Surgical Delay of Thoracodorsal Artery Perforator Flaps for Total Autologous Breast Reconstruction.

BACKGROUND: When abdomen-based free flap reconstruction is contraindicated, the muscle-sparing thoracodorsal artery perforator (TDAP) flap may be considered for total autologous breast reconstruction. The TDAP flap is often limited by volume and is prone to distal flap necrosis. We aim to demonstrate our experience combining the delay phenomenon with TDAP flaps for total autologous breast reconstruction. METHODS: Patients presenting for autologous breast reconstruction between April 2021 and August 2023 were recruited for surgically delayed TDAP flap reconstruction when abdominally based free flap reconstruction was contraindicated because of previous abdominal surgery or poor perforator anatomy. We dissected the TDAP flap except for a distal skin bridge and then reconstructed the breast 1 to 7 days later. Data included flap dimensions (in centimeters × centimeters), delay time (in days), predelay and postdelay perforator caliber (in millimeters) and flow (in centimeters per second), operative time (in minutes), hospital length of stay (in days), complications/revisions, and follow-up time (in days). RESULTS: Fourteen patients and 16 flaps were included in this study. Mean age and body mass index of patients were 55.9 ± 9.6 years and 30.1 ± 4.3 kg/m2, respectively. Average flap skin island length and width were 32.1 ± 3.3 cm (n = 8 flaps) and 8.8 ± 0.7 cm (n = 5 flaps), respectively. Beveled flap width reached 16.0 ± 2.2 cm (n = 3 flaps). Average time between surgical delay and reconstruction was 2.9 days, ranging from 1 to 7 days (n = 18 flaps). Mean predelay and postdelay TDAP vessel caliber and flow measured by Doppler ultrasound increased from 1.4 ± 0.3 to 1.8 ± 0.3 mm (P = 0.03) and 13.3 ± 5.2 to 43.4 ± 18.8 cm/s (P = 0.03), respectively (n = 4 flaps). Complications included 1 donor site seroma and 1 mastectomy skin flap necrosis. Follow-up ranged from 4 to 476 days (n = 17 operations). CONCLUSIONS: We demonstrate surgically delayed TDAP flaps as a viable option for total autologous breast reconstruction. Our series of flaps demonstrated increased perforator caliber and flow and enlarged volume capabilities and had no incidences of flap necrosis.

How anatomic should anatomic total shoulder arthroplasty be? Evaluation of humeral head reconstruction with the best-fit circle.

BACKGROUND: Humeral implant designs for anatomic total shoulder arthroplasty (aTSA) focus on anatomic reconstruction of the articular segment. Likewise, the pathoanatomy of advanced glenohumeral osteoarthritis often results in humeral head deformity. We hypothesized the anatomic reconstruction of the humeral head in aTSA risks overstuffing the glenohumeral joint. METHODS: Ninety-seven cases (52 females) of primary glenohumeral osteoarthritis in patients treated with aTSA were evaluated. Preoperative computed tomography scans were used to classify glenoid morphology according to the Walch classification. Coronal plane images in the plane of the humerus were used to determine the anatomic best-fit circle as described by Youderian et al. Humeral head thinning was determined as the distance from the center of rotation of the best-fit circle to the nearest point along the humeral articular surface. aTSA was modeled with a predicted anatomic humeral head and a simulated 4-mm polyethylene glenoid component. The change in the position of the native humerus was determined. Wilcoxon Rank Sum tests were used to evaluate differences in humeral head thinning and humeral lateralization between monoconcave and biconcave glenoid morphologies. Spearman's rank correlation coefficients were used to assess the relationship between humeral head thinning with preoperative active forward elevation and external rotation. RESULTS: The mean radius of the best-fit circle was 25.0 ± 2.1 mm. There was a mean thinning of 2.4 ± 2.0 mm (range -1.7 to 8.3). The mean percent thinning of the humeral head was 9.4% ± 7.7%. The mean humeral lateralization was 6.4 ± 2.0 mm. Humeral head thinning was not significantly associated with active forward elevation (r = -0.15, P = .14) or active external rotation (r = -0.12, P = .25). There were no significant differences in the percentage of humeral head thinning (P = .324) or humeral lateralization (P = .350) between concentric and eccentric glenoid wear patterns. CONCLUSIONS: Utilization of the best-fit circle as a guide in aTSA may risk excessive lateralization of the humerus and overstuffing the glenohumeral joint. This may have implications for subscapularis repair and healing, as well as glenoid implant and rotator cuff longevity. These findings call into question whether recreation of normal glenohumeral anatomy in aTSA is appropriate for all patients. Humeral head reconstruction in aTSA should account for glenohumeral joint volume and soft tissue contracture.

