No treatment effects were detected.•Lying behavior improved with days of exposure to freestalls.•Some abnormal behavior  persisted. Dairy cattle can experience problems adjusting to freestalls when first introduced, resulting in a marked reduction in lying time and increased abnormal behaviors such as lying in the alley or lying backward in a stall. Our objective was to evaluate 2 strategies to ease the transition of heifers to freestalls: using an older, experienced heifer as a social model [experiment (Exp.) 1] or using brushes mounted in the stalls as an attractant (Exp. 2). In Exp. 1, 44 naïve heifers  were assigned in pairs (n = 11 pairs/treatment) to either the control or social model  treatments. In Exp. 2, 52 naïve heifers (146 ± 9 d of age) were preassigned in pairs (n = 13 pairs/treatment) to either control or brush treatments. In both studies, heifers were initially housed in pens with a bedded pack  before moving to pens in the same barn with sand-bedded freestalls. On d −2, 0, and 4 relative to the move to the freestalls, standing, perching , and lying  were recorded at 5-min intervals. In addition, starting on d 0 relative to the move, latency to first lie down in a stall was recorded continuously. In both studies, time budgets did not differ between treatments. On the day heifers were moved, they spent less time lying relative to d −2  and more time standing and perching. By d 4, lying and standing returned to d −2 baseline levels, although heifers continued to spend more time perching. When heifers moved to freestalls, no treatment differences were observed in the latency to lie down in a stall, but latencies differed between Exp. 1 (averaging approximately 3.8 h across treatments) and Exp. 2 (averaging 31.4 h across treatments). After moving to freestalls, the percentage of total lying time that heifers spent facing forward in a stall increased  or tended to  between d 0 and 4 of exposure, with no effect of treatment. Although lying behavior improved with days of exposure to freestalls, some heifers continued to lie down in the alley or backward in a stall, suggesting the need for additional work to ease the adjustment to freestall housing. Approximately 40, 30, and 13% of US dairy farms use freestalls as the main housing type for their lactating cows, dry cows, and weaned heifers, respectively . In thisAlthough freestalls have benefits, naïve cattle can experience problems in initially adjusting to them, resulting in a marked reduction in lying time when first introduced  and undeLittle research has examined the adjustment of dairy cattle to freestall housing or how this might be improved. One study attempted to make stalls more attractive by covering the stall base with mats and by providing feed in the stalls . Dairy cExp.) 1, we assessed the use of an older, experienced animal as a social model, and in Exp. 2, we assessed the use of stationary brushes mounted inside the stalls as attractants. In both experiments, the objectives were to evaluate effects on stall use and time budgets before and after naïve heifers moved to freestall housing. We hypothesized that the presence of older, experienced heifers and brushes in the stalls would both increase stall use.In the current study, we assessed 2 interventions: in experiment (The experiments were conducted between June and November 2016 (Exp. 1) and June and October 2017 (Exp. 2) at The University of British Columbia (UBC) Dairy Education and Research Centre . All procedures were approved by the UBC Animal Care Committee (protocol A14-0245). The sample sizes in both studies were based on a power analysis using the changes in lying time reported in In Exp. 1, 44 naïve Holstein heifers were enrolled at (mean ± SD) 129 ± 37 d of age. Eleven older heifers (200 ± 24 d of age) served as experienced social companions. These older heifers were selected based on 3 criteria: (1) they had lived in freestall housing for >2 mo, (2) they were 1 to 3 mo older than the naïve heifers, and (3) they were not observed lying in the alley, lying backward in a stall, or displacing other heifers from stalls during 72 h of continuous video observation in freestall housing. Pairs of naïve heifers were assigned pseudo-randomly to either the social model or control treatment (n = 11 pairs/treatment), balancing for age . In the social model treatment, each pair of naïve heifers was housed with 1 older, experienced heifer. In the control treatment, the pair of naïve heifers were the only animals in the pen. To maintain the same number of naïve heifers per treatment, the total number of animals per pen differed. Nonetheless, this confounder meant that in the second phase of the study, fewer stalls would be available per animal in the social model treatment, thus reducing the potential to bias the results in favor of our hypothesis.In the first phase of the study, heifers were housed in bedded-pack experimental pens for 5 d beginning at 0900 h, when naïve heifers in the social model treatment were first introduced to the older, experienced heifers. Two adjacent, mirror-image pens were used, with treatments balanced between the pens. The pens were enclosed on 3 sides with 1.8-m-high plywood to prevent visual and physical contact between treatments. The 4.6- × 7.1-m lying area was bedded with 12 cm of sawdust and separated from the feeding area by a 4.8- × 3.1-m concrete alley covered with texturized rubber. The feed barrier had nonmoving bars slanted at a 60° angle (13 feeding spaces with a center-to-center distance of 0.3 m between bars). Heifers had ad libitum access to long-stemmed fescue grass hay top-dressed with texturized calf grower (Hi-Pro Feeds Ltd.). The previous day's refusals were removed and fresh feed delivered at approximately 0900 h, and the feed was pushed up at approximately 1500 and 2200 h. Heifers had ad libitum access to fresh drinking water from a self-filling trough on a raised platform accessible from the alley. The feed alley was cleaned by an automatic manure scraper 6 times/d, and the lying area was cleaned manually during feeding at 0900 h. Fresh sawdust bedding was added between groups.In the second phase of the study, on the sixth day (d 0 relative to the move) between 0900 and 1100 h, each group of heifers was moved to 1 of 2 adjacent freestall pens within the same barn, enclosed on 3 sides with plywood. No other nonexperimental heifers were housed in these pens. Until this point, the naïve heifers had no exposure to freestalls or feed barriers with self-locking headlocks. Each 6.7- × 12.2-m pen had 13 freestalls in 3 rows of 4, 4, and 5 stalls (from the feed bunk to the back of the pen); the back 2 rows were separated by a 6.7- × 3.1-m alley. Adjacent to the first row of freestalls was a 1.3- × 1.9-m raised crossover alley. The freestalls were deep bedded with sand and measured 1.8 m long, with 0.9 m center-to-center distance between freestall loops, and had 2 neck rails . The 6.7- × 3.1-m feed alley had 13 feeding spaces with headlocks (0.4 m center-to-center distance). Heifers were fed the same diet on the same schedule as in the bedded-pack phase. Water was available ad libitum from an automatically filled trough on a raised platform adjacent to the stalls. All alleys were covered with texturized rubber. The pens were scraped automatically 6 times/d, and the stalls were raked manually once daily during feeding at 0900 h. Heifers were removed from the experimental pen on d 5, relative to the move to freestalls. Between groups, the stalls were topped off with fresh sand and raked.In both phases of the study, behavior was recorded using 2 video cameras/pen (Panasonic WV-CP214 24V) mounted 8 m above each pen along with a 100-W red light to aid nighttime visibility. One observer (G.N.) scored behavior in Excel (Microsoft Corp.) spreadsheets for all heifers at 5-min intervals on d −2, 0, and 4 relative to the move to the freestall pens. The behaviors recorded were posture  and eating . Within lying, the location  was recorded, and on d 0 and 4 in the freestall pens, the direction heifers were facing (forward vs. backward) when lying in a stall was also recorded. In addition, the same observer watched video continuously when heifers moved to the freestall pens to record their latencies to first lie down anywhere in the pen , first lie down in a stall , and to first eat with their heads through the headlocks.In Exp. 2, 52 naïve heifers were enrolled at a mean (± SD) of 146 (± 9) d of age. In the first phase of the study, heifers were housed in groups of 4 (n = 13 groups) for 7 d in a single bedded-pack pen, enclosed on 3 sides with plywood. Groups entered the pen sequentially based on birthdate, which kept age relatively consistent among groups. Housing and management were identical to the first phase of Exp. 1, with 2 exceptions: (1) for the requirements of another study , the lyiOn the eighth day (d 0 relative to the move) at 1030 h, each group of heifers was divided into preassigned treatment pairs and moved to 1 of 2 freestall pens on opposite sides of a 6.3-m-wide center alley within the same barn. Until this point, all heifers were naïve to freestalls and feed bunks with self-locking headlocks. Pairs of heifers had been preassigned pseudo-randomly to either the brush or control treatment (n = 13 pairs/treatment) before phase 1 of the study, balancing for pen location and age . In the brush treatment, each freestall had a brush mounted horizontally on the rail 0.2 m above the stall base in the front of the stall. In the control treatment, the pen contained no brushes. Each experimental pen had access to only the first row of 4 freestalls facing away from the feeding area. The back of the crossover alley adjacent to the freestalls was blocked with a gate to prevent heifers from accessing the rest of the pen, which remained empty. Heifers were fed a TMR, available ad libitum and delivered and pushed up on the same schedule as in the bedded-pack phase. The pens were scraped automatically 6 times/d, and the stalls were raked and the area around the water trough was scraped manually during feeding at 0900 h. Pairs of heifers were removed from the pen at approximately 1100 h on d 6, relative to the move to the freestalls. Between pairs, the stalls were topped off with fresh sand and raked. A video camera and a red light were mounted 8 m above each pen. One observer (J.V.) scored behavior for all heifers in 5-min intervals on d −2, 0, and 4, relative to the move to the freestall pens. The behaviors were the same as in Exp. 1, except that eating was not recorded. The same observer watched video continuously when heifers moved to the freestall