Melodia: A Python Library for Protein Structure Analysis.

SUMMARY: Analysing protein structure similarities is an important step in protein engineering and drug discovery. Methodologies that are more advanced than simple RMSD are available but often require extensive mathematical or computational knowledge for implementation. Grouping and optimising such tools in an efficient open-source library increases accessibility and encourages the adoption of more advanced metrics. Melodia is a Python library with a complete set of components devised for describing, comparing and analysing the shape of protein structures using differential geometry of three-dimensional curves and knot theory. It can generate robust geometric descriptors for thousands of shapes in just a few minutes. Those descriptors are more sensitive to structural feature variation than RMSD deviation. Melodia also incorporates sequence structural annotation and three-dimensional visualisations. AVAILABILITY AND IMPLEMENTATION: Melodia is an open-source Python library freely available on https://github.com/rwmontalvao/Melodia_py, along with interactive Jupyter Notebook tutorials. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Ligament augmentation and reconstruction system (LARS) synthetic grafts are safe and effective for medial collateral ligament and posterolateral corner reconstructions in elite athletes.

PURPOSE: This study documents the efficacy and safety of using a Ligament Augmentation and Reconstruction System (LARS) ligament graft to augment extra-articular knee ligament reconstructions in elite athletes by reporting return-to-play (RTP) rates and levels, career longevity and complications. METHODS: A consecutive series of all extra-articular knee ligament reconstructions augmented by LARS ligaments in elite athletes undertaken by three specialist sports knee surgeons between 2013 and 2020 were reviewed. Seventy-six elite athletes, aged over 16 years old, and more than 2 years postsurgery were included. RTP was defined as competing at professional level or national/international level in amateur sport. RESULTS: There were 64 medial collateral ligament (MCL) and 12 posterolateral corner (PLC) reconstructions. Fifty-two (68.4%) underwent concomitant autograft cruciate ligament(s) reconstruction. The mean age was 25.1 years (SD ± 4.5). Most were football (35, 46.1%) or rugby players (35, 46.1%). Sixty-seven athletes (88.2%) RTP with 65 (97.0%) of these playing at the same or higher Tegner level. Fifty-six (83.6%) of the athletes that RTP were still playing at 2 years postsurgery. Twenty (57.1%) of those who had RTP and were more than 5 years postsurgery were still playing at 5 years. Six (7.9%) players required further surgery relating to the LARS/metalwork. One case had soft tissue inflammation adjacent to the proximal end of the synthetic graft, but it is unknown if this was mechanical irritation or a biological reaction. One MCL reruptured 4 years after RTP. CONCLUSION: Utilising LARS to augment extra-articular knee ligament reconstructions allows 88.2% of athletes with a variety of knee ligament injuries to return to elite sport. The low morbidity rates coupled with 57% of athletes still playing 5 years postsurgery demonstrates that the LARS is safe and effective in these cases. Although there are reports of LARS ligaments being used in MCL and PLC reconstructions, there is very little evidence investigating if they are safe and effective. This study demonstrates that LARS synthetic grafts can be safely used for MCL and PLC reconstructions in elite athletes and they permit a high RTP with a low risk of complications. LEVEL OF EVIDENCE: Level IV.

Rational evolution for altering the ligand preference of estrogen receptor alpha.

Estrogen receptor α is commonly used in synthetic biology to control the activity of genome editing tools. The activating ligands, estrogens, however, interfere with various cellular processes, thereby limiting the applicability of this receptor. Altering its ligand preference to chemicals of choice solves this hurdle but requires adaptation of unspecified ligand-interacting residues. Here, we provide a solution by combining rational protein design with multi-site-directed mutagenesis and directed evolution of stably integrated variants in Saccharomyces cerevisiae. This method yielded an estrogen receptor variant, named TERRA, that lost its estrogen responsiveness and became activated by tamoxifen, an anti-estrogenic drug used for breast cancer treatment. This tamoxifen preference of TERRA was maintained in mammalian cells and mice, even when fused to Cre recombinase, expanding the mammalian synthetic biology toolbox. Not only is our platform transferable to engineer ligand preference of any steroid receptor, it can also profile drug-resistance landscapes for steroid receptor-targeted therapies.

