NSQIP data collection up to 30 postoperative days is sufficient to capture some complications in orthopedic surgeries.

PURPOSE: The primary aim of this study is to determine if the 30-day follow-up period used by the National Surgical Quality Improvement Program (NSIQP) is an appropriate timeframe to capture complications after orthopedic surgeries. METHODS: The 2019 NSQIP data were used. The independent variables were complication type. The dependent variable was days to complication. A Shapiro-Wilk test was used to determine if the data were normally distributed. RESULTS: 271,397 orthopedic cases were included. Myocardial infarction, pneumonia, ventilator over 48 h, progressive renal insufficiency, acute renal failure, stroke, and cardiac arrest had positive skewness and positive kurtosis. Deep incisional surgical site infection (SSI), organ/space SSI, wound disruption, unplanned reoperation one, unplanned reoperation two, readmission two, and readmission three had negative kurtosis and negative skewness. Complications with positive kurtosis and positive skewness are more likely to be confined to the 30-day postoperative period, whereas complications with negative skewness and negative kurtosis may be underreported within the 30-day follow-up. CONCLUSIONS: These findings are useful in their ability to inform future orthopedic research using NSQIP which continues to generate new data for surgeons to consider for their postoperative care and complication management.

Dual Plating of Distal Femoral Fractures.

Background: Dual plating of the distal femur is indicated for the treatment of complex intra-articular fractures, supracondylar femoral fractures, low periprosthetic fractures, and nonunions. The aim of this procedure is anatomical alignment of the articular surface, restoration of the articular block, and prevention of varus collapse. Description: Following preoperative planning, the patient is positioned supine with the knee flexed at 30°. The lateral incision is made first, with a mid-lateral incision that is in line with the femoral shaft. If intra-articular work is needed this incision can be extended by curving anteriorly over the lateral femoral condyle. Next, the iliotibial band is transected in line with its fibers. The vastus lateralis fascia is incised and elevated off the septum, working distal to proximal. Care should be taken to maintain hemostasis when encountering femoral artery perforating vessels. Once there is adequate exposure, several reduction aids can be utilized, including a bump under the knee, Schanz pins, Kirschner wires, and reduction clamps. A lateral precontoured plate is placed submuscularly, and the most proximal holes are filled percutaneously. The medial incision begins distally at the adductor tubercle and is a straight incision made proximally in line with the femoral shaft. The underlying fascia is transected in line with the skin incision, and the vastus medialis is elevated. Care should be taken to avoid the descending geniculate artery, as well as its articular branch and the muscular branch to the vastus medialis. A lateral tibial plateau plate is contoured and placed. Alternatives: Nonoperative treatment of distal femoral fractures is rare, but relative indications for nonoperative treatment include frailty of the patient, lack of ambulatory status, a non-reconstructible fracture, or a stable fracture. These patients are placed in a long-leg cast followed by a hinged knee brace1. There are several other surgical fixation options, including lateral plating, retrograde intramedullary nailing, distal femoral replacement, and augmentation of a retrograde nail with a plate. Rationale: Dual plating has several benefits, depending on the clinical scenario. Biomechanical studies have found that dual plating results in increased stiffness and construct strength2,3. Additional construct stability can be offered through the use of locking plates, particularly in osteoporotic bone. Taken together, this increased stability and construct strength can allow for earlier weight-bearing, which is particularly important for fractures in the geriatric population. Furthermore, the increased stiffness and construct strength make this procedure a favorable treatment option for nonunion, and it has been shown to result in lower rates of postoperative nonunion compared with lateral plating alone4-7. Adjunctive use of a medial plate also has been suggested to prevent varus collapse, particularly with metaphyseal comminution and poor bone quality2,3,8. Finally, in the periprosthetic fracture population, dual plating also removes the concern of incompatibility with a retrograde nail. Expected Outcomes: The outcomes of dual plating are promising, given the severity of the injury. When comparing operative to nonoperative treatment outcomes, nonoperatively managed patients had worse functional outcomes and higher rates of complications related to immobility1. Dual plating of supracondylar fractures and intra-articular distal femoral fractures yields nonunion rates ranging from 0% to 12.5%, lower than the 18% to 20% reported with lateral locking plates4-7,9-12. This reduction in nonunions has been shown to lead to fewer revisions when compared with single-plating techniques7. In prior studies, 95% of nonunions treated with the dual-plating technique achieved union postoperatively11. One concern when utilizing the medial approach is critical damage to medial vascularity; however, this result has not been reported in the literature, and there is a safe operating window13. Despite the benefits of dual plating, there are relatively high rates of infection following dual plating (0% to 16.7%) compared with lateral plating alone (3.6% to 8.5%)5,14-17. However, many of these studies are small case series, highlighting that a surgeon's comfort and skill with these procedures is paramount to patient outcomes. Important Tips: Meticulous placement and contouring of lateral and medial plates are required to prevent malreduction of the articular block that creates a "golf-club deformity."18,19During the medial approach, be aware of descending geniculate artery-particularly its muscular branch, which is ∼5 cm from the adductor tubercle/medial epicondyle, and its root, which enters the compartment at the adductor hiatus at ∼16 cm13.