pens, recording latencies as in Exp. 1, with the exception of feeding behavior, and with the addition of the latency for heifers to first enter a stall with at least 1 hoof.P < 0.05 and tendencies at P < 0.1. Linear mixed models were constructed using PROC MIXED. The assumption of equal variance between treatments was assessed graphically using boxplots (PROC UNIVARIATE). The assumption of normality was evaluated by plotting residuals (PROC PLOT), and when data were not normally distributed , natural log transformations were applied , enter a stall with at least one hoof (Exp. 2 only), lie down anywhere in the pen, and lie down in a stall were evaluated using mixed models with a fixed term for treatment  and a random term for pair of heifers, nested within treatment. We predicted that heifers in the social model (Exp. 1) and brush (Exp. 2) treatments would feed through the headlocks (Exp. 1), enter a stall (Exp. 2), and lie down in a stall (both studies) sooner than those in the control treatments. In addition, the proportion of heifers choosing a stall  as their first lying location and the proportion facing forward (vs. backward) the first time they lay down in a stall are reported descriptively.Not all heifers were observed performing undesirable lying behaviors. Descriptive data for lying in the alley  and lying in a stall backward (d 0 and 4) are reported, along with the proportion of heifers showing only desirable lying behaviors . The percentage of lying time spent lying in a stall facing forward was calculated on each day and evaluated using a mixed model with fixed terms for treatment , day (0 and 4), and the treatment × day interaction, with a random term for pair of heifers, nested within treatment. We predicted that heifers in the social model (Exp. 1) and brush (Exp. 2) treatments would spend a greater percentage of their total lying time in a stall facing forward relative to those in the control treatments, and that this percentage would increase with day of exposure to the freestalls.Finally, overall time budgets were evaluated. In Exp. 1, the amounts of time that inexperienced heifers spent lying, standing, eating (a subset of standing), and perching were evaluated using mixed models with fixed terms for treatment , day , and treatment × day interaction, with a random term for pair of heifers, nested within treatment. In Exp. 2, the amounts of time heifers spent lying, standing, and perching were evaluated using mixed models with fixed terms for treatment (brush vs. control), day , and the treatment × day interaction, with a random term for pair of heifers, nested within treatment. For both studies, where significant effects of day were found, contrasts between days were conducted using the PDIFF function. We predicted that lying and eating time (Exp. 1 only) would be reduced on d 0 relative to d −2 and 4 but that heifers in the social model and brush treatments would show less reduction relative to those in the control treatments.F1,20 ≤ 0.6, P ≥ 0.45). Most heifers chose a stall  for their first lying location , and 86 versus 91% of heifers faced forward when they first lay down in a stall.In Exp. 1, before moving to the freestalls (d −2), few naïve heifers were observed lying in the bedded-pack pen alleys . Upon moving, there was no effect of treatment on the latency to first feed through the headlocks, lie down anywhere in the pen, or lie down in a stall , with 50 versus 64% observed lying in the alley and 36 versus 32% lying backward in a stall. By d 4, 82 versus 55% of heifers in the social model versus control treatments showed only desirable lying behavior, with only 9 versus 23% lying in the alley and 14 versus 27% lying backward in a stall.F1,20 ≤ 2.1; P ≥ 0.16). There was a tendency for a day effect: the percentage of lying time facing forward increased from d 0 to 4 . This difference was driven by a reduction in time spent lying in the alley; heifers in both treatments spent 1.2 versus 0.3 h (SE: 0.3 h) lying in the alley on d 0 versus 4. When lying in a stall, heifers in both treatments were observed lying backward for 0.7 versus 1.0 h (SE: 0.3 h) on d 0 versus 4.Heifers spent most of their total lying time facing forward in a stall, regardless of treatment , but there were effects of day  and eating (P < 0.020) on the day they moved to freestalls (d 0) relative to d −2 or d 4; by d 4, durations for both behaviors were equivalent to baseline values on d −2 (P ≥ 0.49). On d 0, heifers spent more time perching and standing than on d −2 or 4 (P < 0.001); on d 4, durations of perching remained greater than on d −2 (P = 0.011), whereas standing was equivalent between d −2 and 4 (P = 0.11).There was no effect of treatment on time budgets ; one of these heifers spent 0.2 h lying backward in a stall on d 4. No lying in the alley was observed on any day. The older, experienced heifers spent 13.7, 13.8, and 14.1 h lying on d −2, 0, and 4 (SE: 0.5 h/d). They spent 0.0, 0.4, and 0.3 h perching (SE: 0.1 h/d); 10.3, 9.8, and 9.6 h standing (SE: 0.5 h/d); and 6.0, 4.9, and 5.6 h eating  on those days.F1,24 ≤ 1.3, P ≥ 0.26). Few heifers chose a stall  for their first lying location (15 vs. 31% in the brush vs. control treatment) or showed only desirable lying behavior on d 0 (4 vs. 22%); 92 versus 78% were observed lying in the alley, and 13% in each treatment were observed lying backward in a stall.In Exp. 2, before moving to the freestalls (d −2), only one heifer (assigned to the control treatment) was observed lying in the alley of the bedded-pack pen (for 0.6 h). Upon moving heifers to the freestall pen, there were no treatment differences in latency to first enter a stall with at least one hoof or lie down anywhere in the pen . When heifers first lay down in a stall, the majority faced forward (90 vs. 85%). By d 4, 69 versus 54% of heifers in the brush versus control treatment showed only desirable lying behavior, with 23 versus 42% lying in the alley and only 8 versus 12% lying backward in a stall. Heifers spent most of their lying time facing forward in a stall, regardless of treatment . There was a day effect: the percentage of lying time facing forward in a stall increased from d 0 to 4 . This difference was driven by a reduction in time spent lying in the alley; heifers in both treatments spent 4.6 versus 2.6 h (SE: 0.8 h) lying in the alley on d 0 versus 4. When lying in a stall, heifers in both treatments spent on average 0.4 h (SE: 0.2 h) lying backward on d 0 and 4.On average, heifers took more than 1 d to first lie down in a stall, regardless of treatment , but behaviors varied with day . By d 4 in the freestalls, both lying and standing returned to d −2 baseline levels (P ≥ 0.11), although heifers continued to spend more time perching on d 4 versus d −2 (P < 0.001).There was no effect of treatment on time budgets  when heifers moved to freestalls. In Exp. 1, the naïve heifers became familiar with the social models before moving to the freestalls. However, we did not quantify lying proximity during either phase of the study; future work could examine the extent to which younger heifers avoid or choose to lie near older heifers. In Exp. 2, although the heifers used the brushes, they did so for only 4 min, on average, on the day they moved to the freestalls . The comInteresting descriptive similarities and differences emerged between the 2 experiments. In both, the latency for heifers to first lie down anywhere in the freestall pen was similar (approximately 2–3 h), but there was a marked difference in the latency to first lie down in a stall . Similarly, over two-thirds of heifers in Exp. 1 chose a stall as their first lying location, but less than one-third did so in Exp. 2. These differences cannot be easily explained, as the studies were conducted in the same research facility, in a similar season, with inexperienced heifers around the same age (4–5 mo). Stocking density was lower in Exp. 1, in which heifers had access to all 13 stalls in the pen  versus only 4 stalls  in Exp. 2. However, the implication of this difference is unclear because heifers in both studies had access to more than one stall, whereas ≤1 stall is provided per animal on typical commercial dairy farms.In both experiments, lying behavior improved with days of exposure to the freestalls, as characterized by lying duration and the percentage of lying time spent facing forward in the stall. In 2 previous studies, heifers of a similar age showed a marked reduction in lying time when first introduced to freestalls . On the day heifers moved to freestalls, more than half in both Exp. 1 and 2 were observed lying in the alley; by d 4 a minority of heifers were observed still doing so. Previous work reported an increase in lying in the alley when cattle were initially moved to freestall housing . This pr


With the recent global spread of new SARS-CoV-2 variants, there remains an urgent need to develop effective and variant-resistant oral drugs. Recently, we reported in vitro results validating the use of combination drugs targeting both the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and proofreading exonuclease (ExoN) as potential COVID-19 therapeutics. For the nucleotide analogues to be efficient SARS-CoV-2 inhibitors, two properties are required: efficient incorporation by RdRp and substantial resistance to excision by ExoN. Here, we have selected and evaluated nucleotide analogues with a variety of structural features for resistance to ExoN removal when they are attached at the 3′ RNA terminus. We found that dideoxynucleotides and other nucleotides lacking both 2′- and 3′-OH groups were most resistant to ExoN excision, whereas those possessing both 2′- and 3′-OH groups were efficiently removed. We also found that the 3′-OH group in the nucleotide analogues was more critical than the 2′-OH for excision by ExoN. Since the functionally important sequences in Nsp14/10 are highly conserved among all SARS-CoV-2 variants, these identified structural features of nucleotide analogues offer invaluable insights for designing effective RdRp inhibitors that can be simultaneously efficiently incorporated by the RdRp and substantially resist ExoN excision. Such newly developed RdRp terminators would be good candidates to evaluate their ability to inhibit SARS-CoV-2 in cell culture and animal models, perhaps combined with additional exonuclease inhibitors to increase their overall effectiveness.  