The Effect of Primary ACL Reconstruction on Career Longevity in English Premier League and Championship Soccer Players Compared With Uninjured Controls: A Matched Cohort Analysis.

BACKGROUND: Because of the multitude of variables that affect the retirement decisions of professional soccer players, it has proven difficult to isolate the effect of undergoing anterior cruciate ligament (ACL) reconstruction (ACLR) on career longevity. PURPOSE: To compare the career longevity of professional soccer players after a primary ACLR with that of an uninjured matched control cohort. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: A retrospective review of a consecutive series of primary ACLR was performed between 2008 and 2018 in professional male soccer players from the senior author's practice. Each athlete with ACLR was matched to 3 control athletes who had not undergone ACLR according to age, league, playing position, and preinjury game appearances/minutes played. Player career statistics-including league, game appearances, and game minutes-were compiled for each year until retirement or July 1, 2022. RESULTS: A total of 82 soccer players in the English Premier League or Championship at the time of their primary ACLR were matched to 246 control athletes. The mean career length after ACLR was 6 ± 2.6 years, while that of the matched control athletes was 7.6 ± 2.8 years (P < .001). After primary ACLR, an athlete had a 2 times greater chance of retirement compared with the matched control athlete (hazard ratio, 2.19; P < .001). At 5 years after ACLR, 16% of athletes had retired from professional soccer, while 8.5% of the matched cohort were retired (P = .060). By 10 years, 72% of the ACLR cohort had retired compared with 43% of the matched cohort (P < .001). Forwards were more likely to have shortened careers compared with goalkeepers (P = .021); however, no significant differences were observed between midfielders, defenders, and forwards. Within the ACLR cohort, a contralateral ACL tear during the athlete's career caused a 2.30 times (P = .022) increased chance of retirement compared with athletes with only 1 ACL tear during their career. Mechanism of injury, meniscal pathology, graft rerupture, and chondral lesions did not affect career length. CONCLUSION: Professional male soccer players who underwent ACLR had decreased career length by approximately 1.6 years compared with a matched player cohort.

Structural insights into the morpholino nucleic acid/RNA duplex using the new XNA builder Ducque in a molecular modeling pipeline.

The field of synthetic nucleic acids with novel backbone structures [xenobiotic nucleic acids (XNAs)] has flourished due to the increased importance of XNA antisense oligonucleotides and aptamers in medicine, as well as the development of XNA processing enzymes and new XNA genetic materials. Molecular modeling on XNA structures can accelerate rational design in the field of XNAs as it contributes in understanding and predicting how changes in the sugar-phosphate backbone impact on the complementation properties of the nucleic acids. To support the development of novel XNA polymers, we present a first-in-class open-source program (Ducque) to build duplexes of nucleic acid analogs with customizable chemistry. A detailed procedure is described to extend the Ducque library with new user-defined XNA fragments using quantum mechanics (QM) and to generate QM-based force field parameters for molecular dynamics simulations within standard packages such as AMBER. The tool was used within a molecular modeling workflow to accurately reproduce a selection of experimental structures for nucleic acid duplexes with ribose-based as well as non-ribose-based nucleosides. Additionally, it was challenged to build duplexes of morpholino nucleic acids bound to complementary RNA sequences.

Determinants of Performance in Professional Soccer Players at 2 and 5 Years After ACL Reconstruction.

BACKGROUND: A number of studies have investigated return to play after anterior cruciate ligament reconstruction (ACLR) in professional soccer players, but it is unclear which factors are associated with a return to the preinjury performance and ability to play over time. PURPOSE: To identify factors that contribute to a professional soccer player's return to preinjury performance after ACLR, as well as to report their playing performance at 2 and 5 years after ACLR compared with their preinjury performance. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: A consecutive cohort of professional soccer players undergoing primary ACLR were analyzed between 2005 and 2019. A minimum 2-year follow-up was required. The effect of patient, surgical, and postoperative factors on performance rates, defined as a combination of league level and playing time, was evaluated with univariate and multivariate logistic regression models. RESULTS: A total of 200 male professional soccer players were included. When combining league level and playing time, 30% of athletes returned to their preinjury performance at 2 years and 22% at 5 years. However, 53% of athletes returned to their preinjury performance for at least 1 season by year 5. At 2 years, a chondral lesion of grade 3 or 4 decreased the odds of return to preinjury performance (odds ratio [OR], 0.37; P = .010). Athletes receiving an ACLR with the addition of a lateral extra-articular tenodesis procedure were 2.42 times more likely to return to preinjury performance at 2 years than athletes with ACLR alone (P = .004). By 5 years after ACLR, athletes aged ≥25 years at the time of reconstruction were 3 times less likely to be performing at their preinjury performance (OR, 0.32; P < .001), and those with a grade ≥3 chondral lesion were >2 times less likely to be performing at their preinjury performance (OR, 0.43; P = .033). CONCLUSION: The presence of >50% thickness chondral pathology, ACLR without lateral extra-articular tenodesis, and age >25 years at the time of surgery were all significant risk factors of worse performance rates after ACLR. Significant decreases in performance rates were noted at 2 and 5 years postoperatively.

