Complete combinatorial mutational enumeration of a protein functional site enables sequence-landscape mapping and identifies highly-mutated variants that retain activity.

Understanding how proteins evolve under selective pressure is a longstanding challenge. The immensity of the search space has limited efforts to systematically evaluate the impact of multiple simultaneous mutations, so mutations have typically been assessed individually. However, epistasis, or the way in which mutations interact, prevents accurate prediction of combinatorial mutations based on measurements of individual mutations. Here, we use artificial intelligence to define the entire functional sequence landscape of a protein binding site in silico, and we call this approach Complete Combinatorial Mutational Enumeration (CCME). By leveraging CCME, we are able to construct a comprehensive map of the evolutionary connectivity within this functional sequence landscape. As a proof of concept, we applied CCME to the ACE2 binding site of the SARS-CoV-2 spike protein receptor binding domain. We selected representative variants from across the functional sequence landscape for testing in the laboratory. We identified variants that retained functionality to bind ACE2 despite changing over 40% of evaluated residue positions, and the variants now escape binding and neutralization by monoclonal antibodies. This work represents a crucial initial stride toward achieving precise predictions of pathogen evolution, opening avenues for proactive mitigation.

Long-term outcomes in advanced anterior skull base malignancy: a single quaternary institution experience.

BACKGROUND: Advanced skull base malignancies are a heterogenous subset of head and neck cancers, and management is often complex. In recent times, there has been a paradigm shift in surgical technique and the advent of novel systemic options. Our goal was to analyse the long-term outcomes of a single quaternary head and neck and skull base service. METHODS: A retrospective review of 127 patients with advanced anterior skull base malignancies that were treated at our institution between 1999 and 2015 was performed. Multiple variables were investigated to assess their significance on 5 and 10-year outcomes. RESULTS: The mean age was 60.9 (± 12.6 SD). Sixty-four percent were males and 36% were females. Ninety percent of patients had T4 disease. Median survival time was 133 months. The 5-year overall survival (OS) was 66.2%, disease-specific survival (DSS) was 74.7%, and recurrence-free survival (RFS) was 65.0%. The 10-year OS was 55.1%, DSS was 72.1%, and RFS was 53.4%. Histological type and margin status significantly affected OS & DSS. CONCLUSION: Surgical management of advanced skull base tumours has evolved over the last few decades at our institution with acceptable survival outcomes and complication rates. Histological diagnosis and margin status are the main predictors of survival. The addition of neoadjuvant systemic agents in current trials may improve outcomes.

Clinical outcomes in perineural spread of cutaneous squamous cell carcinoma via the ophthalmic nerve.

BACKGROUND: There are no large studies reporting oncological or survival outcomes for patients diagnosed with perineural spread (PNS) of cutaneous squamous cell carcinoma (cSCC) via the ophthalmic nerve (V1). Where orbital exenteration may be necessary for curative treatment, it is critical to have survival data with which the morbidity associated with surgical treatment can be justified. Furthermore, with the emerging treatment option of immunotherapy, current standard of care outcomes are needed to help guide future trial design and eventually changed management guidelines. OBJECTIVE: To determine the oncological and survival outcomes observed in patients with PNS of cSCC via V1. MATERIALS AND METHODS: Retrospective analysis of prospectively maintained cohort of patients with PNS of cSCC via V1 treated in a tertiary Australian head and neck oncology/skull base referral center. Consecutive sample of 53 patients managed between March 1, 1999 and April 30, 2020. Follow-up closure date was September 1, 2021. Curative-intent surgery, curative-intent radiotherapy, or palliative care was undertaken. Endpoints included five-year overall, disease-specific, and disease-free survival from the date of treatment. RESULTS: Five-year Kaplan-Meier overall survival was 61.9% (95% CI 46.2%-74.3%), with disease-specific survival of 74.6% (95% CI 58.8%-85.3%), and disease-free survival 62.1% (95% CI 46.5%-74.3%). Survival was superior in patients treated via surgery and adjuvant radiotherapy than in those receiving surgery alone or definitive radiotherapy. Survival was superior among patients with less advanced disease as assessed by the Williams zonal staging system; patients with Zone 1 disease had disease-specific survival of 94.1% at 5 years with 82.5% disease-free survival. DISCUSSION: Five-year oncological and survival outcomes in this cohort were favorable. Superior survival was observed in patients treated with curative-intent surgery and adjuvant radiotherapy. Less extensive disease as delineated by the Williams zonal staging system was associated with improved survival. CONCLUSION: Surgical resection with adjuvant radiotherapy confers favourable oncological and survival outcome in patients with V1 PNS, particularly with early disease limited to Zone 1.

Skull base surgery for malignant tumors: The 2nd international collaborative study (1995-2015).

BACKGROUND: The current study presents the effort of a global collaborative group to review the management and outcomes of malignant tumors of the skull base worldwide. PATIENTS AND METHODS: A total of 28 institutions contributed data on 3061 patients. Analysis evaluated clinical variables, survival outcomes, and multivariable factors associated with outcomes. RESULTS: The median age was 56 years (IQR 44-67). The open surgical approach was used in 55% (n = 1680) of cases, endoscopic resection was performed in 36% (n = 1087), and the combined approach in 9.6% (n = 294). With a median follow-up of 7.1 years, the 5-year OS DSS and RFS were 65%, 71.7% and 53%, respectively. On multivariable analysis, older age, comorbidities, histology, dural/intracranial involvement, positive margins, advanced stage, and primary site were independent prognostic factors for OS, DSS, and RFS. Adjuvant RT was a protective prognostic factor. CONCLUSION: The progress across various disciplines may have contributed to improved OS and DSS in this study compared to previous reports.

TXNIP-mediated crosstalk between oxidative stress and glucose metabolism.

Thioredoxin-interacting protein (TXNIP) has emerged as a key player in cancer and diabetes since it targets thioredoxin (TRX)-mediated redox regulation and glucose transporter (GLUT)-mediated metabolism. TXNIP consists of two arrestin (ARR, N-ARR and C-ARR) domains at its amino-terminus and two PPxY (PY) motifs and a di-leucine (LL) motif for endocytosis at its carboxyl-terminus. Here, we report that TXNIP shuffles between TRX and GLUTs to regulate homeostasis of intracellular oxidative stress and glucose metabolism. While TXNIP functions as a gatekeeper of TRX by default, it robustly interacted with class I GLUTs through its C-ARR domain upon increase of intracellular reactive oxygen species. This interaction prompted the surface expression downregulation and lysosomal degradation of GLUTs by its carboxyl-terminal LL endocytic signaling motif to attenuate glucose uptake. Consequently, TXNIP expression significantly limited glucose uptake, leading to the suppression of glycolysis, hexosamine biosynthesis, and the pentose phosphate pathway. Our findings establish a fundamental link between ROS and glucose metabolism through TXNIP and provide a promising target for the drug development against GLUT-related metabolic disorders.

Patterns of spread and anatomical prognostic factors of pre-auricular cutaneous squamous cell carcinoma extending to the temporal bone.

BACKGROUND: Improvements can be made in the management and staging of advanced pre-auricular cutaneous squamous cell carcinoma (cSCC). We aimed to analyze radiological patterns of spread and clinico-anatomical prognostic factors. METHODS: Retrospective review of 54 patients with pre-auricular cSCC (cutaneous/nodal) who underwent temporal bone resection with curative intent. RESULTS: Involvement of the cartilaginous external auditory canal (EAC) (79.6%) and retromandibular space (63.0%) was common. Styloid process/anterior carotid sheath (ACS) (11.1%) and bony EAC (7.4%) involvement were rare. ACS involvement resulted in high rates of involved surgical margins (100%) and poor outcomes on univariable analysis. Negative prognostic factors on multivariable analysis included salvage surgery and invasion of the bony EAC, mandible, pterygoid muscle(s), and dura. CONCLUSION: The bony EAC and ACS can form temporary barriers to tumor spread, with the latter representing a potential limit of resectability. Prognostic factors revealed can lead to the development of a more appropriate staging tool.

Inflammatory Bowel Disease: A Concise Review.

Inflammatory bowel disease is a term describing a group of diseases, Crohn's disease and ulcerative colitis, that cause chronic inflammation in the gastrointestinal tract. Both conditions tend to have episodic flares of diarrhea, abdominal pain, fatigue, and unintentional weight loss. This analysis will discuss the etiology, pathophysiology, epidemiology, clinical and histologic features, treatment, and complications of Crohn's disease and ulcerative colitis. Though there are many similarities between these two conditions it is important to recognize their differences for accurate diagnosis, treatment, and surveillance for potential long-term complications.

Structural Insight on Supramolecular Polyion Salts: Inositol Hexaphosphate Enclosed in Cationic Macrocyclic Clusters.

Supramolecular macrocyclic forces have been used to trap phytate, myo-inositol-1,2,3,4,5,6-hexakisphosphate, a key bioanion with multiple roles in metabolic processes. Due to the complex chemistry of six multivalent phosphates surrounding the small, cyclic inositol framework, crystallographic information of simple phytate salts has been elusive. This report represents a combined crystallographic, theoretical, and solution binding investigation of a supramolecular macrocyclic complex of phytate. Together, the results provide significant insight to phytate's intramolecular and intermolecular interactions at the microenvironment level. The macrocycle-phytate aggregates consist of phytate anionic pairs, each partly sandwiched by two 24-membered, amide/amine-based cationic macrocycles. The phytate ion pairs hold the tetrameric macrocyclic array together by six strong intermolecular hydrogen bonds. Both phytates crystallize in 1a5e phosphate conformations (one axial (P2) and five equatorial phosphates). Solution NMR binding studies in 1 : 1 DMSO-d6 : D2 O indicate 2 : 1 macrocycle:phytate associations, suggesting that the sandwich-like nature of the complex holds together in solution. DFT studies indicate the likely occurrence of dynamic intramolecular interchange of phosphate protons, as well as important roles for the axial (P2) phosphate in both intramolecular and intermolecular hydrogen bonding interactions.

The Athletic Intelligence Quotient and performance in the National Basketball Association.

Prior to selecting an NBA player, teams consider multiple factors, including game film and tests of agility, strength, speed, anthropometry, and personality. In recent years, as the other major professional sports have begun to place greater emphasis on the measurement of cognitive abilities, so too have representatives in the NBA. In this study, the predictive validity of an empirically-supported measure of cognitive ability (AIQ) was examined vis-à-vis performance outcomes in the NBA. Specifically, AIQ scores were obtained from 356 NBA prospects prior to their draft between 2014 and 2019. The players' professional status and subsequent performance were assessed through composite and isolated NBA statistics. ANOVAs demonstrated that there were significant differences between NBA and non-NBA players, and subsequent independent samples t-tests revealed that NBA players had significantly higher AIQ scores than non-NBA players for 3 out of 4 factors and the Full Scale AIQ Score. Additionally, using hierarchical multiple regression analyses, it was demonstrated that the AIQ predicted some modest statistically significant relationships with multiple NBA stats (e.g., Player Efficiency Rating, Effective Field Goal Percentage), after controlling for the impact of draft placement. While the effect sizes for these differences and relationships were somewhat small, such findings are consistent with sport analytics and the restricted range when evaluating professional athletes. Given the expanding role of analytics and cognitive assessment in the NBA, the potential importance of the AIQ is considered in the draft process.

OASL phase condensation induces amyloid-like fibrillation of RIPK3 to promote virus-induced necroptosis.

RIPK3-ZBP1-MLKL-mediated necroptosis is a proinflammatory cell death process that is crucial for antiviral host defence. RIPK3 self-oligomerization and autophosphorylation are prerequisites for executing necroptosis, yet the underlying mechanism of virus-induced RIPK3 activation remains elusive. Interferon-inducible 2'-5' oligoadenylate synthetase-like (OASL) protein is devoid of enzymatic function but displays potent antiviral activity. Here we describe a role of OASL as a virus-induced necroptosis promoter that scaffolds the RIPK3-ZBP1 non-canonical necrosome via liquid-like phase condensation. This liquid-like platform of OASL recruits RIPK3 and ZBP1 via protein-protein interactions to provide spatial segregation for RIPK3 nucleation. This process facilitates the amyloid-like fibril formation and activation of RIPK3 and thereby MLKL phosphorylation for necroptosis. Mice deficient in Oasl1 exhibit severely impaired necroptosis and attenuated inflammation after viral infection, resulting in uncontrolled viral dissemination and lethality. Our study demonstrates an interferon-induced innate response whereby OASL scaffolds RIPK3-ZBP1 assembly via its phase-separated liquid droplets to facilitate necroptosis-mediated antiviral immunity.

Nasal Septal Flaps for Repair of Large or Recurrent Palatal Fistula: Report of Technique and 4-Year Experience.

SUMMARY: Large or multiply recurrent oronasal fistulas following cleft palate repair present a challenging problem. Nasal septal mucoperichondrial flaps have been widely used for repair of skull base defects; however, their use in the repair of oronasal cleft palate fistulas has not previously been described. In this pilot study, the authors describe anterior palatal fistula repair using a nasal septal flap and review their experience with this technique over 4 years. Fourteen patients with anterior palatal fistulas not amenable to repair using local palatal flaps were included for analysis. The mean size of the fistula was 12 mm in maximum dimension. Flap healing with complete or near-complete closure of fistula was achieved in 13 patients (93%). Five of these patients had a small, slit-like residual fistula that was asymptomatic. Nasal septal flaps are a new technique for repair of large or recurrent palatal fistulas. The procedure is well-tolerated with minimal side effects, high success rate, and low incidence of recurrence. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Complete Combinatorial Mutational Enumeration of a protein functional site enables sequence-landscape mapping and identifies highly-mutated variants that retain activity.

Understanding how proteins evolve under selective pressure is a longstanding challenge. The immensity of the search space has limited efforts to systematically evaluate the impact of multiple simultaneous mutations, so mutations have typically been assessed individually. However, epistasis, or the way in which mutations interact, prevents accurate prediction of combinatorial mutations based on measurements of individual mutations. Here, we use artificial intelligence to define the entire functional sequence landscape of a protein binding site in silico, and we call this approach Complete Combinatorial Mutational Enumeration (CCME). By leveraging CCME, we are able to construct a comprehensive map of the evolutionary connectivity within this functional sequence landscape. As a proof of concept, we applied CCME to the ACE2 binding site of the SARS-CoV-2 spike protein receptor binding domain. We selected representative variants from across the functional sequence landscape for testing in the laboratory. We identified variants that retained functionality to bind ACE2 despite changing over 40% of evaluated residue positions, and the variants now escape binding and neutralization by monoclonal antibodies. This work represents a crucial initial stride towards achieving precise predictions of pathogen evolution, opening avenues for proactive mitigation.

Epidemiology and treatment outcomes of cutaneous squamous cell carcinoma extending to the temporal bone.

BACKGROUND: Accurate epidemiological and outcomes data regarding cutaneous squamous cell carcinoma (cSCC) extending to the temporal bone is lacking. METHODS: Retrospective analysis of 167 Australian patients with primary and peri-temporal bone cSCC. RESULTS: cSCC extending from secondary subsites (93.4%) was 14 times more frequent than primary temporal bone SCC (6.6%). For patients who underwent curative surgery ± post-operative radiotherapy (n = 146, 87.4%), 5-year disease-free survival, locoregional recurrence-free survival, disease-specific survival, and overall survival was 53.0%, 59.4%, 67.9%, and 44.7%, respectively. External ear and pre-auricular tumors, salvage surgery, tumor size (≥40 mm medial-lateral), nodal disease, and involved margins were negative predictors of survival in multivariable analysis. CONCLUSION: In regions of high sun exposure, cSCCs extending to the temporal bone are more common than primary cancers. Outcomes are improved with clear margins, justifying the need for radical resection. Further research regarding pre-auricular cancers is required given poorer associated survival outcomes.

Updated U.S. Public Health Service Guideline for Testing of Transplant Candidates Aged <12 Years for Infection with HIV, Hepatitis B Virus, and Hepatitis C Virus - United States, 2022.

The U.S. Public Health Service (PHS) has periodically published recommendations about reducing the risk for transmission of HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV) through solid organ transplantation (1-4). Updated guidance published in 2020 included the recommendation that all transplant candidates receive HIV, HBV, and HCV testing during hospital admission for transplant surgery to more accurately assess their pretransplant infection status and to better identify donor transmitted infection (4). In 2021, CDC was notified that this recommendation might be unnecessary for pediatric organ transplant candidates because of the low likelihood of infection after the perinatal period and out of concern that the volume of blood drawn for testing could negatively affect critically ill children.* CDC and other partners reviewed surveillance data from CDC on estimates of HIV, HBV, and HCV infection rates in the United States and data from the Organ Procurement & Transplantation Network (OPTN)† on age and weight distributions among U.S. transplant recipients. Feedback from the transplant community was also solicited to understand the impact of changes to the existing policy on organ transplantation. The 2020 PHS guideline was accordingly updated to specify that solid organ transplant candidates aged <12 years at the time of transplantation who have received postnatal infectious disease testing are exempt from the recommendation for HIV, HBV, and HCV testing during hospital admission for transplantation.

IgG targeting distinct seasonal coronavirus- conserved SARS-CoV-2 spike subdomains correlates with differential COVID-19 disease outcomes.

Despite SARS-CoV-2 being a "novel" virus, early detection of anti-spike IgG in severe COVID-19 patients may be caused by the amplification of humoral memory responses against seasonal coronaviruses. Here, we examine this phenomenon by characterizing anti-spike IgG responses in non-hospitalized convalescent individuals across a spectrum of COVID-19 severity. We observe that disease severity positively correlates with anti-spike IgG levels, IgG cross-reactivity against other betacoronaviruses (ß-CoVs), and FcγR activation. Analysis of IgG targeting ß-CoV-conserved and non-conserved immunodominant epitopes within the SARS-CoV-2 spike protein revealed epitope-specific relationships: IgG targeting the conserved heptad repeat (HR) 2 region significantly correlates with milder disease, while targeting the conserved S2'FP region correlates with more severe disease. Furthermore, a lower HR2-to-S2'FP IgG-binding ratio correlates with greater disease severity, with ICU-hospitalized COVID-19 patients showing the lowest HR2/S2'FP ratios. These findings suggest that HR2/S2'FP IgG profiles may predict disease severity and offer insight into protective versus deleterious humoral recall responses.

Immunotherapy for the treatment of perineural spread in cutaneous head and neck squamous cell carcinoma: Time to rethink treatment paradigms.

BACKGROUND: Immune checkpoint inhibitors have shown promising antitumour activity. Application in head and neck cutaneous squamous cell carcinoma (cSCC) large nerve perineural spread (PNS) is limited. METHODS: Retrospective review of 13 patients with PNS receiving anti-PD-1 therapy from September 2017 to May 2021 is presented. Primary endpoints were objective response (complete or partial response) and median time to progression, determined by Head and Neck Multi-Disciplinary Team (MDT) and independent radiology review of magnetic resonance imaging (MRI) and/or computed tomography/positron emission tomography (CT/PET). RESULTS: Objective response was observed in 9/13 patients (69%), with complete response in 6 (46%) and partial response in 3 patients (23%). Median time to response was 2.1 months (IQR 1.8-2.7 months). There were 3 (23%) patients with progressive disease, with median time to progression of 3.5 months. There were no grade 3-4 treatment related adverse events. CONCLUSIONS: This case series supports developing evidence for anti-PD-1 checkpoint inhibitor therapy for perineural spread, supporting future prospective clinical trials in this patient population.

Workplace Hazards in Orthopaedic Surgery Training: A Nationwide Resident Survey Involving Sharps-related Injuries.

INTRODUCTION: Surgical specialties are at an increased risk for occupational hazards, including sharps-related injuries. The objective of this study was to report the frequency of sharps injuries and evaluate which characteristics influence the number of injuries and reporting behaviors. METHODS: A web-based, anonymous survey was available for 10 weeks to 46 US orthopaedic surgery residency programs (1,207 potential residents) participating in an education research collaborative. The survey was divided into the following areas: demographics, training and attitudes concerning occupational hazards, and sharps injuries and reporting. Logistic regression was used to evaluate the association between the above variables on experiencing sharps injuries with significance threshold set at P < 0.05. RESULTS: In this study, 518 surveys were included yielding a response rate of 42.9% (518/1,207). Nearly 80% of the residents recalled some form of safety training during intern orientation and 62% of the respondents felt that they received adequate occupation safety training specifically related to orthopaedic surgery. Four hundred seventeen residents (80.5%) experienced a sharps injury (mean 2.8). Nearly 20% of the respondents experienced ≥5 sharps injuries. Needle sticks (38.8%) were responsible for the greatest percentage of injuries, followed by Kirschner wires (33.6%), scalpel (22.5%), and bone (17.3%). Only 42% of the residents consistently reported all injuries. Reasons included feelings of no risk (63.1%), too much hassle (58.9%), embarrassment (14.5%), other (8.7%), forgot (5.8%), and unclear what to do (3.3%). Inadequate safety training specific to orthopaedic surgery (odds ratio, 2.32 [95% confidence interval, 1.20 to 4.46]; P = 0.012) and greater training seniority (odds ratio, 2.04 [95% confidence interval, 1.64 to 2.52]; P < 0.0001) were associated with acquiring five or more sharps injuries. DISCUSSION: Sharps injuries are a prevalent and concerning reality for orthopaedic surgical trainees. Despite this common occurrence, only 42% of the residents always reported their injuries. Inadequate training specific to orthopaedic surgery and each subsequent year of postgraduate training are associated with increased sharps injuries. STUDY TYPE: Level III, retrospective observational survey.

Facial nerve perineural spread from cutaneous squamous cell carcinoma of the head and neck: A single institution analysis of epidemiology, treatment, survival outcomes, and prognostic factors.

BACKGROUND: This study aimed to examine patients with facial nerve (VII) perineural spread (PNS) from cutaneous squamous cell carcinoma of the head and neck. METHODS: Retrospective analysis of patients managed by an Australian tertiary center between 2000 and 2019. RESULTS: Seventy three patients were included. Most presented with recurrent disease (89.0%) and simultaneous trigeminal nerve (V) involvement (67.1%). Of the 55 patients (75.3%) who received curative intent treatment, 48 received surgery plus/minus post-operative radiotherapy. In these patients, 5-year disease-free survival, disease-specific survival, and overall survival was 50.7%, 68.7%, and 58.1%, respectively. Pathological nodal disease, involved margins, increasing VII zonal extent, and concurrent zone 2 V PNS significantly worsened outcomes. CONCLUSION: High rates of recurrent disease reflects the importance of adequate treatment of the primary. Surgery and post-operative radiotherapy remains the mainstay treatment. Outcomes are improved in early-stage disease and with clear surgical margins, reinforcing the need for prompt diagnosis and intervention.

Hydrophilic and hydrophobic carboxamide pincers as anion hosts.

Hydrophobic and hydrophilic, monotopic and ditopic carboxamide pincer hosts containing ethyl, hexyl, 2-hydroxyethyl and 2-hydroxyethyl ethyl ether pendant arms were synthesized. Solubility trends indicated that solubilities in water or hydrocarbon solvents varied depending on the nature of the pendant arms. Binding constants for hydrophilic pincers were larger in general than their hydrophobic analogs. Significant synergistic binding effects for the ditopic hosts were not observed.