SARS-CoV-2, the causative agent of COVID-19, is a member of the Nidovirales order of positive-strand RNA viruses ; SARS-Co′-5′ exonuclease (ExoN), consisting of Nsp14 and Nsp10, involved in proofreading and error repair [Because of the large genome size of the coronaviruses (>30 kb), their relatively low fidelity RNA-dependent RNA polymerase (RdRp) would tend to produce a high number of errors during RNA replication and transcription ; the resr repair ,16,17, ar repair . The RdRThe complete structure of the RTC , as well as that of the individual components of the complex, has been resolved by cryo-electron microscopy and X-ray crystallography in order to determine which sites in these proteins are critical for protein-protein interactions or catalytic activity. The structures determined include the replicating RdRp complex in the absence and presence of RNA and specific nucleotide inhibitors ,22,23,24The Nsp14 protein contains an ExoN functional domain that provides replication and transcription error correction, allowing SARS-CoV-2 to maintain its large-sized genome ,32,33,34Recently, various assays have been described to measure the SARS-CoV-2 ExoN activity ,15,39,40Drugs that are based on nucleoside and nucleotide analogues that inhibit the coronavirus RdRp  and ExoN (Nsp14 and Nsp10) fall into multiple categories with respect to their modes of action. Several active forms of nucleoside/nucleotide analogues can be incorporated into the viral RNA by the RdRp where they terminate further incorporation or they lead to mutations in the RNA, either of which may impede various aspects of the viral life cycle ,45,46,47Previously, we demonstrated that the triphosphate forms of numerous nucleotide analogues, including FDA-approved drugs for other viral infections, are capable of being incorporated into RNA by the SARS-CoV-2 RdRp complex and of terminating RNA extension with varying efficiency, in either immediate or delayed fashion ,47. MinsIn this paper, based on published sequence information, we first assess the conservation of protein sequences within the exonuclease functional sites of Nsp14 and Nsp10 in SARS-CoV-2 variants . The hig′-NH2-dUTP, Cordycepin-TP, ddATP, ddCTP, ddGTP, Biotin-16-dUTP), Invitrogen (Biotin-16-UTP), Santa Cruz Biotechnology (Stavudine-TP), Amersham Life Sciences (Zidovudine-TP) or Alfa Chemistry (Tenofovir-DP). RNA oligonucleotides (template-loop-primers) were purchased from Dharmacon. HIV reverse transcriptase, SuperScript IV reverse transcriptase and T7 RNA polymerase were purchased from Millipore Sigma, Thermo Fisher and New England BioLabs, respectively. The expression and purification of the SARS-CoV-2 exonuclease Nsp14/Nsp10 complex have been described in our previous study [Nucleoside triphosphate analogues were purchased from TriLink BioTechnologies  were annealed by heating to 75 °C for 3 min and cooling to room temperature in 1 × HIV reverse transcriptase (RT) or 1 × SuperScript IV RT reaction buffer. Then, 10 μL of the appropriate annealed RNA template-loop-primer solution (10 μM) was added to 8 μL of the reverse transcriptase solution consisting of 54.4 U of HIV RT or 200 U of SuperScript IV RT in 1 × appropriate buffer. Finally, 2 μL of a solution containing 10 mM Biotin-16-dUTP, Stavudine-TP, Cidofovir-DP, Tenofovir-DP, Zidovudine-TP, ddATP, ddCTP or ddGTP was added and incubation was carried out for 4 h at 45 °C. The 20 μL extension reactions contained 54.4 U HIV RT or 200 U SuperScript IV RT, 5 μM RNA template-loop-primer, and 1 mM Biotin-16-dUTP, Stavudine-TP, Cidofovir-DP, Tenfovir-DP, Zidovudine-TP, ddATP, ddCTP or ddGTP. The 1 × HIV RT reaction buffer contained 10 mM Tris-HCl pH 8, 10 mM KCl, 2 mM MgCl2 and 1 mM β-mercaptoethanol. Desalting of the reaction mixture was performed with an Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 μL purified aqueous RNA solutions. Then, 1 μL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 μL RNA extension products were used to test exonuclease activity.The RNA template-loop-primers  were annealed by heating to 75 °C for 3 min and cooling to room temperature in 1 × RNA Pol buffer. Then, 10 μL of the appropriate annealed RNA template-loop-primer solution (10 μM) was added to 8 μL of an RNA polymerase solution consisting of 400 U of T7 RNA polymerase in 1 × RNA Pol buffer. Finally, 2 μL of a solution containing 10 mM of 2′-NH2-dUTP, Biotin-16-UTP or Cordycepin-TP was added and incubated for 4 h at 37 °C. The 20 μL extension reactions contained 400 U T7 RNA polymerase, 5 μM RNA template-loop-primer, and 1 mM 2′-NH2-dUTP, Biotin-16-UTP or Cordycepin-TP. The 1 × RNA polymerase reaction buffer contains 40 mM Tris-HCl, 6 mM MgCl2, 1 mM DTT and 2 mM spermidine. Desalting of the reaction mixture was performed with an Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 μL purified aqueous RNA solutions. Then, 1 μL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 μL RNA extension products were used to test exonuclease activity.The RNA template-loop-primers , 10 μL of exonuclease Nsp14/10 complex (100 nM) in 1 × exonuclease reaction buffer was added and incubated at 37 °C for 5, 10 or 15 min. The final concentrations of reagents in the 20 μL reactions were 50 nM Nsp14/10 and 500 nM different RNAs. The 1 × exonulease reaction buffer contains 40 mM Tris-HCl pH 8, 1.5 mM MgCl2, and 5 mM DTT. After incubation, each reaction was quenched by the addition of 2.2 μL of an aqueous solution of EDTA (100 mM). Following desalting using an Oligo Clean & Concentrator (Zymo Research), the samples were subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis.The synthetic RNA template-loop-primers with A, U, G, C, dC, 2With more than 5 million genomes of SARS-CoV-2 sequenced, naturally occurring mutations have been analyzed in a number of strains, including the most recent variants of concern to public health ,58,59. OThe majority of genomic sequence variations observed in SARS-CoV-2 variants are located within genes encoding Spike, structural and accessory proteins, while the members of the ExoN complex, Nsp14 and Nsp10 genes, accumulate fewer nucleotide changes, most of which are synonymous (not causing changes in the encoded amino acids). When recent variants of SARS-CoV-2 are compared to the original 2019 strain, very few mutations in the replication component, including Nsp14 and Nsp10 proteins, became fixed in the population. Within the portion of Nsp14 that spans the Nsp10-binding site and ExoN domain b, the onIn the relatively short and conservative Nsp10 protein of SARS-CoV-2, naturally occurring mutations are rare . ArtificBased on the conservative nature of components of the ExoN complex, we predict that any inhibitor for this enzymatic function should have broad-spectrum inhibitory potential for most current and possibly future strains of SARS-CoV-2.In our previous study, we reported that Tenofovir-terminated RNA showed high resistance toward excision by the SARS-CoV-2 ExoN compared to RNA terminated with Remdesivir, Molnupiravir, Sofosbuvir or Favipiravir at the 3′ end . We also′-OH, lack of the 3′-OH, or lack of both 2′- and 3′-OH, as well as base-modified nucleotides. The nucleotide analogues lacking the 2′-OH group include 2′-deoxycytosine-5′-triphosphate (2′-dCTP), 2′-fluoro-2′-deoxycytosine-5′-triphosphate (2′-F-dCTP), 2′-O-methylcytosine-5′-triphosphate (2′-OMe-CTP) and 2′-amino-2′-deoxyuridine-5′-triphosphate (2′-NH2-2′-dUTP). Two nucleotide analogues lacking a 3′-OH group, Cordycepin-5′-triphosphate (3′-dATP) and the acyclic nucleotide analogue Cidofovir diphosphate (Cid-DP), were evaluated. We also investigated nucleotide analogues lacking both 2′- and 3′-OH groups: the dideoxynucleoside-5′-triphosphates , a ddUTP derivative , Zidovudine-5′-triphosphate (AZT-TP), as well as another acyclic nucleotide analogue, Tenofovir diphosphate (Tfv-DP). Two nucleotides with modifications at the 5-position of uridine were investigated, biotin-16-aminoallyl-2′-deoxyuridine-5′-triphosphate (Biotin-16-dUTP) and biotin-16-aminoallyl-uridine-5′-triphosphate (Biotin-16-UTP). Cordycepin (3′-deoxyadenosine) has been shown to have biological activities in a wide variety of disease processes [′ ends to perform the following exonuclease assays.We focus on nucleotides and nucleotide analogues with sugar modifications, such as lack of the 2rocesses  but disprocesses . We syntAs shown in A previous study suggested that 3′-deoxynucleotide analogues could potentially resist exonuclease excision . Moreove2-2′-dUTP, Zidovudine-TP, Stavudine-TP, Biotin-16-UTP and Biotin-16-dUTP are shown in 2-2′-dU-terminated RNA is eliminated, with mainly fragments of 18–21 nucleotides remaining  provide low resistance to ExoN, 3′-deoxynucleotide analogues (3′-dN and Cidofovir) at the 3′ position of the RNA display moderate ExoN resistance. Most importantly, appreciable resistance to ExoN was observed for ddN-, 3′-N3-2′-dN-, Tenofovir- and Stavudine-terminated RNAs. These results also indicate that the polarity of the modification group on the sugar ring of the nucleotide analogue attached at the 3′ end of the RNA may have an effect on exonuclease cleavage. Nucleotide analogues containing the 2′- and 3′-OH (polar groups) show no resistance towards excision. However, increasing the hydrophobicity at both the 2′ and 3′ positions of the sugar ring  results in the highest resistance to ExoN excision from RNA. The above results indicate that the structure of the nucleotide analogue at the 3′ terminus of RNA plays an essential role in its excision by the SARS-CoV-2 exonuclease complex. In The nucleotide analogues displaying higher resistance to exonuclease excision may help guide the further development of nucleotide analogues with particular structural features for potential coronavirus therapeutics. The ATP analogue Tenofovir-DP, the UTP analogues Stavudine-TP and AZT-TP are obligate terminators of the RdRp extension reaction though less efficiently incorporated than their corresponding natural nucleotides ,47. As d