No difference in return to play rates between different elite sports after primary autograft ACL reconstruction.

PURPOSE: To demonstrate return to play (RTP) rates, the level of RTP and time taken to RTP in different sports after anterior cruciate ligament reconstruction (ACL-R) and compare the differences between football and rugby. The secondary aims were to compare the differences in intra articular injuries and their treatments and reoperation rates between these sports. METHODS: A retrospective review of a consecutive series of all primary ACL-R undertaken by the senior author between 2005 and 2019 was undertaken. Patients were included if they were elite athletes and were a minimum of 2 year post-primary autograft ACL-R. The outcomes measured were RTP (defined as participation in a professional match or in national/international-level competition in amateur sports), time to RTP after surgery and RTP level (Tegner score). RESULTS: Three hundred and ninety-four elite athletes, with 420 ACL-Rs were included. 235 (55.9%) were in footballers and 125 (29.8%) were in rugby players. 399 (95.0%) of all elite athletes returned to competition at an average of 10.3 months after ACL-R. 386 (90.2% played at the same or higher level post-surgery. Although there was no difference in RTP rates between different sports, rugby players RTP significantly faster than footballers (9.6 vs 10.6 months, (p = 0.027). Footballers were more likely to rupture their ACL during jumping/landing manoeuvres and to receive a PT graft than rugby players. There were no other significant differences between football and rugby players regarding patient characteristics, intraoperative findings, re-rupture and re-operation rates. CONCLUSIONS: Over 95% of all elite athletes RTP after primary ACL-R with 90% able to play at the same level. Rugby players RTP significantly faster than footballers. LEVEL OF EVIENCE: Level IV.

Fracture of the fabella with radiologic and MRI.

The fabella is an inconstant sesamoid bone. A handful of cases have been reported so far describing fabella fracture, usually involving high energy mechanism or stress fractures after total knee arthroplasty. This paper presents a fabella fracture in a man in his 40s after suffering a car crash, the clinical and radiographic presentation, treatment and complete recovery at 4-months follow-up.

A Comparison of All-inside and Inside-out Meniscal Repair in Elite Athletes.

BACKGROUND: The popularization of all-inside (AI) meniscal repair devices has led to a shift away from the historical gold standard of inside-out (IO) meniscal repair without comparative studies to support the change. PURPOSE: To compare the failure rate and time to failure of AI and IO meniscal repair performed in elite athletes. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: A retrospective review was performed of all professional and national- and international-level amateur athletes who underwent meniscal repair, with a minimum of 2-year follow-up between January 2013 and September 2019. Meniscal repair was classified as AI or IO depending on the surgical technique performed. Treatment failure was defined as patients having to undergo subsequent surgery to address a persistent meniscal tear after repair. Cox proportional hazards modeling was used to determine if meniscal repair failure rates differed by the location and technique of meniscal repair. Models were controlled for known risk factors such as age, sex, sport, and concurrent cruciate ligament reconstruction. RESULTS: A total of 192 (135 lateral and 57 medial) meniscal repairs were performed in elite athletes during the study period. Overall, 41 (21%) meniscal repairs met the criteria for failure. Medial meniscal tears repaired with the AI technique failed at a significantly higher rate (18/31 [58%]) than medial meniscal tears repaired with the IO technique (6/26 [23%]) or lateral meniscal tears repaired with the AI (9/76 [12%]) or IO (8/59 [14%]) technique (P < .001). Cox proportional hazards modeling revealed that a medial meniscal tear repaired with the AI technique had an almost 8 times greater hazard of failure than a lateral meniscal tear repaired with the AI technique (P < .001). At 1 year postoperatively, 8% of lateral meniscal repairs had failed (regardless of technique), while medial meniscal tears failed at a rate of 16% with the IO technique and 42% with the AI technique. By 2 years, 53% of medial meniscal tears repaired with the AI technique had failed, and by 5 years, 63% had failed. CONCLUSION: AI repair of medial meniscal tears led to a higher rate of failure than IO repair of medial or lateral meniscal tears in elite athletes. Medial meniscal repair failed at a higher rate than lateral meniscal repair.