Trends in reported outcomes and patient reported outcome measures (PROMs) in humeral shaft fracture literature: a systematic review.

PURPOSE: With a lack of standardization among outcome measures in fracture literature, cross-study comparisons remain limited. This systematic review aimed to identify trends in outcome measures reported by studies of the treatment of humeral shaft fractures. METHODS: A systematic review was performed of studies reporting clinical outcomes of humeral shaft fractures indexed in PubMed. Extracted data included demographics, fracture characteristics, treatment modalities, outcomes, patient reported outcome measures (PROMs), and journal characteristics. Cochran-Armitage tests and linear regressions were used to identify data trends. Pearson chi-square and Kruskal-Wallis tests were used for comparisons between studies. RESULTS: This review included 197 studies with outcomes of 15,445 humeral shaft fractures. 126 studies reported PROMs and 37 different PROMs were used. The Constant Score was most commonly reported (34% of studies), followed by ASES Score (21%), MEPS (21%), and DASH Score (20%). There was a significant increase in PROM usage over time (p = 0.016) and in articles using three or more PROMs (p = 0.005). The number of PROMs were significantly greater in prospective cohort studies and RCTs (p = 0.012) compared to retrospective cohort studies and case series (p = 0.044 for both). Post-treatment shoulder motion was reported in 43% of studies and 34% reported elbow motion. 86% of studies reported complications as an outcome parameter. Time to union and nonunion rate were published in 69% and 88% of studies, respectively. CONCLUSION: This study identified increasing PROM usage over time and disparities in the reporting of outcomes in humeral shaft fracture literature requiring further validation and standardization of available outcome measures.

Surgical Delay-Induced Hemodynamic Alterations of the Superficial Inferior Epigastric Artery Flap for Autologous Breast Reconstruction.

BACKGROUND: The superficial inferior epigastric artery (SIEA) flap allows transfer of tissue without violating the rectus fascia. Traditionally it is best used in single stage reconstruction when vessel caliber is 1.5 mm; 56% to 70% of SIEAs are less than 1.5 mm and, therefore, not reliable. We aim to demonstrate the increased reliability of SIEA through surgical delay by quantifying reconstructive outcomes and delay-induced hemodynamic alterations. METHODS: Patients presenting for autologous breast reconstruction between May 2019 and October 2020 were evaluated with preoperative imaging and received either delayed SIEA or delayed deep inferior epigastric (DIEP) reconstruction based on clinical considerations, such as prior surgery and perforator size/location. Prospective data were collected on operative time, length of stay, and complications. Arterial diameter and peak flow were quantified with Doppler ultrasound predelay and postdelay. RESULTS: Seventeen delayed SIEA flaps were included. The mean age (± SD) was 46.2 ± 10.55 years, and body mass index was 26.7 ± 4.26 kg/m2. Average hospital stay after delay was 0.85 ± 0.90 days, and duration before reconstruction was 6 days to 14.5 months. Delay complications included 1 abdominal seroma (n = 1, 7.7%). Superficial inferior epigastric artery diameter predelay (mean ± 95% confidence interval) was 1.37 ± 0.20 mm and increased to 2.26 ± 0.24 mm postdelay. A significant increase in diameter was noted 0.9 ± 0.22 mm (P < 0.0001). Mean peak flow predelay was 14.43 ± 13.38 cm/s and 44.61 ± 60.35 cm/s (n = 4, P = 0.1822) postdelay. CONCLUSIONS: Surgical delay of the SIEA flap augments SIEA diameter, increasing the reliability of this flap for breast reconstruction. Superficial inferior epigastric artery delay results in low rates of complications and no failures in our series. Although more patients are needed to assess increase in arterial flow, use of surgical delay can expand the use of SIEA flap reconstruction and reduce abdominal morbidity associated with abdominal flap breast reconstruction.