Escherichia coli populations. Quantification of tRNAs suffers additional biases due to differing fractions of tRNAs with base modifications in growing versus starved bacteria. Applying an improved methodology, we measured tRNA levels after starvation for amino acids, glucose, phosphate, or ammonium and transcription inhibition by rifampicin. We report that tRNA levels remain largely unaffected in all tested conditions, including several days of starvation. This confirms that tRNAs are remarkably stable RNAs and serves as a cautionary tale about quantification of RNA from cells cultured outside the steady-state growth regime. rRNA, conversely, is extensively degraded during starvation. Thus, E. coli downregulates the translation machinery in response to starvation by reducing the ribosome pool through rRNA degradation, while a high concentration of tRNAs available to supply amino acids to the remaining ribosomes is maintained.tRNAs and ribosomal RNAs are often considered stable RNAs. In contrast to this view, we recently proposed that tRNAs are degraded during amino acid starvation and drug-induced transcription inhibition. However, reevaluation of our experimental approach revealed that common RNA extraction methods suffer from alarming extraction and size biases that can lead to gross underestimation of RNA levels in starved  Escherichia coli responses to nutrient starvation are manifold, involving redirected enzyme activities and physiological changes such as reduced cell size, altered membrane composition, and increased thickness of the peptidoglycan layer  using [14C]-uracil. Total RNA was extracted from samples harvested before and after isoleucine starvation. For normalization, we spiked all [14C]-labeled cell samples with a small volume of unstarved cells cultured in the presence of [3H]-uridine before extraction. The tested extraction methods were (i) cold phenol extraction, which was originally developed as a mild extraction procedure that maintains the aminoacyl charge on tRNAs (In order to accurately quantitate the relative level of tRNAs (and any RNA) in changing growth conditions, it is crucial that RNA extraction fulfils a number of criteria. First, it must yield total RNA of sufficient quantity and quality for the chosen downstream analysis. Second, the efficiency of RNA extraction must be independent of physiological changes to the cells that occur in response to the change of growth conditions. And third, the relative extraction efficiency of individual RNA species from the same sample must be independent of the state of the culture. To test if RNA extraction methods fulfil these criteria, we conducted a thorough assessment of three commonly used extraction protocols  hosence of H-uridine+) using C-uracil.14C]-uracil in extracted RNA relative to the total incorporated, trichloroacetic acid (TCA)-precipitable [14C]-uracil in RNA from the cultures. The [14C]-TCA-precipitable material is a measure of pyrimidines found in RNA and DNA polymers longer than ~16 nucleotides and can therefore be used as an estimate of total RNA when the alkali-stable DNA component is subtracted , analyzing the overall size distribution, and quantifying changes in extraction efficiency of selected RNA species. As shown in 14C]/[3H]) of distinct bands on the PAGE gels, we cut out the bands of interest for analysis in a scintillation counter . The tRNA bands were analyzed as a whole, and in samples prepared by hot phenol extraction, the tRNA fraction showed an increase to nearly 110% after 80 min of starvation uridine (acp3U) modification in the complementary region of the anticodon targeting probes  due to pausing or stopping of reverse transcriptase at modified nucleotides  after arginine starvation  to approximately 2.4-fold  after 160 min of starvation. Consistent with this, treatment of cells with chloramphenicol, a ribosome-targeting antibiotic that blocks translation and is known to stimulate rRNA and tRNA synthesis by suppression of (p)ppGpp synthesis .Having confirmed that tRNA levels are not reduced during amino acid starvation, we sought to test tRNA stability under other conditions that limit protein synthesis. The half-lives of ribosomal RNAs 16S and 23S are reduced upon starvation for a variety of nutrients, including isoleucine, glucose, phosphate, and nitrogen , 27, 28.de novo RNA synthesis by transcription inhibition would result in a decrease of the existing pool of tRNAs. However, as shown in tyrTUV levels appeared to increase to 1.3-fold relative to the level prior to addition of rifampicin  is inactivated rather than degraded  remained stable during early isoleucine starvation  and G. Idegraded , althougdegraded , 34. TheE. coli cultures in balanced growth  in length and thus only slightly larger than some tRNAs  was strongly reduced after starvation, it cannot be excluded that TRI Reagent extraction of individual, larger tRNAs would be sensitive to the growth state of the cultures. Based on these results, we concluded that the methods used in our previous work did not reliably reflect the actual distribution of RNAs in the cell, and importantly, were strongly affected by starvation. Hot phenol extraction, on the other hand, resulted in an increased overall yield  are degraded, while short RNAs (5S and tRNAs) are not.Prolonged nutrient starvation has repeatedly been shown to lead to extensive degradation of ribosomes and rRNA , 27, 30 gltTUVW consistently reached the highest level after prolonged starvation for various nutrients. While it is generally assumed that tRNA and rRNA synthesis is coregulated, tRNAgltTUVW has been shown to be transcribed during the stationary phase from an internal promoter of the rrn operons containing gltTUVW  (ΔargG ΔleuA ΔpyrE::tetR) was used. SAA21 (MAS1190 ΔrelA::kanR) was generated by P1 transduction using CF1651 (ΔrelA251::kanR) (selc) that produces high amounts of the rare tRNAselC upon IPTG  induction was used as the spike-in strain for normalization of tRNA quantification.The bacterial strains used in this study are listed in +glpR+)  served a1::kanR)  as the d436). Starvation and antibiotic treatment were routinely performed when cultures reached an OD436 of 0.5 to 0.85.For cloning and recombination, cells were grown in yeast extract-tryptone (YT) medium at 37°C with shaking. For all physiological experiments, cells were cultured in MOPS (3-morpholinopropane-1-sulfonic acid) minimal medium  suppleme436 of approximately 0.1 was supplemented with [2-14C]-uracil  to a final concentration of 1 μCi/mL (18.9 μM), and incubation continued until an OD436 of approximately 0.4 was reached, at which point unlabeled uracil (1 mg/mL stock solution) was added to a final concentration of 20 μg/mL (178 μM). At an OD436 of approximately 0.8 three steady-state samples (st-st 1 to 3) were taken. Immediately thereafter, isoleucine starvation was induced by addition of l-valine (50 mg/mL) to a final concentration of 400 μg/mL, and samples were taken at 5, 10, 20, 40, and 80 min after starvation. All samples were taken by transferring 4 to 6 mL of culture into a 1/5 volume of cold stop solution (5% phenol in ethanol)  and harvested by transfer to a 1/5 culture volume stop solution. To each sample, 0.625 mL of the stopped [3H]-labeled spike-in culture was added and mixed by inverting, and the stopped sample/spike-in mixture was divided for RNA purification using the methods described below. To determine the total incorporated radioactivity in the samples, 50 μL of the cell suspension or 5 μL of extracted RNA was precipitated with 0.5 mL of TCA (5%) for approximately 30 min at 0°C. In addition, to obtain an estimate for incorporation of [14C]/[3H] into DNA, identical samples were treated with 0.5 mL of NaOH (0.5 M) and incubated for 2 h at 37°C in a water bath before TCA precipitation. TCA precipitates were collected on glass fiber filters (Advantec) by vacuum filtration, washed three times with 5 mL ice-cold 5% TCA, and dried overnight at room temperature (RT) before the addition of 5 mL Ultima Gold scintillation cocktail (Perkin Elmer) and measurement of disintegrations per minute (dpm) by liquid scintillation counting (LSC).To determine the efficiency of common RNA extraction methods, total cellular RNA was radioactively labeled, and recovery of radioactively labeled RNA after purification was measured. An exponentially growing culture of MAS1081 of an ODethanol)  on ice. 14C] and [3H] counts solely originating from labeled RNA. The overall efficiency of RNA extractions was calculated as the fraction of RNA recovered by the tested extraction method relative to the total, TCA-precipitable RNA. Recovery of total RNA after isoleucine starvation relative to the steady state was calculated by normalizing all samples to the [3H] counts originating from spike-in cells and calculating the fraction of extracted RNA relative to the average RNA recovery of three steady-state samples (st-st 1 to 3).Before calculating the efficiency of RNA extraction, dpm originating from radiolabeled DNA (NaOH-treated samples) were subtracted from all sample counts to obtain [3H] counts in the bands originating from spike-in cells as described for the calculation of total RNA extraction efficiency.Radiolabeled RNA obtained as described above was separated by denaturing polyacrylamide gel electrophoresis (PAGE) on 5% polyacrylamide gels buffered with 1× TBE  and supplemented with 8 M urea. Samples were diluted in loading buffer  prior to loading. Electrophoresis was performed at 15 V/cm, and the resulting gels were fixed by soaking in 50% ethanol and 10% acetic acid, dried, and imaged by phosphor imaging. To determine relative levels of individual RNA species, corresponding bands were cut out, treated with RNase A in TE buffer, and subjected to scintillation counting. Recovery of individual RNA species after starvation was calculated by normalization to  by incubation of 150 pmol of oligonucleotide (1.5 μL of a 100 μM stock) with 10 U T4 polynucleotide kinase  and 60 μCi ɣ-[32P]-ATP  in a total volume of 10 μL at 37°C for 1 h. After 1 h, additional 10 U of PNK was added and incubation continued for 1 h.Denaturing formaldehyde agarose gel electrophoresis was performed as described in reference selC were quantified using ImageQuant TL 8.2 image analysis software . The obtained target tRNA values were corrected by subtraction of the signal originating from endogenous expression of the same tRNA in the spike-in cell sample. The corrected values were then normalized by dividing the corrected value by the value of tRNAselC from the same lane and plotted relative to the average of three samples taken during balanced growth.Normalization of tRNA signals was performed as described in reference 14C] signal was visualized by phosphor imaging. During exposure, blots hybridized with [32P]-labeled oligonucleotide probes were covered with several layers of plastic sheets to minimize exposure of the storage phosphor screen to radioactivity originating from [14C]-labeled RNA. For normalization, the signal intensity of the target tRNA band was divided by the [14C]-signal of the entire tRNA fraction of the same lane, which was shown to be stable by the radiolabeling approach described above. The statistical significance of differences between individual time points after starvation/treatment and the steady-state level was assessed where applicable (n ≥ 3) by a two-tailed Student’s t test assuming unequal variances. To do so, the mean and standard deviation of all available steady-state samples (routinely three steady-state samples per sample series) were used.tRNA levels in radiolabeled RNA samples obtained by hot phenol extraction were determined in the same manner, with the following exceptions. Prior to hybridization with oligonucleotide probes, the [sls@bio.ku.dk).All raw data that are not shown in the figures or the supplementary information file are available upon request from Sine Lo Svenningsen (


