A multi-scale map of protein assemblies in the DNA damage response.

The DNA damage response (DDR) ensures error-free DNA replication and transcription and is disrupted in numerous diseases. An ongoing challenge is to determine the proteins orchestrating DDR and their organization into complexes, including constitutive interactions and those responding to genomic insult. Here, we use multi-conditional network analysis to systematically map DDR assemblies at multiple scales. Affinity purifications of 21 DDR proteins, with/without genotoxin exposure, are combined with multi-omics data to reveal a hierarchical organization of 605 proteins into 109 assemblies. The map captures canonical repair mechanisms and proposes new DDR-associated proteins extending to stress, transport, and chromatin functions. We find that protein assemblies closely align with genetic dependencies in processing specific genotoxins and that proteins in multiple assemblies typically act in multiple genotoxin responses. Follow-up by DDR functional readouts newly implicates 12 assembly members in double-strand-break repair. The DNA damage response assemblies map is available for interactive visualization and query (ccmi.org/ddram/).

High Tumor Mutational Burden Correlates with Longer Survival in Immunotherapy-Naïve Patients with Diverse Cancers.

Higher tumor mutational burden (TMB) has been correlated with response to checkpoint blockade immunotherapy. However, it is unclear whether TMB independently serves as a prognostic biomarker for outcomes in immunotherapy-naïve patients. Here, we evaluated the relationship between TMB and overall survival in 1,415 immunotherapy-naïve patients with diverse advanced malignancies. TMB was studied both as a tiered variable (low ≤5 mutations/Mb, intermediate >5 and <20, high ≥20 and <50, and very high ≥50) and as a continuous variable. Interestingly, we observed a parabolic correlation between TMB and overall survival, in which intermediate-range TMB correlated with decreased survival, whereas low and very high TMB correlated with improved outcomes (median survival: 238, 174, 195, and 350 weeks for low, intermediate, high, and very high TMB, respectively; multivariate P < 0.01). This corresponded to an HR of 1.29 (95% confidence interval, 1.07-1.54; P < 0.01) for intermediate-range TMB on multivariable survival analysis correcting for known confounders, including primary tumor of origin. These results demonstrate that TMB may have utility as a prognostic biomarker in immunotherapy-naïve patients, with a protective effect at higher TMBs, and that studies of survival in immunotherapy-treated patients may need to stratify or randomize by TMB in a nonlinear fashion to account for this confounding.

Cheilectomy Combined With First Tarsometatarsal Joint Arthrodesis for Surgical Treatment of Midstage Hallux Rigidus Complicated by Medial Column Insufficiency: Prospective Evaluation of Outcomes.

The role of metatarsus primus elevatus and first ray hypermobility is under scrutiny with regard to the pathoanatomy of hallux rigidus. Regardless of the underlying biomechanical cause, there is a subset of patients with hallux limitus present with concomitant insufficiency of the medial column identified on clinical exam and lateral imaging as dorsal divergence of the first compared with the second metatarsal. While cheilectomy and decompression metatarsal osteotomy are commonly used to mitigate retrograde forces at the first metatarsophalangeal joint (MPJ) level, traditional hallux limitus procedures do not address more proximal deformity of the medial column. Although the authors prefer to treat this complex condition with cheilectomy combined with tarsometatarsal joint arthrodesis, there is a paucity of literature on this approach. A prospective cohort study of consecutive patients was therefore performed to assess outcomes. Ten patients (3 males, 7 females) and 11 feet (8 right and 3 left) met the inclusion criteria. Mean follow-up was 21.9 months (range 12 to 52). Average age was 50.4 years (range 28 to 61). The average preoperative ACFAS score of 49.6 (range 29 to 61) improved to 78 (range 51 to 92) at 10 weeks postoperatively and 85.4 (range 60 to 100) at 1 year postoperatively. By 1 year postsurgery, 9 of 10 patients (90%) described their satisfaction level as very satisfied, and 1 (10%) was somewhat satisfied.

Prognostic implications of RAS alterations in diverse malignancies and impact of targeted therapies.