Opioid Knowledge and Prescribing Preferences of Orthopaedic Surgery Residents Before and After an Educational Intervention.

Orthopaedic surgeons are among the highest prescribers of opioids. This study explores the effect of an educational intervention on orthopaedic surgery residents' opioid knowledge and prescribing practices. Orthopaedic residents were surveyed at three urban academic institutions. A pre-survey was administered to residents prior to an educational lecture and case-based session. This included background on the opioid epidemic, multimodal analgesia, opioid consumption in common orthopaedic procedures, and state laws regulating prescribing. Following this intervention, residents were given a post-survey to complete. There was a significant increase in resident confidence concerning their opioid prescribing training (p = 0.03) and their knowledge of alternative pain management therapies (p = 0.03). This was accompanied by an objective improvement in knowledge of state prescribing laws and of metrics regarding the opioid epidemic. Hypothetical opioid pills prescribed after common orthopaedic procedures decreased between the pre- and post-tests. The educational session significantly improved orthopaedic surgery residents' knowledge about opioids and prescribing habits. Formal resident education on opioid knowledge and evidence-based prescribing strategies is an area of potential improvement to combat the opioid crisis. (Journal of Surgical Orthopaedic Advances 31(1):030-033, 2022).

Misplaced tracheoesophageal voice prosthesis: A case of foreign body aspiration.

Foreign body aspiration (FBA) is a rare, but potentially fatal condition frequently seen in the emergency department. Bronchoscopy plays a major role in its diagnosis and treatment. In patients with laryngectomy, the strategies for airway maintenance and foreign body retrieval are limited. We describe management of a patient with laryngectomy presenting with aspiration of a tracheoesophageal voice prosthesis (TEP). The TEP was not initially seen in chest radiography; however, computed tomography showed it within the right lower bronchus. Successful extraction of the TEP was achieved through bronchoscopy with forceps and retrieval basket. Otolaryngology placed a larger TEP and secured it with sutures. TEP migration is rare, but represents a risk for FBA. Initial imaging in the emergency department can be misleading, requiring a high degree of suspicion, as the TEP device may not be seen in standard chest radiography. Flexible bronchoscopy under moderate sedation in conjunction with forceps and retrieval basket may be appropriate for treatment of FBA in patients with laryngectomy and can be performed in the emergency department, preventing hospital admission.

A Novel Technique for Radiographic Diagnosis of Sarcopenia that Accurately Predicts Postoperative Complications in Lower Extremity Free Flap Patients.

BACKGROUND: Sarcopenia is linked to poor outcomes throughout the surgical literature and can be assessed on preoperative imaging to potentially aid in risk stratification. This study examined the effects of sarcopenia on surgical morbidity following lower extremity (LE) reconstruction, and also compared two methods of assessment, one of which is novel ("ellipse method"). METHODS: A retrospective cohort study of 50 patients receiving free flap-based reconstruction of the LE was performed. Bilateral psoas density and area were quantified at L4 through tracing ("traditional method") and encircling ("ellipse method") to calculate Hounsfield unit average calculation (HUAC). Logistic regression and receiving operator curve analysis for the primary outcome of any postoperative complication was used to determine HUAC cutoffs (≤ 20.7 vs. ≤ 20.6) for sarcopenia. Risk of complications associated with sarcopenia was evaluated using Fisher's exact tests. RESULTS: Twelve patients (24%) met criteria for sarcopenia via the traditional method and 16 (32%) via the ellipse method. By both methods, sarcopenic patients were older and more often female and diabetic. These patients also had higher American Society of Anesthesiologists scores and lower serum prealbumin levels. The ellipse method was found to be more accurate, sensitive, and specific than the traditional method in predicting postoperative morbidity (p = 0.009). Via the ellipse method, sarcopenic patients were at higher risk for any complication (p = 0.002) and were at a higher risk for a deep vein thrombus or pulmonary embolism via the traditional method (p = 0.047). CONCLUSION: Sarcopenia is associated with greater pre- and postoperative morbidity in LE reconstruction. The novel ellipse method is a simplified and accurate method of assessing sarcopenia that can be easily performed in the clinical setting.

Interspecific variation in hypoxia tolerance and hypoxia acclimation responses in killifish from the family Fundulidae.

Hypoxia is a pervasive stressor in aquatic environments, and both phenotypic plasticity and evolutionary adaptation could shape the ability to cope with hypoxia. We investigated evolved variation in hypoxia tolerance and the hypoxia acclimation response across fundulid killifishes that naturally experience different patterns of hypoxia exposure. We compared resting O2 consumption rate (MO2 ), and various indices of hypoxia tolerance [critical O2 tension (Pcrit), regulation index (RI), O2 tension (PO2 ) at loss of equilibrium (PLOE) and time to LOE (tLOE) at 0.6 kPa O2] in Fundulus confluentus, Fundulus diaphanus, Fundulus heteroclitus, Fundulus rathbuni, Lucania goodei and Lucania parva We examined the effects of chronic (28 days) exposure to constant hypoxia (2 kPa) or nocturnal intermittent hypoxia (12 h normoxia:12 h hypoxia) in a subset of species. Some species exhibited a two-breakpoint model in MO2  caused by early, modest declines in MO2  in moderate hypoxia. We found that hypoxia tolerance varied appreciably across species: F. confluentus was the most tolerant (lowest PLOE and Pcrit, longest tLOE), whereas F. rathbuni and F. diaphanus were the least tolerant. However, there was not a consistent pattern of interspecific variation for different indices of hypoxia tolerance, with or without taking phylogenetic relatedness into account, probably because these different indices are underlain by partially distinct mechanisms. Hypoxia acclimation generally improved hypoxia tolerance, but the magnitude of plasticity and responsiveness to different hypoxia patterns varied interspecifically. Our results therefore suggest that hypoxia tolerance is a complex trait that is best appreciated by considering multiple indices of tolerance.

Prevalence of Accessory Branches and Other Anatomical Variations in the Radial Artery Encountered during Radial Forearm Flap Harvest: A Systematic Review and Meta-analysis.

BACKGROUND: Harvest of the radial forearm flap (RFF) for reconstructive surgery is proceeded by the Allen test to assess for adequate contralateral perfusion of the hand, yet the Allen test may fail to detect anatomical variations in the radial artery such as aberrant branching. Therefore, the goal of this study was to systematically review the literature regarding anatomical abnormalities of the radial artery that can affect flap harvest and to perform a meta-analysis to estimate the prevalence of such abnormalities. METHODS: A systematic review of the literature was conducted using five online databases to identify all instances of radial artery anatomical variations. Abstracts were reviewed and categorized into either (1) large cohort studies of anatomical variations identified by angiogram or (2) case reports specifically mentioning anomalous or accessory branches of the radial artery. Data from the large cohort studies were included in a random effect meta-analysis to estimate the prevalence of such variations. RESULTS: Eighteen angiogram cohort studies containing a total of 18,115 patients were included in the meta-analysis. Accessory branches were the least common anatomical variant reported, with an estimated average prevalence of 0.5%. Prevalence estimates for more common anatomical variants, including radial artery loops (0.9%), stenosis (1.3%), hypoplasia (1.9%), tortuosity (4.3%), and abnormal origin (5.6%), were also calculated. Thirteen case reports detailing anomalous branches of the radial artery were identified, seven of which involved accessory branches encountered during RFF harvest with no incidence of flap loss. CONCLUSION: Radial artery accessory branches are exceedingly rare, but the prevalence of other anatomical variations that can affect harvest of the RFF warrants consideration. We recommend surgeons consider comprehensive screening prior to RFF harvest to avoid intraoperative discovery of anatomical variants and suggest a low threshold for repeat perfusion testing intraoperatively if radial artery accessory branches are encountered.

Mild thermal stress affects Steinernema carpocapsae infective juvenile survival but not protein content.

Steinernema nematodes and their Xenorhabdus symbionts are a malleable model system to study mutualistic relations. One of the advantages they possess is their ability to be disassociated under in vitro rearing conditions. Various in vitro methods have been developed to produce symbiont colonized and aposymbiotic (symbiont-free) nematodes. Until now, there has been no investigation on how in vitro rearing conditions may have an impact on the storage ability and the protein content of the infective juvenile at different storage temperatures. Thus, in this study, we investigated how infective juvenile longevity and protein content are impacted when the nematodes were reared with two in vitro methods (lipid and liver kidney agar) considering colonized and uncolonized nematodes, and under two different temperatures: 15 °C and 20 °C (mild stress). Infective juveniles reared in vitro (with or without their symbionts) had lower 8-week survival rates. No in vitro reared, colonized IJs survived to the desired 16-week time point. Survival of infective juveniles stored under mild stress temperature (20 °C) was lower than that observed at 15 °C. However, when comparing the interaction between rearing condition and storage temperature, there were not significant differences. With respect to protein content, in vivo, colonized infective juveniles maintained a static protein content over time, suggesting symbiont colonization may influence protein metabolism and/or turnover in infective juveniles.

Identifying Risk Factors for 30-Day Readmissions After Triple Arthrodesis Surgery.

Rigid flatfoot deformity is a debilitating condition that can be managed by triple arthrodesis surgery. Triple arthrodesis has the potential to restore health-related quality of life, but it is also associated with several complications. Few studies have examined the 30-day readmission rates after triple arthrodesis. The objective of this study was to investigate risk factors for 30-day all-cause readmissions after triple arthrodesis. The nationwide readmission database was queried from 2013. By using International Classification of Disease, Ninth Revision, procedure codes, all triple arthrodesis procedures were identified. Demographic factors, comorbidities, insurance status, and hospital characteristics were statistically compared between patients who experienced a 30-day readmission and those who did not. Multivariable logistic regression was used to identify independent risk factors for 30-day readmission. Overall, 1916 triple arthrodesis cases were identified. The overall 30-day readmission rate after triple arthrodesis was 4.6%. Univariate analysis revealed a statistically higher proportion of patients with electrolyte abnormalities (13.8% vs 4.6%; p < .01) in the patients who were readmitted within 30 days compared with those who were not. Multivariable analysis demonstrated Medicaid insurance, relative to private insurance, as the only statistically significant predictor of 30-day readmission with an odds ratio of 4.43 (p < .05). These results suggest that patients of lower socioeconomic status may be at a greater risk for development of a short-term readmission after triple arthrodesis surgery. These findings are important for surgeon and patient communication, counseling, and postoperative care when choosing to pursue triple arthrodesis surgery.

Machine Learning Accurately Predicts Short-Term Outcomes Following Open Reduction and Internal Fixation of Ankle Fractures.

Ankle fractures are common orthopedic injuries with favorable outcomes when managed with open reduction and internal fixation (ORIF). Several patient-related risk factors may contribute to poor short-term outcomes, and machine learning may be a valuable tool for predicting outcomes. The objective of this study was to evaluate machine-learning algorithms for accurately predicting short-term outcomes after ORIF for ankle fractures. The Nationwide Inpatient Sample and Nationwide Readmissions Database were queried for adult patients ≥18 years old who underwent ORIF of an ankle fracture during 2013 or 2014. Morbidity and mortality, length of stay >3 days, and 30-day all-cause readmission were the outcomes of interest. Two machine-learning models were created to identify patient and hospital characteristics associated with the 3 outcomes. The machine learning models were evaluated using confusion matrices and receiver operating characteristic area under the curve values. A total of 16,501 cases were drawn from the Nationwide Inpatient Sample and used to assess morbidity and mortality and length of stay >3 days, and 33,504 cases were drawn from the Nationwide Readmissions Database to assess 30-day readmission. Older age, Medicaid, Medicare, deficiency anemia, congestive heart failure, chronic lung disease, diabetes, hypertension, and renal failure were the variables associated with a statistically significant increased risk of developing all 3 adverse events. Logistic regression and gradient boosting had similar area under the curve values for each outcome, but gradient boosting was more accurate and more specific for predicting each outcome. Our results suggest that several comorbidities may be associated with adverse short-term outcomes after ORIF of ankle fractures, and that machine learning can accurately predict these outcomes.

Comparing 30-day all-cause readmission rates between tibiotalar fusion and total ankle replacement.

BACKGROUND: End-stage ankle arthritis is a debilitating condition that negatively impacts patient quality of life. Tibiotalar fusion and total ankle replacement are treatment options for managing ankle arthritis. Few studies have examined short term readmission rates of these two procedures. The objective of this study was compare all-cause 30-day readmission rates between patients undergoing tibiotalar fusion vs. total ankle replacement. METHODS: This study queried the Nationwide Readmission Database (NRD) from 2013-2014 and used international classification of disease, 9th revision (ICD-9) procedure codes to identify all patients who underwent a tibiotalar fusion or a total ankle replacement. Comorbidities, insurance status, hospital characteristics, and readmission rates were statistically compared between the two cohorts. Risk factors were then identified for 30-day readmission. RESULTS: A total of 5660 patients were analyzed with 2667 in the tibiotalar fusion cohort and 2993 in the total ankle replacement cohort. Univariate analysis revealed that the readmission rate after tibiotalar fusion (4.4%) was statistically greater than after total ankle replacement (1.4%). Multivariable regression analysis indicated that deficiency anemia (OR 2.18), coagulopathy (OR 3.51), renal failure (OR 2.83), other insurance relative to private (OR 3.40), and tibiotalar fusion (OR 2.51) were all statistically significant independent risk factors for having a readmission within 30-days. CONCLUSIONS: These findings suggest that during the short-term period following discharge from the hospital, patients who received a tibiotalar fusion are more likely to experience a 30-day readmission. These findings are important for decision making when a surgeon encounters a patient with end stage ankle arthritis. LEVEL OF EVIDENCE: Level III, cohort study.

Return to Play Criteria Following Surgical Stabilization for Traumatic Anterior Shoulder Instability: A Systematic Review.

PURPOSE: To identify and describe in the existing literature any criteria used for return to play following surgical stabilization for traumatic, anterior shoulder instability. METHODS: We performed a systematic review evaluating surgical stabilization for primary traumatic anterior shoulder instability in skeletally mature patients with a minimum of 1-year follow-up using Level I to IV studies in PubMed and EMBASE from January 1994 to January 2017. RESULTS: Fifty-eight studies with at least 1 explicitly stated criterion for return to play were identified from a review of more than 5,100 published articles. Seven different categories of return to play criteria were identified, the most common of which were time from surgery (89.6%), strength (18.9%), and range of motion (13.8%). Pain, stability, proprioception, and postoperative radiographic evaluation were also used. As hypothesized, in 75.8% of the included studies (44/58), time was the only criterion explicitly used. The most commonly used time for return to play was 6 months. CONCLUSIONS: This systematic review identifies 7 criteria that have been used in the available literature to determine when patients are ready to return to play; however, consistent with our hypothesis, 75% of studies used time from surgery as the sole listed criterion, with the most commonly used time point of 6 months postoperative. All of these criteria can be used in future research to develop a comprehensive checklist of functional criteria in hopes of reducing recurrent injury. LEVEL OF EVIDENCE: Level IV, systematic review.

Human vascular progenitor cells derived from renal arteries are endothelial-like and assist in the repair of injured renal capillary networks.

Vascular progenitor cells show promise for the treatment of microvasculature endothelial injury. We investigated the function of renal artery progenitor cells derived from radical nephrectomy patients, in animal models of acute ischemic and hyperperfusion injuries. Present in human adventitia, CD34positive/CD105negative cells were clonal and expressed transcription factors Sox2/Oct4 as well as surface markers CXCR4 (CD184)/KDR(CD309) consistent with endothelial progenitor cells. Termed renal artery-derived vascular progenitor cells (RAPC), injected cells were associated with decreased serum creatinine after ischemia/reperfusion, reduced albuminuria after hyperperfusion, and improved blood flow in both models. A small population of RAPC integrated with the renal microvasculature following either experimental injury. At a cellular level, RAPC promoted local endothelial migration in co-culture. Profiling of RAPC microRNA identified high levels of miRNA 218; also found at high levels in exosomes isolated from RAPC conditioned media after cell contact for 24 hours. After hydrogen peroxide-induced endothelial injury, RAPC exosomes harbored Robo-1 transcript; a gene known to be regulated by mir218. Such exosomes enhanced endothelial cell migration in culture in the absence of RAPC. Thus, our work shows the feasibility of pre-emptive pro-angiogenic progenitor cell procurement from a targeted patient population and potential therapeutic use in the form of autologous cell transplantation.

A common solution to group 2 influenza virus neutralization.

The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.

Photoacoustic Probes for Ratiometric Imaging of Copper(II).

Photoacoustic tomography has emerged as a promising alternative to MRI and X-ray scans in the clinical setting due to its ability to afford high-resolution images at depths in the cm range. However, its utility has not been established in the basic research arena owing to a lack of analyte-specific photoacoustic probes. To this end, we have developed acoustogenic probes for copper(II)-1 and -2 (APC-1 and APC-2, a water-soluble congener) for the chemoselective visualization of Cu(II), a metal ion which plays a crucial role in chronic neurological disorders such as Alzheimer's disease. To detect Cu(II), we have equipped both APCs with a 2-picolinic ester sensing module that is readily hydrolyzed in the presence of Cu(II) but not by other divalent metal ions. Additionally, we designed APC-1 and APC-2 explicitly for ratiometric photoacoustic imaging by using an aza-BODIPY dye scaffold exhibiting two spectrally resolved NIR absorbance bands which correspond to the 2-picolinic ester capped and uncapped phenoxide forms. The normalized ratiometric turn-on responses for APC-1 and APC-2 were 89- and 101-fold, respectively.

RGS4 inhibits angiotensin II signaling and macrophage localization during renal reperfusion injury independent of vasospasm.

Vascular inflammation is a major contributor to the severity of acute kidney injury. In the context of vasospasm-independent reperfusion injury we studied the potential anti-inflammatory role of the Gα-related RGS protein, RGS4. Transgenic RGS4 mice were resistant to 25 min injury, although post-ischemic renal arteriolar diameter was equal to the wild type early after injury. A 10 min unilateral injury was performed to study reperfusion without vasospasm. Eighteen hours after injury, blood flow was decreased in the inner cortex of wild-type mice with preservation of tubular architecture. Angiotensin II levels in the kidneys of wild-type and transgenic mice were elevated in a sub-vasoconstrictive range 12 and 18 h after injury. Angiotensin II stimulated pre-glomerular vascular smooth muscle cells (VSMCs) to secrete the macrophage chemoattractant RANTES, a process decreased by angiotensin II R2 (AT2) inhibition. However, RANTES increased when RGS4 expression was suppressed implicating Gα protein activation in an AT2-RGS4-dependent pathway. RGS4 function, specific to VSMC, was tested in a conditional VSMC-specific RGS4 knockout showing high macrophage density by T2 MRI compared with transgenic and non-transgenic mice after the 10 min injury. Arteriolar diameter of this knockout was unchanged at successive time points after injury. Thus, RGS4 expression, specific to renal VSMC, inhibits angiotensin II-mediated cytokine signaling and macrophage recruitment during reperfusion, distinct from vasomotor regulation.

Characterization of a broadly neutralizing monoclonal antibody that targets the fusion domain of group 2 influenza A virus hemagglutinin.

UNLABELLED: Due to continuous changes to its antigenic regions, influenza viruses can evade immune detection and cause a significant amount of morbidity and mortality around the world. Influenza vaccinations can protect against disease but must be annually reformulated to match the current circulating strains. In the development of a broad-spectrum influenza vaccine, the elucidation of conserved epitopes is paramount. To this end, we designed an immunization strategy in mice to boost the humoral response against conserved regions of the hemagglutinin (HA) glycoprotein. Of note, generation and identification of broadly neutralizing antibodies that target group 2 HAs are rare and thus far have yielded only a few monoclonal antibodies (MAbs). Here, we demonstrate that mouse MAb 9H10 has broad and potent in vitro neutralizing activity against H3 and H10 group 2 influenza A subtypes. In the mouse model, MAb 9H10 protects mice against two divergent mouse-adapted H3N2 strains, in both pre- and postexposure administration regimens. In vitro and cell-free assays suggest that MAb 9H10 inhibits viral replication by blocking HA-dependent fusion of the viral and endosomal membranes early in the replication cycle and by disrupting viral particle egress in the late stage of infection. Interestingly, electron microscopy reconstructions of MAb 9H10 bound to the HA reveal that it binds a similar binding footprint to MAbs CR8020 and CR8043. IMPORTANCE: The influenza hemagglutinin is the major antigenic target of the humoral immune response. However, due to continuous antigenic changes that occur on the surface of this glycoprotein, influenza viruses can escape the immune system and cause significant disease to the host. Toward the development of broad-spectrum therapeutics and vaccines against influenza virus, elucidation of conserved regions of influenza viruses is crucial. Thus, defining these types of epitopes through the generation and characterization of broadly neutralizing monoclonal antibodies (MAbs) can greatly assist others in highlighting conserved regions of hemagglutinin. Here, we demonstrate that MAb 9H10 that targets the hemagglutinin stalk has broadly neutralizing activity against group 2 influenza A viruses in vitro and in vivo.

Antibody recognition of the pandemic H1N1 Influenza virus hemagglutinin receptor binding site.

Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in antigenicity but fortunately was not nearly as deadly. 5J8 is a human antibody that potently neutralizes a broad spectrum of H1N1 viruses, including the 1918 and 2009 pandemic viruses. Here, we present the crystal structure of 5J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the A/California/07/2009 (H1N1) pandemic virus. 5J8 recognizes a conserved epitope in and around the receptor binding site (RBS), and its HCDR3 closely mimics interactions of the sialic acid receptor. Electron microscopy (EM) reconstructions of 5J8 Fab in complex with an HA trimer from a 1986 H1 strain and with an engineered stabilized HA trimer from the 2009 H1 pandemic virus showed a similar mode of binding. As for other characterized RBS-targeted antibodies, 5J8 uses avidity to extend its breadth and affinity against divergent H1 strains. 5J8 selectively interacts with HA insertion residue 133a, which is conserved in pandemic H1 strains and has precluded binding of other RBS-targeted antibodies. Thus, the RBS of divergent HAs is targeted by 5J8 and adds to the growing arsenal of common recognition motifs for design of therapeutics and vaccines. Moreover, consistent with previous studies, the bacterially expressed H1 HA properly refolds, retaining its antigenic structure, and presents a low-cost and rapid alternative for engineering and manufacturing candidate flu vaccines.