The research and application of immune checkpoint inhibitors (ICIs) have enormously promoted the progression of tumor treatment. Gradual implementation of ICIs in clinical practice is largely limited as they exert uncontrolled collateral effects on the immune system, such as immune-related adverse events (irAEs); this includes rarely reported glomerular diseases. This study aimed to describe the clinical and pathological manifestation of ICIs-induced glomerular diseases and focused on the mechanism and therapeutic strategy for glomerular diseases associated with ICIs. The data of 53 patients with glomerular diseases related to ICIs were retrieved from the PubMed database. The most frequently reported ICIs-related glomerular diseases were pauci-immune glomerulonephritis (28.3%), podocytopathies (26.4%), and immune-complex glomerulonephritis (18.9%). Moreover, anti-PD1 antibodies were the most commonly used ICIs (71.4%). Most patients receiving ICIs discontinued the treatment (89.4%) and were initiated with steroids (87.2%). Rituximab was also useful in the treatment, especially for renal vasculitis. Rechallenging ICIs could be considered for cancer progression or as salvage therapy, where rechallenging ICI therapy with steroids may be beneficial. We believe the treatment should be personalized based on the degree of renal pathology, serum creatinine (Scr), and tumor progression to obtain a good prognosis. ICIs are monoclonal antibodies targeting inhibitory receptors that block the co-inhibitory molecules expressed on T cells and tumor cells for improving anti-tumor immunity . These iThe side effects of ICIs have been demonstrated with the increasing use of ICIs . The inc2.A total of 33 articles were retrieved, including individual case reports and serial reports. These articles consisted of data from 53 patients with glomerular disease related to ICIs therapy  ,11,13–43Classification of glomerular diseases and glomerulonephritis (GN) was carried out according to the KDIGO 2021 Guideline for the Management of Glomerular Diseases  and the The malignancies involved consisted of melanoma (32.6%), non-small cell lung cancer , renal cell carcinoma , gastrointestinal cancer (6.1%), lymphoma (6.1%), and malignant pleural mesothelioma. The mean age of the patients was 63.78 ± 1.61. The majority of patients were male (76.1%), owing to the high prevalence rate of skin cancer or lung cancer in males. Widely used ICIs were anti-PD1 antibodies (71.4%). Glomerular diseases associated with the use of ICIs therapy included 15 cases of renal pauci-immune GN (28.3%), 14 cases of podocytopathies (26.4%), 10 cases of immune-complex GN (18.9%), 4 cases of MN (7.5%), 4 cases of AA amyloidosis (7.5%), 4 cases of C3 glomerulopathy (7.5%), and 2 cases of anti-glomerular basement membrane GN (3.8%) . Most of2.1.n = 6) while the total number of female is 11 (Supplementary Table 1). High-dose methylprednisolone was used in 6 cases, including 2 receiving cyclophosphamide and 1 receiving mycophenolate together [Supplementary Table 1).Pauci-immune GN and renal vasculitis are normally regarded as the common types of glomerular lesions related to ICI use. Compared with other types of glomerular diseases, pauci-immune GN and renal vasculitis have a relatively higher incidence in female . Generally, compared with Pauci-immune GN and renal vasculitis, the prognosis of nephrotic syndrome (NS) related to ICIs is well.Podocytopathies are the second most reported glomerular diseases correlated with ICIs, which included 12 cases of MCD and 2 cases of focal segmental glomerulosclerosis (FSGS). Our statistics suggested that most cases (8/12) were related to anti-PD-1 antibodies (including pembrolizumab and nivolumab). Only one case was treated with combination medication. Glutsch et al.  reported2.3.The incidence of immune-complex GN associated with ICIs therapy was also high, with 10 related cases consisting of 6 cases of IgAN ,35,36,383.CTLA-4, a key negative regulator of T cell activation, is located on the T cell surface and prevents T cell activation by outcompeting CD28 for its ligand, B7, thereby inhibiting T cell co-stimulation. PD-1, which protects against autoimmune diseases, is a cell surface receptor on the T cell. PD-L1, a protein expressed on cancer cells, can bind to PD-1 and inhibit the T cell from attacking the cancer cell . BesidesThe mechanisms and manifestations of irAEs caused by ICIs are based on the type of ICI therapy. CTLA-4 inhibitors can induce many cellular changes, such as T-cell activation and proliferation, survival of impaired CD4+ CD25+ regulatory T cell (Treg cell), and an increase in the number of type 17 T helper cells. Besides, cross-reactivity between anti-tumor T cells and antigens is induced in healthy cells with the production of autoantibodies. PD-1 and PD-L1 inhibitors lower the survival and inhibitory function of Treg cells while increasing cytokine production . It was 3.1.ICIs enhance T cell immune function without selectivity between tumor tissue and normal tissue, down-regulate its tolerance to its antigens, and hence cause nonspecific activation of the immune system. Glomerular disease is induced by the ‘reprogramming’ of the immune system . CTLA-4 3.2.ICIs induce the production of autoimmune antibodies, leading to the deposition of antibody-antigen complexes in the kidney. Lute et al. generated human CTLA4 gene knock-in mice. They used them to compare a panel of anti-human CTLA-4 antibodies for their ability to induce autoimmunity. The group suggested that the treatment with anti-CTLA4 antibodies revealed circulating anti-double-stranded DNA antibodies as well as glomerular IgG and C3 deposits . Fadel e3.3.ICIs enhance proinflammatory cytokines/chemokines release in kidney tissue, further enhancing podocyte foot-process effacement. It has been hypothesized that by adopting therapies modulating T lymphocyte regulation, cytokine release was increased, thereby influencing VPF/VEGF secretion and promoting subsequent proteinuria . Additio3.4.ICIs can assist in forming new or reactivated T cells against tumor antigens (Ags) which are capable of cross-reacting with off-target kidney tissues . Johnson3.5.The complement alternative pathway could be a target of autoimmune dysregulation (AP) caused by ICIs. Ville et al. presented a case of C3 GN related to pembrolizumab use. The patient was screened for a wide range of autoantibodies with all negative except for anti-C3b .The diagram of the plausible mechanism is illustrated in 4.The treatment of ICIs-related kidney injury is aggressive. Notably, Scr level was normal in a few patients, such as MCD, and early manifestations may present with edema, oliguria, or elevation of blood pressure ,14. Thes4.1.In most of the 53 cases, ICIs were discontinued immediately. Irrespective of whether the treatment continues, the positive effect remains. The benefit of stopping exposure to ICIs is significant. Kishi et al.  discoverHowever, it is controversial whether ICI therapy should be discontinued when glomerular disease occurs. For patients with mild renal pathologic changes and Scr of grade 1–2, ICI therapy may be considered for persistent use, requiring close monitoring of renal function, electrolytes, and urinary abnormalities. Additionally, the combination therapy with corticosteroid or rituximab might allow continued use of the ICIs despite the occurrence of renal irAEs ,34. Irif4.2.Steroid therapy was employed in most ICIs-related glomerular diseases. The effect of steroid therapy is effective and remarkable . All of 4.3.MMF is adopted to prevent organ transplant rejection and treat autoimmune diseases like systemic lupus erythematosus and steroid-resistant NS. With respect to the renal irAEs, MMF would be adopted for patients with hormone insensitivity or drug resistance. Findings about MMF remain controversial. It is deleterious for TIN patients as it can cause pancytopenia and fatal septic complications , while i4.4.Considering the specificity in targeting B-lymphocytes, rituximab may provide a good treatment option for glomerular disease and cannot directly impact cancer therapy. Rituximab should be considered for treating ICI-induced vasculitis. Mamlouk et al. presented five cases of ICI-induced vasculitis, and all the cases received the treatment of prednisone and rituximab . Moreove4.5.Supportive care should be initiated when severe and persistent AKI occurs. It was reported that the RRT rate is 9% in all ICI-related AKI patients , while 24.6.Angiotensin-converting enzyme inhibitor (ACEI) treatment and diuretics may be initiated with volume control in NS . Cycloph5.Over one-third of patients suffering from preexisting rheumatic or autoimmune diseases  have experienced flares of their prior disorder ascribed to the use of ICIs for malignancy . Anti-PD6.Among the reported cases of ICIs-associated glomerular diseases, a total of five cases 10.2%) were found with a rechallenge of ICIs therapy .2% were ,28,31,387.In conclusion, researchers have reported various pathologies of glomerular disease associated with ICIs. The most frequently reported were pauci-immune GN, podocytopathies, and immune-complex GN. Anti-PD1 antibodies were the most widely used ICIs. Various ICI-causing renal manifestations suggested that it is necessary to further elucidate its complicated mechanisms. The time of AKI or proteinuria occurrence is inconsistent and even occurs months after discontinuing ICIs. Therefore, regular monitoring of urine routine and Scr is required. It is emphasized in this paper that kidney biopsy is suitable for diagnosing ICIs-induced kidney disease. Discontinuation of ICIs and glucocorticoid treatment can improve the renal outcome of most patients. The treatment should be personalized according to the degree of renal pathology, Scr, and tumor progression to obtain a good prognosis. Rechallenge with ICIs should be considered for cancer progression or as salvage therapy. This requires the collaboration of several specializations, such as the oncology, nephrology, and urological surgical fields. Proper biomarkers for toxicity prediction should be discovered to improve the management of patients with ICI therapy . HoweverClick here for additional data file.


Solute carrier transporters are integral membrane proteins, and are important for diverse cellular nutrient transports, metabolism, energy demand, and other vital biological activities. They have recently been implicated in pancreatic cancer and other cancer metastasis, angiogenesis, programmed cell death and proliferation, cell metabolism and chemo-sensitivity. Here we report the study of 13 human solute carrier membrane transporters using the highly accurate AlphaFold2 predictions of 3D protein structures. In the native structures, there are hydrophobic amino acids leucine (L), isoleucine (I), valine (V) and phenylalanine (F) in the transmembrane alpha-helices. These hydrophobic amino acids L, I, V, F are systematically replaced by hydrophilic amino acids glutamine (Q), threonine (T) and tyrosine (Y), thus the QTY code. Therefore, these QTY variant transporters become water-soluble without requiring detergents. We present the superposed structures of these native solute carrier transporters and their water-soluble QTY variants. The superposed structures show remarkable similarity with RMSD ~ 1 Å–< 3 Å despite > 46% protein sequence substitutions in transmembrane alpha-helices. We also show the differences of surface hydrophobicity between the native solute carrier transporters and their QTY variants. Our study may further stimulate designs of water-soluble transmembrane proteins and other aggregated proteins for drug discovery and biotechnological applications. Many active basic and clinical research efforts have been made, but no effective treatment is yet in sight. Recently, several solute carrier (SLCs) transporters have come to light that are involved in six crucial routes of pancreatic cancer resulting in very poor prognoses including: (i) cancer cell proliferation, (ii) programmed cell death, (iii) invasion and metastasis, (iv) angiogenesis, (v) cellular metabolism and (vi) chemo-sensitivity. These SLC transporters move a broad range of key substrates including (a) amino acids, (b) nucleotides, (c) essential metal ions including zinc, magnesium, sodium, lithium, copper, other organic ions, NH4+, HCO3−, (d) short chain fatty acids, (e) co-factors, vitamins, and (f) other organic ions and compounds into cells. Some of these SLCs are involved in pancreatic cancer metastasis and angiogenesis, others are involved in proliferation and programmed cell death, as well as cellular metabolism and chemo-sensitivity  helices. Most have 10TM-12TM helices depending on their transporting substrates with the longest having 13TM (SLC5A8 for short chain fatty acids). Several of these SLC transporters have large N-termini and extracellular loops , and with high confidence for another 45% (~ 96.3 million). AlphaFold2 has truly started a new era of digital biology. Nevertheless, academic investigators, the pharmaceutical and biotech companies must still ultimately study the physical structures of proteins, including SLC membrane transporters since the structures are vital to understanding how substrates including sugars, amino acids, essential ions, organic molecules, drugs, or other essential nutrients are transported across highly regulated and controlled cell membranes.On 15 July 2021, Google DeepMind announced the AlphaFold2, and at the same time David Baker’s lab introduced RoseTTAFold as machine learning revolutionary tool for the very accurate prediction of protein structures35. The expressed and purified water-soluble proteins exhibited predicted characteristics and retained ligand-binding activity35. In July 2021, we prepared QTY variant protein structure predictions using AlphaFold2, achieving better results in 1–2 hours37, rather than ~ 5 weeks for each molecular simulation using GOMoDo, AMBER and YASARA programs33. We also produced a program and website for generating the membrane protein water-soluble QTY variants38.We previously applied the QTY  code to design several detergent-free transmembrane (TM) protein chemokine receptors and cytokine receptors for various uses using conventional computing programs to simulate several G protein-coupled receptors. Each took ~ 5 weeks to complete the simulationHere, we report using AlphaFold2 to design water-soluble QTY variants of the 13 solute carrier transporters, and to directly compare with their counterpart native structures. In addition to targeting the key nutrients and ion uptake activity of cancer cells, these QTY variant water-soluble SLC transporters can prospectively find many additional applications. Working with water-soluble QTY variants may substantially accelerate the discovery and development of therapeutic and diagnostic biologicals.We aligned the native SLC transporters with their QTY variants. Despite significant QTY replacement of hydrophobic residues in the transmembrane domains (~ 44–55%) in the SLC transporters, the isoelectric focusing point pI and molecular weight remain rather similar Figs. , Table 2Since the electron density maps share remarkable structure similarities between leucine (L) versus glutamine (Q); isoleucine (I), valine (V) versus threonine (T); and phenylalanine (F) versus tyrosine (Y), the QTY code selects 3 neutrally polar amino acids: glutamine, threonine and tyrosine to replace 4 hydrophobic amino acids leucine, isoleucine, valine and phenylalanine. After applying the QTY code, the hydrophobic amino acids in the transmembrane segments are replaced by Q, T, and Y, therefore the transmembrane segments have significantly reduced hydrophobicity. For example, SLC39A3 and SLC29A1 differ > 54 and > 45% from their water-soluble QTY variants, respectively, in their transmembrane alpha-helical segments.  vary, some in the acidic and some in the basic range. For example, native SLC7A11 has a basic pI of 9.29. On the other hand, SLC1A5 has an acidic pI of 5.34. Others including SLC9A1 have a near neutral pI of 6.74  and Thr (T), and b) the addition of OH- on Tyr (Y). These increase the protein molecular weights  the substitutions of CHts Figs. , Table 239, SLC7A11 (PDB: 7PV9)40, SLC4A4 (PDB: 6CAA)41, SLC1A5 (PDB: 5LMM)42, SLC7A5 (PDB: 6IRS)43, SLC29A1 (PDB: 6OB6)44. The superposed structures are performed for: SLC2A1Crystal versus SLC2A1QTY, SLC7A11CryoEM versus SLC7A11QTY, SLC4A4CryoEM versus SLC4A4QTY, SLC1A5Crystal versus SLC1A5QTY, SLC7A5CryoEM versus SLC7A5QTY and SLC29A1Crystal versus SLC29A1QTY QTY (2.281 Å); for SLC7A11 versus SLC7A11QTY (0.933 Å); SLC4A4 versus SLC4A4QTY (0.445 Å); SLC1A5 versus SLC1A5QTY(0.965 Å); SLC7A5 versus SLC7A5QTY (1.815 Å); and SLC29A1 versus SLC29A1QTY (1.512 Å). ; SLC39A3 versus SLC39A3QTY (1.567 Å); SLC9A1 versus SLC9A1QTY (0.905 Å); SLC6A14 versus SLC6A14QTY (0.521 Å); SLC4A7 versus SLC4A7QTY (0.426 Å); SLC5A8 versus SLC5A8QTY (1.593 Å); and SLC41A1 versus SLC41A1QTY (1.914 Å)  for these superposed structures are displayed  as water-soluble antigens to generate useful monoclonal antibodies in animals since they have many extracellular loops including few large loops, and (2) these anti-SLCxxx antibodies could be useful as research reagents for an assay system to study the transporters in tissue cell cultures and in vivo. These specific monoclonal antibodies can perhaps also be useful (a) as therapeutics to treat diseases including pancreatic cancer, (b) as diagnostic reagent to monitor cancer treatments and perhaps (c) for early pancreatic cancer detection.Our current study using the AlphaFold 2 demonstrates that the water-soluble QTY-variant structures of SLC transporters are substantially similar to the native structures. AlphaFold 2 is a very useful approach to predict other membrane embedded transmembrane proteins. QTY is a useful approach for working with difficult-to-study hydrophobic proteins. The SLC transporter water-soluble QTY variants not only could be used for designs of molecular machines, but also as water-soluble antigens for generating therapeutic monoclonal antibodies and for accelerating drug discovery.34. The website Expasy (https://web.expasy.org/compute_pi/) was used to calculate the molecular weights (MW) and pI values of the proteins.The native protein sequences for SLC transporters and their QTY-variant sequences are aligned using the same methods previously described30 Program https://github.com/sokrypton/ColabFold was used for the structure predictions of the QTY variants following the instructions at the website on 2 × 20 Intel Xeon Gold 6248 cores, 384 GB RAM, and a Nvidia Volta V100 GPU. The European Bioinformatics Institute  has all AlphaFold2-predicted structures. The Uniprot website https://www.uniprot.org has each protein ID, entry name, description, and FASTA sequence. The data was taken from UniProt using a custom Python code. The QTY method website (https://pss.sjtu.edu.cn/) can convert the FASTA protein sequences into their water-soluble versions. These steps were optimized using Python libraries for web applications such as requests and splinter.AlphaFold2https://www.rcsb.org. They include SLC2A1 (PDB: 6THA)39, SLC7A11 (PDB: 7P9U)40, SLC4A4 (PDB: 6CAA)41, SLC1A5 (PDB: 5LMM)42, SLC7A5 (PDB: 6IRS)43, SLC29A1 (PDB: 6OB6)44. AlphaFold2 predictions of 8 native SLC transporters and their QTY variants were carried out using the AlphaFold2 program at https://github.com/sokrypton/ColabFold. Uniprot https://www.uniprot.org is the source for all 13 SLC transporter protein sequences and AlphaFold2 was performed to predict QTY variant structures. These structures are superposed using PyMOL https://pymol.org/2/.The molecular structures are taken from PDB https://pymol.org/2/ and UCSF Chimera https://www.rbvi.ucsf.edu/chimera/. PyMOL program is used for the superposed models, while hydrophobicity models were rendered using Chimera.Two key programs were used for structure visualization: PyMOL (1) All methods were carried out in accordance with relevant guidelines and regulations. (2) All experimental protocols were approved by a named institutional and licensing committee. (3) Neither human biological samples, nor human subjects were used in the study. This is a completely digital structural biology study using the publicly available AlphaFold2 machine learning program.