Rebranding the 'anatomic' ACL reconstruction: Current concepts.

The anterior cruciate ligament (ACL) is a complex ribbon-like structure, which is approximately 3.5 times larger at the tibial and femoral insertions than at the midpoint. Accordingly, it is impossible to recreate with a single cylindrical graft. However, this has not stopped surgeons from using the term "anatomic" to describe multiple ACL reconstruction techniques inserting at a number of different locations within the original ACL footprint, causing confusion. The term "anatomic" should be discarded and replaced by an anatomic description of the tunnel placements on the tibia and femur. Current ACL reconstruction techniques cite anatomical studies that identified "direct and indirect fibres" of the ACL. The "direct fibres" bear 85-95% of the load and provide the main resistance to both anterior tibial translation and internal rotation/pivot shift. On the femur, these fibres insert in a line just posterior to the intercondylar ridge and comprise the portion of the ACL that surgeons should strive to restore. Placement of the graft just posterior to the intercondylar ridge creates a line of placement options from the anteromedial bundle to the "central" position and finally to the posterolateral bundle position. The authors prefer placing the femoral tunnel in the isometric anteromedial position and addressing a high-grade pivot shift at the IT-band with a lateral extra-articular tenodesis. As with the femoral tunnel, the native ACL footprint on the tibia is much larger than the ACL graft and thus can be placed in multiple "anatomic" locations. The authors prefer placement of the tibial tunnel in the anterior most position of the native footprint that does not cause impingement in the femoral notch. Additional research is needed to determine the ideal tunnel positions on the femur and tibia and validating the technique with patient outcomes. However, this cannot be accomplished without describing tunnel placement with specific anatomical locations so other surgeons can replicate the technique.

Rates and Levels of Elite Sport Participation at 5 Years After Revision ACL Reconstruction.

BACKGROUND: There is a paucity of data regarding return to play (RTP), level of competition, and longevity of play after revision of anterior cruciate ligament (ACL) reconstruction (ACLR) in elite athletes. PURPOSE: To report RTP rates and competition levels in elite athletes at the point of RTP, as well as at 2 and 5 years after revision ACLR, and the effect of meniscal and chondral pathology at revision surgery on these outcomes. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: A retrospective review of a consecutive series of all revision ACLRs undertaken by the senior author between 2009 and 2019, with a minimum 2-year follow-up, was carried out. Outcome measures were RTP rates and competition level. RESULTS: A total of 49 knees in 48 elite athletes met the inclusion criteria. After revision ACLR, 43 (87.8%) elite athletes achieved RTP, of whom 75.5% were at the same level. At 2 years after surgery, 39 (79.6%) were still playing, 25 (51%) at the same level; at 5 years after surgery, 20 (44.4%) were still playing, 9 (20%) at the same level. Elite athletes with <50% thickness or no articular cartilage lesions were more likely to RTP (94.6% vs 66.7%; P = .026), as well as return to the same competition level (83.8% vs 50%; P = .047), compared with those with ≥50% thickness chondral lesions. Those without medial meniscal pathology were more likely to RTP at the same level after revision surgery (94.4% vs 64.5%; P = .036). The median time elite athletes continued to play after revision ACLR was 73 months (95% CI, 43.4-102.6); 23 months at the same level (95% CI, 13.6-32.4). The probability of still playing at 5 years after surgery was 55.9%, with a 22.5% chance of maintaining preinjury competition level. CONCLUSION: In elite athletes, RTP rates and competition level decreased over time after revision ACLR. The presence of >50% thickness chondral pathology was associated with lower RTP rates and competition level at RTP time, while medial meniscal pathology was associated with lower competition level at RTP.