RAS alterations are often found in difficult-to-treat malignancies and are considered "undruggable." To better understand the clinical correlates and coaltered genes of RAS alterations, we used targeted next-generation sequencing (NGS) to analyze 1,937 patients with diverse cancers. Overall, 20.9% of cancers (405/1,937) harbored RAS alterations. Most RAS-altered cases had genomic coalterations (95.3%, median: 3, range: 0-51), often involving genes implicated in oncogenic signals: PI3K pathway (31.4% of 405 cases), cell cycle (31.1%), tyrosine kinase families (21.5%) and MAPK signaling (18.3%). Patients with RAS-altered versus wild-type RAS malignancies had significantly worse overall survival (OS; p = 0.02 [multivariate]), with KRAS alterations, in particular, showing shorter survival. Moreover, coalterations in both RAS and PI3K signaling or cell-cycle-associated genes correlated with worse OS (p = 0.004 and p < 0.0001, respectively [multivariate]). Among RAS-altered patients, MEK inhibitors alone did not impact progression-free survival (PFS), while matched targeted therapy against non-MAPK pathway coalterations alone showed a trend toward longer PFS (vs. patients who received unmatched therapy) (HR: 0.79, 95% CI: 0.61-1.03, p = 0.07). Three of nine patients (33%) given tailored combination therapies targeting both MAPK and non-MAPK pathways achieved objective responses. In conclusion, RAS alterations correlated with poor survival across cancers. The majority of RAS alterations were accompanied by coalterations impacting other oncogenic pathways. MEK inhibitors alone were ineffective against RAS-altered cancers while matched targeted therapy against coalterations alone correlated with a trend toward improved PFS. A subset of the small number of patients given MEK inhibitors plus tailored non-MAPK-targeting agents showed responses, suggesting that customized combinations warrant further investigation.

Surgical Outcomes After Minimally Invasive Release of Stroke-Related Equinovarus Contracture of the Foot and Ankle.

Cerebrovascular accident frequently causes spastic equinovarus contracture of the foot and ankle, for which traditional surgical correction involves tendon transfer, osteotomy, and hindfoot fusion, which can be challenging for patients after cerebrovascular accident. We prospectively assessed the efficacy of a minimally invasive, ambulatory approach to correct this complex deformity in 12 consecutive patients. Surgery included Achilles tendon lengthening, lengthening of the posterior tibial tendon, and flexor tenotomy of all 5 digits. The 10-cm visual-analog scale and the Bristol Foot Score were used to assess pain and subjective foot-related quality of life, respectively. The mean patient age was 61.5 ± 5.68 years, and the duration of follow-up was 29.3 ± 18.5 (range 12.2 to 63.3) months. All patients had a preoperative equinovarus foot structure and all had a rectus foot in weightbearing stance at the 1-year postoperative evaluation. Nine (75.0%) patients showed no residual or recurrent deformity, whereas 3 (25.5%) displayed incomplete release of digital contractures; all patients were treated with in-office flexor tenotomy. Preoperative maximum ankle dorsiflexion was ≤90° in 12 (100%) patients and >90° in 9 (75.0%) patients postoperatively. The mean visual-analog scale score decreased in 10 (83.3%) patients, although a statistically significant decrease was not observed (p = .0535). The Bristol Foot Score improved from 55.17 ± 11.10 preoperatively to 36.83 ± 13.26 postoperatively, and this improvement was statistically significant (p = .0022). These outcomes demonstrate the effectiveness of the minimally invasive, ambulatory surgical approach to spastic equinovarus contracture without identified patient harm.

Combined Medial Column Arthrodesis with Open Reduction Internal Fixation of Central Column for Treatment of Lisfranc Fracture-Dislocation: A Review of Consecutive Cases.