Chroicocephalus ridibundus) are common migratory birds inhabiting in most urban and rural wetlands of Yunnan Province, China, which is a typical habitat for black-headed gulls to overwinter. Whether Yunnan black-headed gulls carry CoV has never been determined. In this study, we identified three strains of DCoVs in fecal samples of Yunnan black-headed gulls by reverse-transcriptional PCR and sequenced their whole genomes. Genomic analysis revealed that these three strains shared genomic identity of more than 99%, thus named DCoV HNU4-1, HNU4-2, and HNU4-3; their NSP12 showed high similarity of amino acid sequence to the homologs of falcon coronavirus UAE-HKU27 (HKU27), houbara coronavirus UAE-HKU28 (HKU28), and pigeon coronavirus UAE-HKU29 (HKU29). Since both HKU28 and HKU29 were found in Dubai, there might be cross-border transmission of these avian DCoVs through specific routes. Further coevolutionary analysis supported this speculation that HNU4 (or its ancestors) in black-headed gulls originated from HKU28 (or its homologous strain) in houbara, which was interspecies transmission between two different avian orders. In addition, interspecies transmission of DCoV, from houbara to falcon, pigeon and white-eye, from sparrow to common-magpie, and quail and mammal including porcine and Asian leopard cat, from munia to magpie-robin, was predicted. This is the first report of black-headed gull DCoV in Asia which was highly homolog to other avian DCoVs, and the very “active” host-switching events in DCoV were predicted, which provides important reference for the study of spread and transmission of DCoVs.Deltacoronavirus (DCoV) is a genus of coronavirus (CoV) commonly found in avian and swine, but some DCoVs are capable of infecting humans, which causes the concern about interspecies transmission of DCoVs. Thus, monitoring the existence of DCoVs in animals near communities is of great importance for epidemic prevention. Black-headed gulls ( Orthocoronavirinae in the family of Coronaviridae. In the recent two decades, CoVs have caused three intercontinental epidemics, including the ongoing worldwide pandemic of COVID-19 with more than 500 million infections and almost 6 million deaths. It has been reported that human epidemic CoVs were originated from zoonotic CoVs which were reserved in wild animals and infected humans via occasional interspecies transmission (Coronaviridae Study Group (CSG) of ICTV classified coronaviruses into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus, by analyzing five essential protein domains  combined with phylogeny and genomic structure (Coronavirus (CoV) usually refers to the viruses of the sub-family of smission . A variesmission . So far,smission . The Cortructure . Alphacotructure . Gammacotructure .Although birds are major hosts for DCoV, this virus also infects swine, indicating potential interspecies transmission from birds to mammals which is supported by the evolutionary relationship between sparrow DCoV and porcine DCoV (PDCoV) . NotablyChroicocephalus ridibundus) is a migratory bird widely distributed in Eurasia and on the East Coast of North America (rdrp gene of black-headed gull DCoV (BHG-DCoV) has been reported in Finland (Black-headed gull ( America . Xinjian America . Accordi America . As far  Finland . HoweverCharadriiformes) may spread from HKU28 (or its homologous strain) in houbara (from Gruiformesor). According to the sequence of the five essential domains for CoV taxonomy, HNU4 could be classified into the same species of White-eye CoV-HKU16, and further analysis showed that White-eye CoV-HKU16 like coronavirus had been found in six orders of birds, suggesting the activity of host jumping ability of this coronavirus. Our finding reveals the widespread of DCoV as well as its potential transboundary and interspecies transmission. Therefore, more attention should be paid on DCoV in wide birds to prevent potential epidemics.In this study, we collected the fecal samples of black-headed gulls in Kunming and Dali, two major cities in Yunnan, for DCoV detection. As a result, three strains of BHG-DCoV strains  were identified with whole-genome identity of more than 99%. Further genome analysis revealed that the genome of HNU4 showed high identity of more than 96% to those of HKU27, HKU28, and HKU29 found in United Arab Emirates, indicating a potential transboundary transmission. The differentiation time analysis and coevolution analysis support that HNU4 (or its ancestors) in black-headed gull  and kept in dry ice for transportation to the laboratory and stored at −80°C until use.rdrp gene of CoVs using primers DCoV-F (5′-GTGGVTGTMTTAATGCACAGTC-3′) and -R (5′-TACTGYCTGTTRGTCATRGTG-3′) . To confirm the bird species, the mitochondrial cytochrome b (cytb) gene was amplified and sequenced of the samples.Viral RNA was extracted from the fecal samples using MagaBio plus Virus DNA/RNA Purification Kit II  following the manufacturer’s instructions. RNA extracted from fecal was washed off with 70-μl RNase-free water and stored in −80°C as a template for reverse-transcriptional PCR (RT-PCR). Initial CoV screening was performed by amplifying a 440-bp fragment of the RGTG-3′) . Primescrdrp genes of CoVs in the GenBank database.The PCR products were gel-purified and sequenced with an ABI Prism 3,700 DNA analyzer (Applied Biosystems), using the PCR primers. The sequences of the PCR products were compared with known sequences of the Three complete genomes of BHG-DCoVs were amplified and sequenced using RNA extracted from black-headed gull feces as templates. RNA was amplified with degenerate primers, which were designed by multiple alignments of other coronavirus genomes with the complete genome, using Primescript one-step RT-PCR kit version 2. Additional primers were designed according to the results of the first and subsequent rounds of sequencing. The 5′ and 3′ genome end sequences were obtained by 5′ and 3′ race (Roche), respectively. The expected size of PCR products was purified by gel and directly sequenced. The sequence was assembled to obtain the full-length genome sequence.Multiple sequence alignment was performed by MAFFT v7.149 in BioAider v1.334 . The maxrdrp gene was aligned using MAFFT program with codon method in BioAider v1.334. Then, we detected the temporal structure in these rdrp gene sequences by TreeTime program  according to the latest report of evolution rate of rdrp gene in DCoV  were reserved as the representative for these highly similar sequences with the same host. The host evolutionary tree and topologies were obtained from the TimeTree websiteanalyzed . Then, w program . We run According to previous research on the evolutionary history of RNA virus , we examdeltacoronavirus  and identified 10 typical open reading frames (ORFs) of coronavirus which were aligned as 5′ untranslated region (5′ UTR)-replicase ORF1ab-spike (S)-envelope (E)-membrane (M)-Nucleocapsid (N)-3′ UTR. Moreover, additional ORFs coding non-structural (NS) proteins  were identified . The ideWhite-eye coronavirus HKU16 (WECoV-HKU16), HKU27, HKU28, and HKU29. The result showed that all the eight viruses could be classified into the same species under the genus of deltacoronavirus  was compared . The S pTo assess the impact of mutations on S protein structure and function, we compared the differences in primary and tertiary structures of HNU4-1 and HKU28. In primary sequence, there were a total of 50 aa mutations and one aa insertion in S protein of HNU4-1 compared to HKU28 . Of thesArenaria interpres DCoV (ARDCoV) found in Canada . The time of the most recent common ancestor (tMRCA) differentiation time of DCoV was about 2977 B.C . The tMRCA of White-eye coronavirus HKU16 species was about 1200 A.D . The tMRCA of HNU4-1, HNU4-2, and HNU4-3 was about 1964 A.D . However, the tMRCA of HKU27, HKU28, and HKU29 was about 1952 A.D (1896 A.D–1983 A.D), and it suggests that they may differentiate earlier than BHG-DCoV   . Frequen > 0.01) . This inGruiformesor) looks “restless” and spread to other hosts, such as falcon , pigeon (Columba livia), and white-eye (Zosterops japonicus). Consistent with the previous speculation, the host transfer between houbara and falcons may be caused by the food chain (Montifringilla taczanowskii), sparrow, and munia shared co-divergence with three hosts. Then, the sparrow DCoV spread to common-magpie (Pica pica), quail (Coturnix coturnix), and mammal (porcine and Asian leopard cat). The munia DCoV may spread to magpie-robin (Copsychus saularis). In general, many host-switching events were predicted in DCoV, which imply that DCoV was very “active” in their hosts. However, the current sample of bird DCoV is still limited, and only a single host species has been detected in some orders. Therefore, more convincing evolutionary relationships require more samples to support in future. The large number of potential host-switching events indicates that the prevalence of DCoV in birds is wide and complex, and it is necessary to strengthen monitoring.Besides, the DCoV in houbara (od chain . It is wrdrp gene (461bp) identity between BHG-DCoV in Yunnan and BHG-CoV found in Finland is 94.794–95.662%. In view of the high conservation of rdrp gene, BHG-DCoV in black-headed gulls may also be diverse. According to the hallmark domain sequences for CoV classification proposed by ICTV, these newly found BHG-DCoV strains could be classified into the species White-eye coronavirus HKU16, but the overall genome sequences of the BHG-DCoV showed low identity with White-eye coronavirus HKU16, the prototypical virus of the species. In comparison, these BHG-DCoVs genomes were highly similar (>96%) to the genomes of HKU27, HKU28, and HKU29 found in Dubai, indicating the close evolutionary relationship of these avian DCoVs and potential interspecies and transboundary transmission of them.Increasing studies have shown that the outbreak of human epidemic is closely related to pathogens reserved in wildlife . EspeciaDespite the high similarity in the whole-genome level, HNU4, HKU27, HKU28, and HKU29 showed a few amino acid variations in several structural and non-structural proteins, including M, N, NSP3, NSP6, NS7a, NS7b, and NS7c. These proteins are critical for CoV life cycle. For instance, NSP3 is a scaffold protein with activity of deubiquitination (DUB) and deISGylation which is necessary for immune evasion ; NSP6 isSpike protein is the key factor determining the receptor recognition of coronavirus . CoronavMontifringilla taczanowskii), sparrow (Passer montanus), and munia (Lonchura striata), or their common ancestor, was not clear. Of note, the evolutionary relationship between the ancestors of Night heron coronavirus HKU19, Wigeon coronavirus HKU20, and their hosts was not clear due to the lower support value, and the host transfer from houbara to thrush (Turdus hortulorum) also lacked sufficient support.The estimation of differentiation date showed that tMRCA of HKU27, HKU28, and HKU29 was earlier than BHG-DCoV, which supports the possibility of cross-species transmission of them together with the host-switching event. In addition, the host–virus mapping model constructed in this study also supported that the origin of mammalian DCoVs was bird DCoVs . HoweverWhite-eye coronavirus HKU16 like coronavirus has been found in six orders of birds, suggesting the active interspecies transmission ability of White-eye coronavirus HKU16 like coronavirus, ornithological studies have shown that the black-headed gulls in Yunan usually migrate for long distance from Russia, Mongolia, and Xinjiang Uygur Autonomous Region  can be found below: K-KC, Z-JZ, Y-ZZ, and X-YG contributed to the conception and design of the study. K-KC and LG collected the samples. Z-JZ organized the database. K-KC and Z-JZ performed the data analysis. K-KC and YQ wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Electronic patient-reported outcomes measures (e-PROMs) are a valuable tool for the monitoring and management of chronic conditions over time. However, there are few validated tools available that capture symptoms across body systems in telehealth settings. The Parsley Symptom Index (PSI) is a recently developed symptom assessment for adults with chronic disease in telehealth settings. A previous study demonstrated the feasibility and acceptability of the PSI in a clinical telehealth setting.The purpose of this study was to assess convergent validity between the PSI and the self-rated health (SRH) item.This prospective cohort study took place from January 15, 2021, to December 15, 2021, among a sample of 10,519 adult patients at Parsley Health, a subscription-based holistic medical practice. The PSI and the SRH