Effect of Lateral Extra-articular Tenodesis on the Rate of Revision Anterior Cruciate Ligament Reconstruction in Elite Athletes.

BACKGROUND: There is growing evidence that anterolateral procedures can reduce the risk of rerupture in high-risk recreational athletes undergoing primary anterior cruciate ligament (ACL) reconstruction (ACLR). However, this effectiveness has never been evaluated in elite athletes. PURPOSE: The purpose of this study was to evaluate the effectiveness of lateral extra-articular tenodesis (LET) in reducing revision rates in primary ACLR in elite athletes. Additionally, this study evaluated whether LET had a greater effect when combined with ACLR utilizing a hamstring or patellar tendon graft. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: A consecutive cohort of elite athletes with an isolated ACL tear undergoing autograft patellar or hamstring tendon reconstruction with or without Lemaire LET were analyzed between 2005 and 2018. A minimum 2-year follow-up was required. The association between the use of LET and ACL graft failure as defined by revision ACLR was evaluated with univariate and multivariate logistic regression models. RESULTS: A total of 455 elite athletes (83% men and overall age 22.5 ± 4.7 years) underwent primary ACLR with (n = 117) or without (n = 338) a LET procedure. Overall, 36 athletes (7.9%) experienced ACL graft failure, including 32 (9.5%) reconstructions without a LET and 4 (3.4%) with a LET. Utilization of LET during primary ACLR reduced the risk of graft failure by 2.8 times, with 16.5 athletes needing LET to prevent a single ACL graft failure. Multivariate models showed that LET significantly reduced the risk of graft rupture (relative risk = 0.325; P = .029) as compared with ACLR alone after controlling for sex and age at ACLR. Including graft type in the model did not significantly change the risk profile, and although a patellar tendon graft had a slightly lower risk of failure, this was not statistically significant (P = .466). CONCLUSION: The addition of LET reduced the risk of undergoing revision by 2.8 times in elite athletes undergoing primary ACLR. This risk reduction did not differ significantly between the patellar tendon and hamstring tendon autografts. With these results, status as an elite athlete should be included in the indications for a LET, as they are at increased risk for ACL graft failure.

Directed evolution of colE1 plasmid replication compatibility: a fast tractable tunable model for investigating biological orthogonality.

Plasmids of the ColE1 family are among the most frequently used in molecular biology. They were adopted early for many biotechnology applications, and as models to study plasmid biology. Their mechanism of replication is well understood, involving specific interactions between a plasmid encoded sense-antisense gene pair (RNAI and RNAII). Due to such mechanism, two plasmids with the same origin cannot be stably maintained in cells-a process known as incompatibility. While mutations in RNAI and RNAII can make colE1 more compatible, there has been no systematic effort to engineer new compatible colE1 origins, which could bypass technical design constraints for multi-plasmid applications. Here, we show that by diversifying loop regions in RNAI (and RNAII), it is possible to select new viable colE1 origins compatible with the wild-type one. We demonstrate that sequence divergence is not sufficient to enable compatibility and pairwise interactions are not an accurate guide for higher order interactions. We identify potential principles to engineer plasmid copy number independently from other regulatory strategies and we propose plasmid compatibility as a tractable model to study biological orthogonality.

Long-term serological SARS-CoV-2 IgG kinetics following mRNA COVID-19 vaccine: real-world data from a large cohort of healthcare workers.

OBJECTIVES: This study aimed to assess kinetics and predictive variables of humoral immune response to mRNA SARS-CoV-2 vaccine administration. METHODS: We collected blood samples before (T0) and 15, 90, and 180 days after vaccination (T1, T2, and T3, respectively). The Quant SARS-CoV-2 Immunoglobulin (IgG) II Chemiluminescent Microparticle Immunoassay was used to determine anti-spike IgG. RESULTS: In almost 3000 healthcare-collected blood samples at the three time points, we found the following: at 15 days postvaccination, 97.6% of subjects presented a robust IgG anti-spike response (>4160 AU/ml); then, at three and six months, it decreased in median 6.5-fold to 35.0% and 3.0-fold to 3.3%, respectively. A linear mixed-effects model supported that female gender, younger age groups, and being seropositive prevaccination maintained higher antibody titers. Curves became tighter with time progression, although titers from seropositive subjects decrease at a slower rate than seronegative ones. CONCLUSION: These findings strengthen the case for a steep decrease of anti-SARS-CoV-2 antibodies up to six months, suggesting that serological evaluation might guide the need for periodic booster vaccinations in specific groups prone to lower antibody titers.

