with patients self-selecting whether to be in the intervention or control group. Thus, a bias could affect our findings. As the project was limited to a single RM platform, our results may not be applicable to other systems. Moreover, we cannot exclude possible differences in the implementation of the initiative among centers, with an impact on the degree of success.The MyLATITUDE™ Patient App proved to be well accepted by CIED patients. It offered support for communicator installation and management and reassured patients. Its use allowed patients to remain connected with greater continuity during follow-up.