Improved detection of low-frequency within-host variants from deep sequencing: A case study with human papillomavirus.

High-coverage sequencing allows the study of variants occurring at low frequencies within samples, but is susceptible to false-positives caused by sequencing error. Ion Torrent has a very low single nucleotide variant (SNV) error rate and has been employed for the majority of human papillomavirus (HPV) whole genome sequences. However, benchmarking of intrahost SNVs (iSNVs) has been challenging, partly due to limitations imposed by the HPV life cycle. We address this problem by deep sequencing three replicates for each of 31 samples of HPV type 18 (HPV18). Errors, defined as iSNVs observed in only one of three replicates, are dominated by C→T (G→A) changes, independently of trinucleotide context. True iSNVs, defined as those observed in all three replicates, instead show a more diverse SNV type distribution, with particularly elevated C→T rates in CCG context (CCG→CTG; CGG→CAG) and C→A rates in ACG context (ACG→AAG; CGT→CTT). Characterization of true iSNVs allowed us to develop two methods for detecting true variants: (1) VCFgenie, a dynamic binomial filtering tool which uses each variant's allele count and coverage instead of fixed frequency cut-offs; and (2) a machine learning binary classifier which trains eXtreme Gradient Boosting models on variant features such as quality and trinucleotide context. Each approach outperforms fixed-cut-off filtering of iSNVs, and performance is enhanced when both are used together. Our results provide improved methods for identifying true iSNVs in within-host applications across sequencing platforms, specifically using HPV18 as a case study.

Femoral rotational osteotomy for femoroacetabular impingement: A systematic review.

Purpose: To synthesize existing literature regarding the indications and outcomes of femoral rotational osteotomies (FDO) for femoroacetabular impingement (FAI) due to. Methods: Medline, Cochrane, and Embase were searched using keywords "femoroacetabular impingement", "rotational osteotomy" and others to identify FAI patients undergoing FDO. Double-screened studies were reviewed by blinded authors according to inclusion criteria. Data from full texts was extracted including study type, number of patients, sex, mean age, surgical indication, type of dysplasia, associated pathology, surgical technique, follow-up, and pre-op/post-op evaluations of the following: impingement test, femoral version (FV), 'other angles measured', outcome scores, range of motion (ROM). Results: 7 studies including 91 patients (97 FDO surgeries), 73 females (80 %) with mean age of 28.3 years, and follow-up mean of 2.44 ± 2.83 years. Pain or impingement was the most common clinical indication, while others included aberrant FV and ROM measurements for both anteverted and retroverted femurs. There were reports of FDO being performed with concomitant procedures addressing other pathology. Various outcome scores and ROM measurements showed postoperative improvement after FDO. Complication data was sparse, preventing aggregation. The rate of unplanned reoperation was 40 % (where reported), with 'hardware removal' being the most common. Conclusions: FDO is effective in treating FAI due to increased FV, improving clinical symptoms, and potentially delaying articular degeneration. Hardware removal surgery remains an inherent risk in undergoing FDO. Further work is needed to discover indications warranting FDO as a primary treatment versus hip arthroscopy. Level of evidence: This review contains 4 studies with Level IV evidence and 3 studies with Level III evidence.