High return to play rate following treatment of multiple-ligament knee injuries in 136 elite athletes.

PURPOSE: The purpose of the study is to assess return to play rate and timing of elite athletes treated for multi-ligament knee injuries with modern surgical techniques. METHODS: A retrospective review was performed of all professional or national/international level athletes treated for a multi-ligament knee injury by the senior author in the period from February 2001 to October 2019, with a minimum of 2-year follow-up. The study cohort consisted of 136 elite athletes who underwent surgery of two or more knee ligaments. Outcomes measures were return to play (RTP), defined as return to elite level sport, return to play time, and further surgical procedures. RESULTS: One hundred and twenty (88.2%) athletes returned to elite level sport at an average of 12.8 months from operation. In unicruciate injuries, ACL and PCL-based injuries had similar return to play rates (89.1% vs. 87.5% respectfully, n.s.) although mean RTP time was longer in those with PCL-based injuries (15.2 vs. 11.9 months, p < 0.01). Bicruciate injuries had longer RTP times compared to unicruciate injuries (mean RTP 16.0vs. 12.4 months, p < 0.05) but were able to return at a similar rate (83.3% vs. 88.9%, n.s.). Medial and lateral sided injuries had similar RTP rates (89.3% vs. 87.9%, n.s.) though lateral sided injuries took longer before returning to play (13.4 vs. 11.6 months, p < 0.05). Further surgery was required in 54 (39.9%) patients including 25 (18.4%) manipulations under anaesthesia. Four (2.9%) of the athletes underwent revision ligament surgery. CONCLUSION: Modern surgical techniques used in the treatment of multi-ligament knee injuries can lead to high return to play rates in elite athletes. LEVEL OF EVIDENCE: IV.

Fast quantification of gut bacterial species in cocultures using flow cytometry and supervised classification.

A bottleneck for microbial community experiments with many samples and/or replicates is the fast quantification of individual taxon abundances, which is commonly achieved through sequencing marker genes such as the 16S rRNA gene. Here, we propose a new approach for high-throughput and high-quality enumeration of human gut bacteria in a defined community, combining flow cytometry and supervised classification to identify and quantify species mixed in silico and in defined communities in vitro. We identified species in a 5-species in silico community with an F1 score of 71%. In addition, we demonstrate in vitro that our method performs equally well or better than 16S rRNA gene sequencing in two-species cocultures and agrees with 16S rRNA gene sequencing data on the most abundant species in a four-species community. We found that shape and size differences alone are insufficient to distinguish species, and that it is thus necessary to exploit the multivariate nature of flow cytometry data. Finally, we observed that variability of flow cytometry data across replicates differs between gut bacterial species. In conclusion, the performance of supervised classification of gut species in flow cytometry data is species-dependent, but is for some combinations accurate enough to serve as a faster alternative to 16S rRNA gene sequencing.

Factors affecting return to play and graft re-rupture after primary ACL reconstruction in professional footballers.