were completed by patients via an online portal. The association between the PSI and SRH was assessed via polyserial and polychoric correlations, while weighted κ scores provided information related to agreement between the PSI and SRH.r=0.51; polychoric r=0.52) and agreement (weighted κ=0.46) between the PSI and SRH. In total, 74.13%  of responses between the PSI and SRH were relatively congruent while 36.17% (n=8229) were literally congruent.From 22,748 responses, there were moderate levels of association (polyserial The PSI demonstrates convergent validity with the SRH for adults with chronic disease in a telehealth setting. This finding further supports the validation of the PSI in a real-world clinical setting. Although it is conceptually similar to the 1-question SRH, the PSI is a 45-item PROM designed to capture quality of life and specific symptoms by body system. Future studies will compare the PSI to multi-item PROMs. Health crisis triaging during the COVID-19 pandemic further increased demand for telehealth care [Providing telehealth options has become indispensable to health care delivery in the United States. Even before the COVID-19 pandemic fundamentally altered the health care landscape, claim lines in the United States for nonhospital-based clinicians to patient telehealth grew 1393% [lth care ,3, accellth care ,5.Having access to affordable care is especially important for the 60% of Americans that live with at least 1 chronic disease, and this group spends 2 to 4 times more on health care than do those without any chronic conditions , which rDespite the fact that many Americans with chronic diseases are currently being treated via telehealth, there are limited e-PROM tools available to telehealth providers and clinics for assessing and tracking a patient’s health status over time. Tools like the Patient-Reported Outcomes Measurement Information System (PROMIS) ,12, the As part of a larger effort to leverage new tools like e-PROMs to make the telehealth experience engaging and effective for patients with chronic diseases, a research team at Parsley Health (a subscription-based holistic medicine practice) built the Parsley Symptom Index (PSI). The PSI is a 45-item e-PROM designed specifically for use in telehealth settings to function as an ROS. When used strategically, a patient-reported outcome–driven approach can shift an ROS to a cooperative dialogue between patients and clinicians . Like anIn an initial feasibility and acceptability study that assessed construct and face validity, the PSI was deployed, completed, and found helpful to both patients and clinicians . Having This prospective cohort study took place at Parsley Health from January 15, 2021, to December 15, 2021, among a sample of 10,519 adult patients. Patients completed the PSI and the SRH via an online portal. The average monthly PSI completion rate was 77.21% (range 69.23%-83.44%) over the study period.Ethics ApprovalThis study used patient-reported survey data that were recorded in such a manner that participants were unidentifiable to the researchers. The institutional review board at Stony Brook University considered this study exempt (IRB2020-00429) from Code of Federal Regulations Title 45 requirements .Parsley Health is a subscription-based membership model for delivering primary care and proactive chronic disease management through a holistic-medicine lens. Patients receive care from Parsley Health clinicians and health coaches in-person and remotely, with additional access to their care team via email and an online portal. Prior to the COVID-19 pandemic, Parsley Health further increased their telehealth availability to over 45 states. Inclusion criteria for this study were Parsley Health patients that had an active subscription membership plan between January 15, 2021, and December 15, 2021, and a minimum of 1 clinical encounter within their membership period. Exclusion criteria were severe psychiatric disorders (particularly psychosis and depression requiring a change in treatment in the last 30 days), age under 18 years, and being unable to speak or read English.The PSI is a 45-item, ROS-style PROM tool designed to capture chronic disease symptoms . The PSIThe SRH item was administered alongside the PSI. The SRH was a mandatory item at the end of the PSI, and only complete questionnaires were included in this study. The SRH is a single question, with a 5-item Likert scale answer that reads as follows: “In general, would you say that your health is excellent, very good, good, fair, or poor?” The SRH is validated and is commonly used to demonstrate construct validity of PROMs -21 and aAfter patients scheduled a visit, they were instructed to log into an online patient portal and complete the PSI 24 to 48 hours before each clinical visit. Initial visits were rescheduled if all forms were not completed, but follow-up visits were not postponed for an incomplete PSI. For follow-up visits, patients who had not completed the PSI received an automated reminder 48 hours before the clinical visit. If the PSI was not completed after the automated prompt, another prompt was sent from the clinician or clinical operations coordinator.When clinicians prepared for an online visit, they used a standardized note template within the electronic health record to pull the most recent PSI score into the visit note. The PSI design allowed for the results to be immediately usable: once a PSI was completed, patients received instant feedback, and clinicians could quickly import the data into the note to prepare for the patient visit. With the PSI template integrated into the beginning of the encounter note, clinicians were subtly prompted to use the PSI to discuss patient-reported symptoms and provide positive feedback to the patient for completing the PSI.During the telehealth patient visit, the PSI score was used as a touchpoint for the patient-clinician discussion. As the PSI was previously completed, clinicians were able to ask targeted questions about symptoms and had more time to focus on burden and distribution of illness. The longitudinal PSI graph further deepened the provider’s ability to identify triggers and mediators that influenced disease trajectory over time. To test the hypothesis that the SRH item would correlate with the PSI, 2 measures of association were calculated. First, a polyserial correlation was performed on the raw continuous score of the PSI (range 0-500) with the ordinal SRH categories . Next, the PSI’s responses were scored and translated into ordinal categories  to compare directly with the SRH categories and generate polychoric correlation coefficients . This seTo determine agreement, weighted κ (quadratic) scores incorporated information about the distance between the transformed ordinal PSI and SRH ratings: ratings that were 1 category apart counted as “less disagreement” than did a pair of ratings 2 categories apart. The weighted κ method partially contributes to responses that are “near” the rating category; for example, “Very good” and “Excellent” are categorically closer than are “excellent” and “poor.” To interpret the κ score, the following guidelines are used to suggest agreement -28: 0=agIn addition, a binary interpretation of agreement results as “literally congruent” or “relatively congruent” was calculated. If the PSI and the SRH were an exact match  the congruence type was scored as literal, while if an individual’s responses to the PSI and SRH were not an exact match but consistent in terms of their position as either good  or bad health , the congruence type was scored as relative .All analyses were carried out in SAS version 9.4 (SAS Institute) .There were a total of 22,732 observations from 10,519 unique patients from January 15, 2021, to December 15, 2021. Only completed sociodemographic data for patients are represented in The distribution of responses for each scale item was skewed toward the positive . Of the r=0.51, suggesting moderate association. When the PSI scores were treated as ordinal , the polychoric correlation coefficient was nearly identical at r=0.52, also suggesting moderate association. The weighted κ coefficient between the transformed PSI and SRH was 0.46, suggesting moderate agreement  between the PSI and SRH. When the PSI was scored as an ordinal, it did not perfectly match the 5 health categories in the SRH; however, they were consistent in terms of their position as good health  versus bad health . In other words, the PSI and SRH generally point in the same directions for self-reported health categorization.t tests showed no significant difference between these correlations. We also explored whether agreement between PSI and SRH were different between the first patient visit versus subsequent visits. Agreement between PSI and SRH for patients with repeated assessments remained consistent over time, suggesting consistency for the PSI from first visit to follow-up visits.Various analyses were performed to explore association and agreement. First, we analyzed whether collapsing PSI scores into ordinal categorical variables (vs continuous) would change the association with SRH. The results were similar between continuous  and categorical (polychoric correlation) when compared to the categorical SRH. We noted that patients tended to report better health on the PSI than on the SRH. In this study, the SRH question was asked at the end of the PSI. It is possible that while answering the PSI questions, patients were reminded of their health symptoms leading them to be more likely to rate their health poorly in the SRH. The order of administration may play a role in the agreement level . Future Although the focus of this study was not to assess or describe longitudinal changes between the PSI and SRH, we did observe that the PSI captured improvement in symptoms over time with treatment . In compBeyond the effects of administration order, there are conceptual differences between the PSI and SRH that may contribute to the degree of agreement. Although they address the same broad clinical concepts, the 45-item PSI captures more information than does the single-item SRH . We woulThe PSI was created because a short-form e-PROM to capture a review of systems did not exist. Other PROMs like the PROMIS ,12, SF-3However, these results suggest that further validation of the PSI would benefit from comparing it to a PROM with similar granularity  even if this PROM would not be a perfect conceptual match. The PROMIS, SF-36, and MSQ are similar enough that we hope to compare these tools to the PSI in future studies to better understand the PSI as a conceptually valid yet distinctly useful tool.The majority of Parsley Health members are White and female, so the study population was skewed in that direction, limiting the ecological validity of our results. Additionally, there was no randomization of PSI and SRH item presentation to address response biases. As the SRH was nested within the existing PSI, the infrastructure of the electronic health record could not support randomization. Future studies should consider randomization or A/B testing. There was also a lack of conceptually and operationally similar PROMs which we could use to validate the PSI. This is the reason that we created the PSI. In this study, we chose to compare the PSI to the SRH, a single-item questionnaire, to demonstrate convergent validity. Future studies will compare the PSI to PROMs that are similar in item length if not perfect matches in their design and intent. This convergent validation study compared the best available questionnaire (SRH) to the PSI. Although the SRH and PSI fall under the same conceptual umbrella, they are different in their level of granularity. Further validation studies should compare the PSI to other multi-item, short-form PROMs of similar scope to continue the validation process. As telehealth will inevitably continue to grow, PROMs will be increasingly used and built as exclusively digital tools. Therefore, PROMs being used in the digital space must be researched and validated within the telehealth environment. This is a paradigm shift in the world of PROM development and validation. As this field evolves, we will need to assess what it means to validate a tool that is no longer administered to a captive audience in a physical waiting room, but rather, one that is engaged with remotely. Measures of engagement and “stickiness” will need to be considered as we build tools that can be completed anywhere and at any time of the day. These digital PROMs will need to be validated against previously validated tools while also being able to stand up to the test of our modern, all-access world.