Lisfranc fracture-dislocations can be devastating injuries with significant long-term sequelae with or without surgical intervention. The main goal of treatment is to minimize the common long-term complications including pain, progressive arch collapse, degenerative joint disease, hardware failure, and reoperation. Partial primary fusion involving the first, second, and third tarsometatarsal joints has become a common approach for primarily dislocation injuries, with open reduction and internal fixation (ORIF) favored for Lisfranc injuries involving fracture. ORIF commonly requires revision surgery for hardware removal or delayed fusion. Major revision creates hardship for the patient due to the prolonged recovery required, and even "simple" hardware removal can be traumatic to local nerve, artery, and tendon structures. A common injury pattern includes the findings of primary dislocation and instability of the first tarsometatarsal joint with oftentimes comminuted fracture to the second and third tarsometatarsal joints, which does not fit the standard surgical approach. We report a review of our preferred surgical approach consisting of medial column primary arthrodesis combined with central column ORIF and lateral column temporary pinning. We undertook an institutional review board-approved review of 35 consecutive Lisfranc injuries treated with this hybrid approach. Mean follow-up time was 22.14 ± 22.39 (range 2.5 to 84) months. All but 2 (5.71%) patients had radiographic evidence of union at 10 weeks. Complications included 3 with neuritis, 1 with medial column nonunion that was treated with a bone stimulator, and 1 with revision of second metatarsal nonunion. The present retrospective series highlights our experience with isolated primary fusion of the medial column in both subtle and obvious Lisfranc injuries.

Pharmacological validation of individual animal locomotion, temperature and behavioural analysis in group-housed rats using a novel automated home cage analysis system: A comparison with the modified Irwin test.

BACKGROUND: The ActualHCA™ system continuously monitors the activity, temperature and behavior of group-housed rats without invasive surgery. The system was validated to detect the contrasting effects of sedative and stimulant test agents (chlorpromazine, clonidine and amphetamine), and compared with the modified Irwin test (mIT) with rectal temperature measurements. METHODS: Six male Han Wistar rats per group were used to assess each test agent and vehicle controls in separate ActualHCA™ recordings and mIT. The mIT was undertaken at 15, 30 mins, 1, 2, 4 and 24 h post-dose. ActualHCA™ recorded continuously for 24 h post-dose under 3 experimental conditions: dosed during light phase, dark phase, and light phase with a scheduled cage change at the time of peak effects determined by mIT. RESULTS: ActualHCA™ detected an increase stimulated activity from the cage change at 1-2 h post-dose which was obliterated by chlorpromazine and clonidine. Amphetamine increased activity up to 4 h post-dose in all conditions. Temperature from ActualHCA™ was affected by all test agents in all conditions. The mIT showed effects on all 3 test agents up to 4 h post-dose, with maximal effects at 1-2 h post-dose. The maximal effects on temperature from ActualHCA™ differed from mIT. Delayed effects on activity were detected by ActualHCA™, but not on mIT. CONCLUSIONS: Continuous monitoring has the advantage of capturing effects over time that may be missed with manual tests using pre-determined time points. This automated behavioural system does not replace the need for conventional methods but could be implemented simultaneously to improve our understanding of behavioural pharmacology.

Vitamin D Levels Do Not Predict Risk of Metatarsal Fractures.

INTRODUCTION: In the literature, there is conflicting data regarding the relationship between vitamin D and fractures. Reports on the effects of vitamin D levels on pathologies of the foot and ankle are limited. The purpose of this study is to assess the prevalence of vitamin D insufficiency in patients who have sustained low-energy metatarsal fractures compared to foot or ankle sprains without osseous involvement. METHODS: Between May 2012 and August 2014, vitamin D levels and demographic data were collected prospectively in a total of 99 patients; 71 with metatarsal fractures and 28 with sprains, both from a low-energy mechanism of injury. Data between the metatarsal fracture group and sprain group were compared through univariate and multivariate analyses. RESULTS: Mean vitamin D in the fracture group was 26.9 ng/mL (range = 78.0-4.3), and in the sprain group it was 27.1 ng/mL (range = 64.1-8.3; P = .93). Vitamin D insufficiency (<30 ng/mL) was present in 47 (66%) of fracture patients and 20 (71%) of sprain patients ( P = .81). CONCLUSION: A high incidence of hypovitaminosis D was seen in all foot and ankle patients. There was no difference in mean vitamin D level or incidence of vitamin D insufficiency between patients with metatarsal fractures or sprains resulting from similar low-energy mechanisms. LEVELS OF EVIDENCE: Level III: Prospective, case-control study.

Development and characterisation of a novel glucagon like peptide-1 receptor antibody.

AIMS/HYPOTHESIS: Glucagon like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion by binding to GLP-1 receptors (GLP1Rs) on pancreatic beta cells. GLP-1 mimetics are used in the clinic for the treatment of type 2 diabetes, but despite their therapeutic success, several clinical effects of GLP-1 remain unexplained at a mechanistic level, particularly in extrapancreatic tissues. The aim of this study was to generate and characterise a monoclonal antagonistic antibody for the GLP1R for use in vivo. METHODS: A naive phage display selection strategy was used to isolate single-chain variable fragments (ScFvs) that bound to GLP1R. The ScFv with the highest affinity, Glp1R0017, was converted into a human IgG1 and characterised further. In vitro antagonistic activity was assessed in a number of assays: a cAMP-based homogenous time-resolved fluorescence assay in GLP1R-overexpressing cell lines, a live cell cAMP imaging assay and an insulin secretion assay in INS-1 832/3 cells. Glp1R0017 was further tested in immunostaining of mouse pancreas, and the ability of Glp1R0017 to block GLP1R in vivo was assessed by both IPGTT and OGTT in C57/Bl6 mice. RESULTS: Antibodies to GLP1R were selected from naive antibody phage display libraries. The monoclonal antibody Glp1R0017 antagonised mouse, human, rat, cynomolgus monkey and dog GLP1R. This antagonistic activity was specific to GLP1R; no antagonistic activity was found in cells overexpressing the glucose-dependent insulinotropic peptide receptor (GIPR), glucagon like peptide-2 receptor or glucagon receptor. GLP-1-stimulated cAMP and insulin secretion was attenuated in INS-1 832/3 cells by Glp1R0017 incubation. Immunostaining of mouse pancreas tissue with Glp1R0017 showed specific staining in the islets of Langerhans, which was absent in Glp1r knockout tissue. In vivo, Glp1R0017 reversed the glucose-lowering effect of liraglutide during IPGTTs, and reduced glucose tolerance by blocking endogenous GLP-1 action in OGTTs. CONCLUSIONS/INTERPRETATION: Glp1R0017 is a monoclonal antagonistic antibody to the GLP1R that binds to GLP1R on pancreatic beta cells and blocks the actions of GLP-1 in vivo. This antibody holds the potential to be used in investigating the physiological importance of GLP1R signalling in extrapancreatic tissues where cellular targets and signalling pathways activated by GLP-1 are poorly understood.

Comparing Different Surgical Techniques for Addressing the Posterior Malleolus in Supination External Rotation Ankle Fractures and the Need for Syndesmotic Screw Fixation.

Trimalleolar ankle fractures are unstable injuries with possible syndesmotic disruption. Recent data have described inherent morbidity associated with screw fixation of the syndesmosis, including the potential for malreduction, hardware irritation, and post-traumatic arthritis. The posterior malleolus is an important soft tissue attachment for the posterior inferior syndesmosis ligament. We hypothesized that fixation of a sizable posterior malleolar (PM) fracture in supination external rotation type IV (SER IV) ankle fractures would act to stabilize the syndesmosis and minimize or eliminate the need for trans-syndesmotic fixation. A retrospective review of trimalleolar ankle fractures surgically treated from October 2006 to April of 2011 was performed. A total of 143 trimalleolar ankle fractures were identified, and 97 were classified as SER IV. Of the 97 patients, 74 (76.3%) had a sizable PM fragment. Syndesmotic fixation was required in 7 of 34 (20%) and 27 of 40 (68%), respectively, when the PM was fixed versus not fixed (p = .0002). When the PM was indirectly reduced using an anterior to posterior screw, 7 of 15 patients (46.7%) required syndesmotic fixation compared with none of 19 patients when the PM fragment was fixated with direct posterior lateral plate fixation (p = .0012). Fixation of the PM fracture in SER IV ankle fractures can restore syndesmotic stability and, thus, lower the rate of syndesmotic fixation. We found that fixation of a sizable PM fragment in SER IV or equivalent injuries through posterolateral plating can eliminate the need for syndesmotic screw fixation.

Two-View Gravity Stress Imaging Protocol for Nondisplaced Type II Supination External Rotation Ankle Fractures: Introducing the Gravity Stress Cross-Table Lateral View.

Assessing ankle stability in nondisplaced Lauge-Hansen supination external rotation type II injuries requires stress imaging. Gravity stress mortise imaging is routinely used as an alternative to manual stress imaging to assess deltoid integrity with the goal of differentiating type II from type IV injuries in cases without a posterior or medial fracture. A type II injury with a nondisplaced fibula fracture is typically treated with cast immobilization, and a type IV injury is considered unstable and often requires operative repair. The present case series (two patients) highlights a standardized 2-view gravity stress imaging protocol and introduces the gravity stress cross-table lateral view. The gravity stress cross-table lateral view provides a more thorough evaluation of the posterior malleolus owing to the slight external rotation and posteriorly directed stress. External rotation also creates less bony overlap between the tibia and fibula, allowing for better visualization of the fibula fracture. Gravity stress imaging confirmed medial-sided injury in both cases, confirming the presence of supination external rotation type IV or bimalleolar equivalent fractures. Open reduction and internal fixation was performed, and both patients achieved radiographic union. No further treatment was required at 21 and 33 months postoperatively.

Surgical Treatment Guidelines for Digital Deformity Associated With Intrinsic Muscle Spasticity (Intrinsic Plus Foot) in Adults With Cerebral Palsy.

Intrinsic plus foot deformity has primarily been associated with cerebral palsy and involves spastic contracture of the intrinsic musculature with resultant toe deformities. Digital deformity is caused by a dynamic imbalance between the intrinsic muscles in the foot and extrinsic muscles in the lower leg. Spastic contracture of the toes frequently involves curling under of the lesser digits or contracture of the hallux into valgus or plantarflexion deformity. Patients often present with associated pressure ulcers, deformed toenails, shoe or brace fitting challenges, and pain with ambulation or transfers. Four different patterns of intrinsic plus foot deformity have been observed by the authors that likely relate to the different patterns of muscle involvement. Case examples are provided of the 4 patterns of intrinsic plus foot deformity observed, including global intrinsic plus lesser toe deformity, isolated intrinsic plus lesser toe deformity, intrinsic plus hallux valgus deformity, and intrinsic plus hallux flexus deformity. These case examples are presented to demonstrate each type of deformity and our approach for surgical management according to the contracture pattern. The surgical approach has typically involved tenotomy, capsulotomy, or isolated joint fusion. The main goals of surgical treatment are to relieve pain and reduce pressure points through digital realignment in an effort to decrease the risk of pressure sores and allow more effective bracing to ultimately improve the patient's mobility.

Lateral Stress Dorsiflexion View: A Case Series Demonstrating Clinical Utility in Midterm Hallux Limitus.

The lateral hallux stress dorsiflexion view is part of our standard workup for midterm hallux limitus (HL)/hallux rigidus (HR). It provides a functional radiographic examination of the first metatarsal phalangeal joint. Midterm HL primarily involves degenerative changes in the upper one third of the metatarsal phalangeal joint involving formation of bone spurs, dorsal bone impingement, joint space narrowing with cartilage degeneration, and fragmentation of the bone spurs. The lateral hallux stress dorsiflexion view provides diagnostic information not visible on a standard weightbearing lateral view in patients with midterm HL/HR, including joint space narrowing on the dorsal third of the joint despite intact cartilage through the center one third of the joint, the extent of maximum first metatarsal phalangeal joint dorsiflexion, and direct visualization of dorsal bone spur impingement. This functional radiographic examination also appears to provide improved patient understanding regarding why their joint is stiff and painful. Improved patient understanding of their condition positively influences the shared decision making regarding the treatment objectives and options. The cases of 5 patients with stage II or III HL/HR are presented to depict the utility of this radiographic view, including objective measurement of maximum first metatarsal phalangeal joint dorsiflexion, confirmation of a bony block at the end range of dorsiflexion, the presence or absence of joint space narrowing at the dorsal third of the joint, evaluation of the excursion of the sesamoid apparatus, a tool to help the patient understand, an intraoperative assessment of procedure effectiveness, and a comparison of maximum dorsiflexion before and after surgery.

Minimally invasive soft tissue release of foot and ankle contracture secondary to stroke.

Lower extremity contracture associated with stroke commonly results in a nonreducible, spastic equinovarus deformity of the foot and ankle. Rigid contracture deformity leads to gait instability, pain, bracing difficulties, and ulcerations. The classic surgical approach for stroke-related contracture of the foot and ankle has been combinations of tendon lengthening, tendon transfer, osteotomy, and joint fusion procedures. Recovery after traditional foot and ankle reconstructive surgery requires a period of non-weightbearing that is not typically practical for these patients. Little focus has been given in published studies on minimally invasive soft tissue release of contracture. We present the case of a 61-year-old female with an equinovarus foot contracture deformity secondary to stroke. The patient underwent Achilles tendon lengthening, posterior tibial tendon Z lengthening, and digital flexor tenotomy of each toe with immediate weightbearing in a walking boot, followed by transition to an ankle-foot orthosis. The surgical principles and technique tips are presented to demonstrate our minimally invasive approach to release of foot and ankle contracture secondary to stroke. The main goal of this approach is to improve foot and ankle alignment for ease of bracing, which, in turn, will improve gait, reduce the risk of falls, decrease pain, and avoid the development of pressure sores.

Near total calcanectomy with rotational flap closure of large decubitus heel ulcerations complicated by calcaneal osteomyelitis.

Osteomyelitis is a common late-stage sequela of deep heel ulceration and frequently results in leg amputation, because few options exist to effectively treat this challenging problem. A technique that has been successful at our institution is near total calcanectomy with rotational flap closure of large decubitus heel ulcers complicated by calcaneal osteomyelitis. A case series is presented of 3 patients with differing locations or "zones" of heel decubitus ulceration with acute osteomyelitis. A description of our preferred flap choice, given the zone of the ulceration, is demonstrated, as are the surgical principles for the treatment approach. This procedure is an effective option, provided specific principles are followed to achieve complete wound coverage and minimize pressure points both when in bed and when walking.

Surgical technique for combined Dwyer calcaneal osteotomy and peroneal tendon repair for correction of peroneal tendon pathology associated with cavus foot deformity.

Peroneal tendon pathology is commonly seen in patients with underlying pes cavus. The Dwyer calcaneal osteotomy is a useful adjunctive procedure to address the heel varus component of the cavus foot deformity, especially in the presence of concomitant peroneal tendon pathology. The lateralizing heel osteotomy using a wedge resection can effectively reduce future stress on the repaired peroneal tendons, although technical challenges arise when attempting to perform both tendon repair and heel osteotomy through the same incision. Specific principles must be followed to achieve adequate exposure of the desired structures, obtain desired correction of the deformity, and avoid complications such as sural neuritis. In the present report, the surgical principles and technical pearls are highlighted in a pictorial demonstration of preoperative planning for calcaneal wedge resection, incision placement, osteotomy guide pin technique, fixation pearls, and peroneal tendon repair and transfer.

Impaired lower extremity wound healing secondary to sirolimus after kidney transplantation.

PURPOSE: Sirolimus is an immunosuppressive drug used as part of the drug regimen after kidney, liver, and heart transplantation. There have been numerous reports of transplant surgical wound healing complications secondary to sirolimus. The authors present a case of impaired wound healing in the lower extremity after kidney transplantation for a patient on sirolimus. This is one of the few reported cases that we are aware of that demonstrates the effects of sirolimus on wound healing at a nontransplant site. METHODS: A case highlighting aggressive limb-salvage modalities for a kidney transplant patient on sirolimus is presented. The subject was informed that data concerning the case would be submitted for publication. A brief review of the literature shows the wound healing problems previously associated with sirolimus. RESULTS: Despite all salvage techniques employed, including aggressive early debridement, forefoot offloading, IV antibiotics, negative pressure wound therapy, and hyperbaric oxygen therapy, the patient eventually went on to failure and a transtibial amputation. CONCLUSION: Physicians specializing in wound care and limb salvage must be aware of the effects of sirolimus on wound healing and should consider modifications to these patients' immunosuppressive regimens.