Surgical Outcomes and Associated Injuries of Anterior Labroligamentous Periosteal Sleeve Avulsion (ALPSA) Lesions: A Systematic Review.

Introduction: Anterior Labroligamentous Periosteal Sleeve Avulsion (ALPSA) lesions can lead to chronic shoulder instability and repetitive dislocations in active populations. Objective: The purpose of this systematic review was to evaluate associated injuries and postoperative outcomes following ALPSA lesion repairs. Methods: Medline, Embase, Cochrane, and Web of Science were searched through May 2022 for studies that investigated management and surgical outcomes of ALPSA lesion repair. Data was extracted on the following topics: surgical management, surgical complications, associated injuries, follow-up duration, and outcome parameters, including recurrence rates, functional outcome scores, range-of-motion (ROM), and return to activity. Results: A total of 6 studies covering 202 patients met the inclusion criteria. In the included studies, 79% of patient were male with a mean age of 25.1 years. A total of 192 associated injuries were reported amongst 176 patients with the most common being Hill Sachs lesions (84, 43.8%), synovitis (35, 18.2%), SLAP tears (32, 16.7%) and glenoid erosions or lesions (30, 15.6%). All 202 patients were treated arthroscopically with no reported complications. 26 patients (12.9%) experienced operative failure as evidenced by recurrence of shoulder instability over a mean follow-up of 4.3 years. Various clinical outcome scores showed postoperative functional improvement and one study reported a 100% return to activity rate in 26 patients. Conclusion: Our findings suggest a high 12.9 % risk of recurrence following ALPSA repair but satisfactory functional outcomes, both of which should be weighed by physicians when considering arthroscopic repair. Physicians should also be cognizant of co-pathologies when examining patients with suspected ALPSA lesions.

Return to sport and active military duty after cervical disc arthroplasty: A systematic review.

Introduction: Cervical disc arthroplasty (CDA) has been established as an effective treatment for cervical disc degeneration or herniation in the general population. Return to sport (RTS) outcomes in athletes remain unclear. Objective: The purpose of this review was to evaluate RTS following single-level, multi-level, or hybrid CDA, with additional return to activity context provided by return to duty (RTD) outcomes in active-duty military. Methods: Medline, Embase, and Cochrane were searched through August 2022 for studies that reported RTS/RTD after CDA in athletic or active-duty populations. Data was extracted on the following topics: surgical failures/reoperations, surgical complications, RTS/RTD, and postoperative time to RTS/RTD. Results: Thirteen papers covering 56 athletes and 323 active-duty members were included. Athletes were 59% male with a mean age of 39.8 years and active-duty members were 84% male with a mean age of 40.9 years. Only 1 of 151 cases required reoperation and only 6 instances of surgical complications were reported. Classified as return to general sporting activity, RTS was observed in 100% of patients (n = 51/51) after an average of 10.1 weeks to training and 30.5 weeks to competition. RTD was observed in 88% of patients (n = 268/304) after an average of 11.1 weeks. Average follow-up was 53.1 months for athletes and 13.4 months for the active-duty population. Conclusion: CDA displays excellent RTS and RTD rates in physically demanding populations at rates superior or equivalent to alternative treatments. Surgeons should consider these findings when determining the optimal cervical disc treatment approach in active patients.

Minimal Clinically Important Differences of Patient-Reported Outcomes Measurement Information System Physical Function in Patients With Tibial Shaft Fracture.

OBJECTIVE: To calculate the minimal clinically important differences (MCIDs) of patient-reported outcomes measurement information system physical function (PROMIS PF) scores for patients with operatively treated tibial shaft fractures. DESIGN: Retrospective Cohort Study. SETTING: A Level 1 trauma center. PATIENTS: All operatively treated tibial shaft fractures identified by Current Procedural Terminology codes. INTERVENTION: Enrolled patients treated acutely with operative fixation of their tibia. MAIN OUTCOME MEASUREMENTS: MCIDs were calculated by distribution-based and anchor-based methods, calculated from PROMIS PF scores completed at least at two-time points postoperatively. MCIDs were calculated at different time points including overall, 7-12 weeks, 3-6 months, and 6-24 months. MCIDs were calculated for different subgroups including open fractures, closed fractures, any complications, and no complications. RESULTS: MCID for PROMIS PF scores was 5.7 in the distribution-based method and 7.84 (SD 18.65) in the anchor-based method. At 6-24 postoperatively, the months the distribution-based MCID was 5.95 from a postoperative baseline 27.83 (8.74) to 42.85 (9.61), P < 0.001. At 6-24 months, the anchor-based MCID was 10.62 with a score difference between the improvement group of 16.03 (10.73) and the no improvement group of 5.41 (15.75), P < 0.001. Patients with open fractures (distribution-based 6.22 and anchor-based 8.05) and any complications (distribution-based 5.71 and anchor-based 9.29) had similar or higher MCIDs depending on the methodology used than the overall cohort MCIDs. CONCLUSION: This study identified distribution-based MCID of 5.7 and anchor-based MCID of 7.84 calculated from PROMIS PF scores in operative tibial shaft fractures. Distribution-based methods yielded smaller MCIDs than anchor-based methods. These MCID scores provide a standard to compare clinical and investigational outcomes.

Human papillomavirus genomics: Understanding carcinogenicity.

Human papillomavirus (HPV) causes virtually all cervical cancers and many cancers at other anatomical sites in both men and women. However, only 12 of 448 known HPV types are currently classified as carcinogens, and even the most carcinogenic type - HPV16 - only rarely leads to cancer. HPV is therefore necessary but insufficient for cervical cancer, with other contributing factors including host and viral genetics. Over the last decade, HPV whole genome sequencing has established that even fine-scale within-type HPV variation influences precancer/cancer risks, and that these risks vary by histology and host race/ethnicity. In this review, we place these findings in the context of the HPV life cycle and evolution at various levels of viral diversity: between-type, within-type, and within-host. We also discuss key concepts necessary for interpreting HPV genomic data, including features of the viral genome; events leading to carcinogenesis; the role of APOBEC3 in HPV infection and evolution; and methodologies that use deep (high-coverage) sequencing to characterize within-host variation, as opposed to relying on a single representative (consensus) sequence. Given the continued high burden of HPV-associated cancers, understanding HPV carcinogenicity remains important for better understanding, preventing, and treating cancers attributable to infection.

The Role of Cryotherapy After Total Knee Arthroplasty: A Systematic Review.

BACKGROUND: Previous research shows conflicting evidence regarding the postoperative role of cryotherapy after total knee arthroplasty (TKA). This systematic review aims to further investigate the effect of various methods of cryotherapy on the following: (1) pain; (2) swelling; (3) postoperative opioid use; and (4) range of motion (ROM). METHODS: A strategic keyword search of Medline, Cochrane, Embase, and CINAHL retrieved randomized controlled trials examining cryotherapy following TKA published between February 1, 2017, and February 24, 2022. The studied outcomes included pain ratings, knee/limb swelling, opioid use, and ROM. Six studies were selected for inclusion in this review. RESULTS: Opioid use was significantly decreased in cryotherapy groups compared to noncryotherapy groups within the first postoperative week only (P < .05). This effect may be augmented by the use of computer-assisted (temperature regulated) cryotherapy devices, compared to other modalities including ice packs. Pain ratings also decrease, but this decrease may not be clinically relevant. Cryotherapy appears to confer no consistent benefit to ROM and swelling at any time point. Computer-assisted cryotherapy may be associated with decreased opioid consumption after TKA compared to traditional ice packs. CONCLUSION: Cryotherapy's role after TKA appears to be in decreasing opioid consumption primarily in the first postoperative week. Pain ratings also decrease consistently with cryotherapy use, but this decrease may not be clinically relevant. Study heterogeneity requires further research focusing on optimizing cryotherapy modalities within the first postoperative week, and analyzing cost associated with modern outpatient postoperative TKA protocols.

Posterior Approach for Open Reduction and Internal Fixation for Scapular Fractures.

Background: This technique utilizes a full-thickness flap to provide a posterior approach to the scapula for open reduction and internal fracture fixation. The present video article outlines the Judet approach along with an incision modification tip for the surgeon's consideration. Description: Prior to making the incision, perform preoperative planning, patient and C-arm positioning, and identification of the primary fragments of the fracture that necessitate fixation on imaging. The Judet incision is made, and the full-thickness flap is retracted laterally (also described as a "boomerang-shaped" incision, allowing for the flap to be reflected medially). Next, detach and reflect the deltoid off the scapular spine superolaterally to reveal the internervous plane between the infraspinatus and teres minor. Utilize this interval to access the fracture sites while making sure to reflect the infraspinatus cranially, carefully minding the suprascapular neurovascular bundle, and the teres minor inferiorly, protecting the axillary nerve. A longitudinal arthrotomy may then be created parallel to the posterior border of the glenoid, with careful attention paid toward protecting the labrum from iatrogenic injury. The arthrotomy will allow for intra-articular evaluation of the reduction if needed. Primary fractures are then reduced. Reduction is confirmed with use of fluoroscopy, and fixation is applied to maintain the reduction. Alternatives: Most scapular fractures do well with nonoperative treatment, and this has been well documented in the literature. Open reduction and internal fixation has been shown to offer good-to-excellent clinical outcomes with minimal risk of complications in patients with traumatic scapular fractures that necessitate operative treatment1. In certain fractures of the glenoid fossa, operative treatment is necessary to restore normal anatomy, provide stability to the glenohumeral joint, and facilitate functional rehabilitation. Operative treatment is typically reserved for injuries with intra-articular involvement that results in joint incongruity or joint instability2,3. When operative treatment is indicated, an open posterior approach is utilized for some fractures. The posterior Judet approach is the best-known operative technique for such fractures, while other modifications of the Judet technique have also been described in the literature3-5. Rationale: Reports state that scapular body or neck and glenoid fossa fractures account for up to 80% of scapular fractures6. Open reduction and internal fixation of the scapula is an invasive procedure, requiring large incisions and manipulation of soft tissues to expose the various possible fracture sites on the scapula. Thus, numerus surgical techniques have been described that allow surgeons to best tailor treatment to their patients on a case-by-case basis. However, the Judet approach is the workhorse approach for the operative treatment of scapular fractures and is a technique that should be mastered7. The Judet approach allows access to the posterior scapula and provides excellent exposure for fractures that require posterior fixation. The alternative boomerang-shaped incision represents a mirrored version of the Judet incision, with the skin flap reflected medially. The benefit of this modified approach is that it increases the degree of lateral surgical exposure of the scapula and provides easier access to the glenohumeral joint. Expected Outcomes: With this technique for open reduction and internal fixation of scapular fractures, patients can expect comparable outcomes to those described in the literature for the standard Judet technique. These outcomes have been reported as clinical scores and defined as good-to-excellent in a few retrospective case series1,2. Given the variability in scapular fracture morphology, a trauma surgeon should have a strong repertoire of approaches to address these fractures on a case-by-case basis. The Judet approach is one of these necessary approaches and has been shown in the literature to have acceptable outcomes1-3,7. Important Tips: Placing the vertical limb of the boomerang incision too medial can limit lateral exposure of the scapula and make glenohumeral joint access difficult. To avoid this, be sure that the vertical limb of the incision remains in line with the posterior axillary fold.Wound-healing complications can occur following such an extensive surgical approach. A thorough and secure wound closure with repair of the deltoid back to the scapular spine may avoid these problems.Difficulty with intra-articular visualization may occur. Placing a threaded pin into the humeral head or a small distractor across the glenohumeral joint (with a pin in the extra-articular proximal humerus) may improve visualization. Manipulation of the arm can also be beneficial in this regard.Lateral positioning offers easier imaging and allows for exposure to the coracoid or clavicle if these structures are also injured and require operative fixation.Drawing a boomerang-shaped incision with the horizontal limb paralleling the scapular spine and vertical limb along the posterior axillary fold of the arm allows the skin flap to be reflected medially, increasing the degree of lateral surgical exposure of the scapula.After identifying the internervous plane between the infraspinatus and teres minor, take care to reflect the infraspinatus cranially, protecting the suprascapular neurovascular bundle, and the teres minor inferiorly, protecting the axillary nerve. Acronyms and Abbreviations: ORIF = open reduction and internal fixationK-wire = Kirschner wire.

An Arthroscopic-assisted AC Joint Reconstruction and Hook Plate Fixation for the Treatment of AC Joint Dislocation: A Unique Surgical Technique.

Introduction: Acromioclavicular Joint (ACJ) dislocations can lead to increased pain and disability. On failure of conservative management, surgical intervention is often recommended. At present, there is no gold standard surgical technique to treat this pathology. The current surgical options include coracoclavicular screw, hook plate (HP), endobutton coracoclavicular fixation, ligament reconstruction, and among others. Unfortunately, all these techniques have been reported to have increased complications such as recurrent dislocation, impingement, and among others. Case Report: A 41-year-old healthy male presented to the clinic with a symptomatic chronic right Grade 3 ACJ dislocation. He failed conservative treatment, including NSAID's, rest, and physical therapy. We agreed to pursue an arthroscopic assisted ACJ reconstruction with allograft and joint fixation with a HP. Using a combined technique, we performed an arthroscopic-assisted ACJ reconstruction with an allograft and supplemental HP for additional fixation. This case report details the unique technique. Using this technique, the patient had improved pain and ACJ stability long-term. After 6 months, the HP was removed and the patient continued to remain asymptomatic and without instability. Conclusion: An arthroscopic-assisted ACJ reconstruction and HP fixation provide orthopedic surgeons a novel technique for the treatment of an ACJ dislocation. Further prospective studies are still recommended for further evaluation.

Indications, management, and outcomes of humeral avulsions of the glenohumeral ligament: a systematic review.

Purpose: To provide an updated overview of recent literature regarding the clinical indications, management, and outcomes of humeral avulsions of the glenohumeral ligament (HAGL) lesion. Methods: Medline, Cochrane and Embase were systematically searched using keywords "humeral avulsion of the glenohumeral ligament" and others to identify studies reporting on management of HAGL lesions. Studies were double-screened and reviewed by two blinded authors according to selection criteria. Data was systematically extracted on the following topics: demographics, mechanism of injury, indication for treatment, treatment, post-operative complications, associated injuries, and outcomes including clinical outcome scores, clinical evaluations, and return to activity. Results: There were 14 studies included that evaluated a total of 119 patients with HAGL lesions. In these studies, 69% of patients were male with a mean age of 24 years. Sports (23.5%) and fitness training (41.2%) were the most common injury mechanisms. Instability was the most common indication for treatment, followed by pain and recurrent dislocations. 85 associated lesions were reported, with non-descript labral tears (34.5%) and Bankart lesions (22.4%) being the most common. Out of patients treated, 117 of 119 patients were treated operatively, with 60 open and 57 arthroscopic procedures. A variety of clinical outcome scores showed postoperative functional shoulder improvement and 97% of patients were able to return to some level of activity. Conclusions: HAGL lesions commonly present with associated injuries, necessitating a high clinical awareness of their presentation and management options to prevent undiagnosed lesions that can lead to persistent instability and pain. Surgical management yields good clinical outcomes.

Plant ecological genomics at the limits of life in the Atacama Desert.

The Atacama Desert in Chile-hyperarid and with high-ultraviolet irradiance levels-is one of the harshest environments on Earth. Yet, dozens of species grow there, including Atacama-endemic plants. Herein, we establish the Talabre-Lejía transect (TLT) in the Atacama as an unparalleled natural laboratory to study plant adaptation to extreme environmental conditions. We characterized climate, soil, plant, and soil-microbe diversity at 22 sites (every 100 m of altitude) along the TLT over a 10-y period. We quantified drought, nutrient deficiencies, large diurnal temperature oscillations, and pH gradients that define three distinct vegetational belts along the altitudinal cline. We deep-sequenced transcriptomes of 32 dominant plant species spanning the major plant clades, and assessed soil microbes by metabarcoding sequencing. The top-expressed genes in the 32 Atacama species are enriched in stress responses, metabolism, and energy production. Moreover, their root-associated soils are enriched in growth-promoting bacteria, including nitrogen fixers. To identify genes associated with plant adaptation to harsh environments, we compared 32 Atacama species with the 32 closest sequenced species, comprising 70 taxa and 1,686,950 proteins. To perform phylogenomic reconstruction, we concatenated 15,972 ortholog groups into a supermatrix of 8,599,764 amino acids. Using two codon-based methods, we identified 265 candidate positively selected genes (PSGs) in the Atacama plants, 64% of which are located in Pfam domains, supporting their functional relevance. For 59/184 PSGs with an Arabidopsis ortholog, we uncovered functional evidence linking them to plant resilience. As some Atacama plants are closely related to staple crops, these candidate PSGs are a "genetic goldmine" to engineer crop resilience to face climate change.

Transmission dynamics of SARS-CoV-2 within-host diversity in two major hospital outbreaks in South Africa.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes acute, highly transmissible respiratory infection in humans and a wide range of animal species. Its rapid global spread has resulted in a major public health emergency, necessitating commensurately rapid research to improve control strategies. In particular, the ability to effectively retrace transmission chains in outbreaks remains a major challenge, partly due to our limited understanding of the virus' underlying evolutionary dynamics within and between hosts. We used high-throughput sequencing whole-genome data coupled with bottleneck analysis to retrace the pathways of viral transmission in two nosocomial outbreaks that were previously characterised by epidemiological and phylogenetic methods. Additionally, we assessed the mutational landscape, selection pressures, and diversity at the within-host level for both outbreaks. Our findings show evidence of within-host selection and transmission of variants between samples. Both bottleneck and diversity analyses highlight within-host and consensus-level variants shared by putative source-recipient pairs in both outbreaks, suggesting that certain within-host variants in these outbreaks may have been transmitted upon infection rather than arising de novo independently within multiple hosts. Overall, our findings demonstrate the utility of combining within-host diversity and bottleneck estimations for elucidating transmission events in SARS-CoV-2 outbreaks, provide insight into the maintenance of viral genetic diversity, provide a list of candidate targets of positive selection for further investigation, and demonstrate that within-host variants can be transferred between patients. Together these results will help in developing strategies to understand the nature of transmission events and curtail the spread of SARS-CoV-2.

Discovering unknown human and mouse transcription factor binding sites and their characteristics from ChIP-seq data.

Transcription factor binding sites (TFBSs) are essential for gene regulation, but the number of known TFBSs remains limited. We aimed to discover and characterize unknown TFBSs by developing a computational pipeline for analyzing ChIP-seq (chromatin immunoprecipitation followed by sequencing) data. Applying it to the latest ENCODE ChIP-seq data for human and mouse, we found that using the irreproducible discovery rate as a quality-control criterion resulted in many experiments being unnecessarily discarded. By contrast, the number of motif occurrences in ChIP-seq peak regions provides a highly effective criterion, which is reliable even if supported by only one experimental replicate. In total, we obtained 2,058 motifs from 1,089 experiments for 354 human TFs and 163 motifs from 101 experiments for 34 mouse TFs. Among these motifs, 487 have not previously been reported. Mapping the canonical motifs to the human genome reveals a high TFBS density ±2 kb around transcription start sites (TSSs) with a peak at -50 bp. On average, a promoter contains 5.7 TFBSs. However, 70% of TFBSs are in introns (41%) and intergenic regions (29%), whereas only 12% are in promoters (-1 kb to +100 bp from TSSs). Notably, some TFs (e.g., CTCF, JUN, JUNB, and NFE2) have motifs enriched in intergenic regions, including enhancers. We inferred 142 cobinding TF pairs and 186 (including 115 completely) tethered binding TF pairs, indicating frequent interactions between TFs and a higher frequency of tethered binding than cobinding. This study provides a large number of previously undocumented motifs and insights into the biological and genomic features of TFBSs.

Lack of transgenerational effects of ionizing radiation exposure from the Chernobyl accident.

Effects of radiation exposure from the Chernobyl nuclear accident remain a topic of interest. We investigated germline de novo mutations (DNMs) in children born to parents employed as cleanup workers or exposed to occupational and environmental ionizing radiation after the accident. Whole-genome sequencing of 130 children (born 1987-2002) and their parents did not reveal an increase in the rates, distributions, or types of DNMs relative to the results of previous studies. We find no elevation in total DNMs, regardless of cumulative preconception gonadal paternal [mean = 365 milligrays (mGy), range = 0 to 4080 mGy] or maternal (mean = 19 mGy, range = 0 to 550 mGy) exposure to ionizing radiation. Thus, we conclude that, over this exposure range, evidence is lacking for a substantial effect on germline DNMs in humans, suggesting minimal impact from transgenerational genetic effects.

Genetic variation of staphylococcal LukAB toxin determines receptor tropism.

Staphylococcus aureus has evolved into diverse lineages, known as clonal complexes (CCs), which exhibit differences in the coding sequences of core virulence factors. Whether these alterations affect functionality is poorly understood. Here, we studied the highly polymorphic pore-forming toxin LukAB. We discovered that the LukAB toxin variants produced by S. aureus CC30 and CC45 kill human phagocytes regardless of whether CD11b, the previously established LukAB receptor, is present, and instead target the human hydrogen voltage-gated channel 1 (HVCN1). Biochemical studies identified the domain within human HVCN1 that drives LukAB species specificity, enabling the generation of humanized HVCN1 mice with enhanced susceptibility to CC30 LukAB and to bloodstream infection caused by CC30 S. aureus strains. Together, this work advances our understanding of an important S. aureus toxin and underscores the importance of considering genetic variation in characterizing virulence factors and understanding the tug of war between pathogens and the host.

Conflicting and ambiguous names of overlapping ORFs in the SARS-CoV-2 genome: A homology-based resolution.

At least six small alternative-frame open reading frames (ORFs) overlapping well-characterized SARS-CoV-2 genes have been hypothesized to encode accessory proteins. Researchers have used different names for the same ORF or the same name for different ORFs, resulting in erroneous homological and functional inferences. We propose standard names for these ORFs and their shorter isoforms, developed in consultation with the Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. We recommend calling the 39 codon Spike-overlapping ORF ORF2b; the 41, 57, and 22 codon ORF3a-overlapping ORFs ORF3c, ORF3d, and ORF3b; the 33 codon ORF3d isoform ORF3d-2; and the 97 and 73 codon Nucleocapsid-overlapping ORFs ORF9b and ORF9c. Finally, we document conflicting usage of the name ORF3b in 32 studies, and consequent erroneous inferences, stressing the importance of reserving identical names for homologs. We recommend that authors referring to these ORFs provide lengths and coordinates to minimize ambiguity caused by prior usage of alternative names.

Dynamically evolving novel overlapping gene as a factor in the SARS-CoV-2 pandemic.

Understanding the emergence of novel viruses requires an accurate and comprehensive annotation of their genomes. Overlapping genes (OLGs) are common in viruses and have been associated with pandemics but are still widely overlooked. We identify and characterize ORF3d, a novel OLG in SARS-CoV-2 that is also present in Guangxi pangolin-CoVs but not other closely related pangolin-CoVs or bat-CoVs. We then document evidence of ORF3d translation, characterize its protein sequence, and conduct an evolutionary analysis at three levels: between taxa (21 members of Severe acute respiratory syndrome-related coronavirus), between human hosts (3978 SARS-CoV-2 consensus sequences), and within human hosts (401 deeply sequenced SARS-CoV-2 samples). ORF3d has been independently identified and shown to elicit a strong antibody response in COVID-19 patients. However, it has been misclassified as the unrelated gene ORF3b, leading to confusion. Our results liken ORF3d to other accessory genes in emerging viruses and highlight the importance of OLGs.