PURPOSE: Modern ACL reconstruction (ACL-R) techniques have led to improved outcomes in professional footballers. The aim of this study was to identify and assess patient, surgical and post-operative factors that affected rates and time to return to play (RTP) as well as ACL re-rupture rates. METHODS: A retrospective review of consecutive ACL-R undertaken in professional footballers between 2005 and 2018. RESULTS: Two-hundred and thirty-two knees in 215 professional footballers (17 bilateral) were included. 205 (88.9%) were male and average age at surgery was 23.3 ± 4.4 years. Two-hundred and twenty-two (96.1%) returned to professional football, with 209 (90.1%) returning to the same or higher Tegner level. Subgroup analysis revealed three factors that independently affected RTP rate: (1) Players under 25 years had a higher rate of RTP (99.3% vs 90.2%. p = 0.001); (2) a subsequent operation prior to RTP decreased RTP rate from 98.2 to 89.7% (p = 0.009).; (3) undergoing meniscal surgery at ACL-R decreased RTP rate (p = 0.002). The mean time to RTP from surgery was 10.5 ± 3.6 months. Factors found to increase RTP time included age under 25 (11.0 vs 9.7 months, p = 0.005), recurrent effusions (11.4 vs 10.2 months, p = 0.035), and medial meniscal repair at ACL-R compared to meniscectomy (12.5 vs 9.6 months, p = 0.022). The surgical technique varied over the study period in relation to graft type, femoral tunnel position and addition of lateral extra-articular tenodesis (LET). Overall, the re-rupture rate was 8.2% at 2 years. Patella tendon autograft in an anteromedial bundle femoral tunnel position with addition of LET has the lowest re-rupture rate (2.0%). CONCLUSION: Primary ACL-R in professional footballers yields high rates of RTP (96.1%), with 90.1% at the same level or higher, at a mean 10.5 months. Patients under 25 years not only had a significantly higher RTP rate, but also had a lengthier period of rehabilitation. LEVEL OF EVIDENCE: Level IV.

Humoral response to the SARS-CoV-2 BNT162b2 mRNA vaccine: Real-world data from a large cohort of healthcare workers.

BACKGROUND: The SARS-CoV-2 pandemic was responsible for the death of millions of people around the world, which accelerated the study of vaccines. The BNT162b2 mRNA COVID-19 is a messenger RNA vaccine that encodes the spike protein of the virus. However, the duration of the protection conferred by this vaccine and factors associated with immune responses require validation in large cohorts. METHODS: Here, we present data of humoral immune response to vaccination in4264 healthcare workers, tested before (T0) and 15 and 90 days (T1 and T2, respectively) following vaccination.Peripheral blood was collected for immunological analysis using the Quant SARS-CoV-2 IgG II Chemiluminescent Microparticle Immunoassay (CMIA) to determine anti-spike IgG, receptor binding domain (RBD), S1 subunit of SARS-CoV-2. FINDINGS: At T0, 96·8% (n = 4129) of participants had IgG antibodies non-reactive to anti-SARS-CoV-2. Fifteen days after completing the vaccination, the IgG overall median titer was significantly elevated (21·7x103AU/mL). Both for uni- and multivariate logistic regression analyses women presented higher antibody levels than men, independent of age. Titers were significantly altered among age groups, decreasing by each increase in 10-year of age. At 3 months after completing the vaccination, anti-SARS-CoV-2 IgG titers were 6·3-fold diminished. This real-world post-vaccination data confirmed production of a frequent and elevated anti-SARS-CoV-2 IgG titers, associated with high protection rates. Females and younger participants had higher titer 15 days after vaccination, and despite the significant reduction from 15-to-90 days, those with higher pre-vaccination titers maintained higher levels throughout the remaining timepoints. INTERPRETATION: These findings support the need to track humoral immunity kinetics to uncover viral susceptibility and eventually implement re-vaccination, particularly in groups prone to lower humoral immune response. FUNDING: No external funding was received to conduct this study.

A novel framework for engineering protein loops exploring length and compositional variation.

Insertions and deletions (indels) are known to affect function, biophysical properties and substrate specificity of enzymes, and they play a central role in evolution. Despite such clear significance, this class of mutation remains an underexploited tool in protein engineering with few available platforms capable of systematically generating and analysing libraries of varying sequence composition and length. We present a novel DNA assembly platform (InDel assembly), based on cycles of endonuclease restriction digestion and ligation of standardised dsDNA building blocks, that can generate libraries exploring both composition and sequence length variation. In addition, we developed a framework to analyse the output of selection from InDel-generated libraries, combining next generation sequencing and alignment-free strategies for sequence analysis. We demonstrate the approach by engineering the well-characterized TEM-1 ß-lactamase Ω-loop, involved in substrate specificity, identifying multiple novel extended spectrum ß-lactamases with loops of modified length and composition-areas of the sequence space not previously explored. Together, the InDel assembly and analysis platforms provide an efficient route to engineer protein loops or linkers where sequence length and composition are both essential functional parameters.

Structural Studies of HNA Substrate Specificity in Mutants of an Archaeal DNA Polymerase Obtained by Directed Evolution.

Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants.