A phosphoinositide switch controls the maturation and signaling properties of APPL endosomes.

The recent identification of several novel endocytic compartments has challenged our current understanding of the topological and functional organization of the endocytic pathway. Using quantitative single vesicle imaging and acute manipulation of phosphoinositides we show that APPL endosomes, which participate in growth factor receptor trafficking and signaling, represent an early endocytic intermediate common to a subset of clathrin derived endocytic vesicles and macropinosomes. Most APPL endosomes are precursors of classical PI3P positive endosomes, and PI3P plays a critical role in promoting this conversion. Depletion of PI3P causes a striking reversion of Rab5 positive endosomes to the APPL stage, and results in enhanced growth factor signaling. These findings reveal a surprising plasticity of the early endocytic pathway. Importantly, PI3P functions as a switch to dynamically regulate maturation and signaling of APPL endosomes.

Next generation sequencing aids diagnosis and management in a case of encephalocraniocutaneous lipomatosis.

Encephalocraniocutaneous lipomatosis (ECCL) is a rare neurocutaneous disorder caused by somatic FGFR1 and KRAS variants. It shares significant phenotypic overlap with several closely related disorders caused by mutations in the RAS-MAPK pathway (mosaic RASopathies). We report a diagnostically challenging case of ECCL in which next-generation sequencing of affected tissue identified a pathologic FGFR1 p.K656E variant, thereby establishing a molecular diagnosis. Patients with FGFR1-associated ECCL carry a risk of developing malignant brain tumors; thus, genetic testing of patients with suspected ECCL has important management implications.

Non-vascular intracranial lesions in three children with PHACE association.

PHACE (posterior fossa malformations, hemangiomas, arterial anomalies, cardiac anomalies, eye anomalies) association has many recognized clinical features. A link between PHACE and non-vascular intracranial lesions has not been well-described. We report three pediatric patients with PHACE and non-vascular intracranial lesions.

Hospital Variation and Resource Use for Infants with Craniosynostosis Undergoing Open, Endoscopic, and Distraction Osteogenesis Surgical Techniques.

BACKGROUND: Craniosynostosis is treated with endoscopic, open, and/or distraction surgical techniques. We assessed institutional variation in the use these techniques for craniosynostosis and compared hospital resource use. METHODS: Retrospective analysis of 5249 infants age <18 months old undergoing surgical procedures for all types of craniosynostosis in 2016-2020 in 39 freestanding children's hospitals in the Pediatric Health Information System (PHIS) database. Endoscopic vs. open cranial vault surgery (with and without distraction osteogenesis) was identified using ICD-10-CM codes. Inpatient cost and length of stay (LOS) were compared by surgery type with Wilcoxon Rank Sum. RESULTS: There was significant (p < .001) variation in the percentage of infants who underwent endoscopic repair across hospitals [median 23.6% (interquartile range (IQR): 7.6%-37.5%), range: 0% to 80.4%] and across regions [range: 22.1% (southeast) to 42.5% (northeast)]. For endoscopic procedures, median LOS and inpatient cost were lower (p < .001) without vs. with distraction [1 day (IQR 1-1) vs. 2 days (IQR 2-2); $14,617 (IQR 11,823-22178) vs. $33,599 (IQR 22,800-38,619)]. For open interventions, median LOS and inpatient cost were also lower (p < .001) without vs. with distraction [3 days (IQR 2-4) vs. 5 days (IQR 4-6) and $37,251 (IQR 27,114-50.320) vs. $62,247 (IQR 42,124-91,620)]. CONCLUSIONS: Substantial variation in the surgical approach for craniosynostosis exists across hospitals and regions. Endoscopic repair without distraction had the lowest hospital resource use, while open repair with distraction had the highest hospital resource. Subsequent analysis of short- and long-term outcomes as well as patient-and-family costs is necessary to assess the true cost-effectiveness of each approach.

Lymphoscintigraphy Using Tilmanocept Detects Multiple Sentinel Lymph Nodes in Melanoma Patients.

BACKGROUND: Technetium-99m-labeled Tilmanocept, a multivalent mannose, is readily internalized by the CD206 surface receptor on macrophages and dendritic cells which are abundantly present in lymph nodes. We want to examine the drainage patterns of Technetium-99m-labeled Tilmanocept to sentinel lymph nodes (SLNs) in melanoma patients following the 10% rule. METHODS: Multi-center retrospective review of patients with cutaneous melanoma undergoing SLN biopsy using Technetium-99m-labeled Tilmanocept between 2008 and 2014 was conducted. Statistical methods were used for data analyses. RESULTS: Of the 564 patients (mean age of 60.3 and 62% male) with preoperative lymphoscintigraphy showing at least one SLN, several primary tumor sites were included: 27% head/neck, 33% trunk, 21% upper extremity and 19% lower extremity. For the head/neck primary site, 36.5% of patients had multiple draining basins; for the trunk site, 36.4% of patients; for the upper extremity site, 13% of patients; and for the lower extremity, 27.4% of patients. A median of 3 (range 1-18) SLNs were identified and resected. Overall, 78% of patients had >1 SLN identified by Technetium-99m-labeled Tilmanocept. In a multivariate model, patients with >1 SLN were significantly associated with age, Breslow depth, tumor location and higher AJCC tumor stage. A total of 17.7% of patients (100/564) had a positive SLN identified. A total of 145 positive SLNs were identified out of 1,812 SLNs with a positive SLN rate of 8%. Positive SLN status was significantly associated with younger age, greater Breslow depth, mitosis rate, higher AJCC tumor stage, presence of ulceration and angiolymphatic invasion. CONCLUSIONS: Using the 10% rule, Technetium-99m-labeled Tilmanocept detects multiple SLNs in most melanoma patients.

Metopic ridge presenting to pediatric dermatology and vascular anomalies clinics.

Metopic ridge (MeR) is a midline osseous forehead prominence resulting from physiologic closure of the underlying metopic suture. This mass-like ridge can be mistaken for serious conditions such as a craniosynostosis or vascular anomaly, prompting concern and workup. We reviewed patients presenting for a forehead mass to Vascular Anomalies and Dermatology clinics and diagnosed with MeR to increase familiarity with this finding and to encourage MeR in the differential diagnosis of pediatric midline forehead masses.

Videofluoroscopy Versus Nasopharyngoscopy of Lateral Pharyngeal Wall Movement for Assessment of Velopharyngeal Insufficiency.

BACKGROUND: Competent speech requires closure of the velopharyngeal sphincter by dynamic apposition of the velum and posterior and lateral pharyngeal walls. An accurate estimation of lateral pharyngeal wall motion is an important determinant in the planning and the outcome of any operation to correct velopharyngeal insufficiency (VPI). The purpose was to compare the assessment of lateral pharyngeal wall movement by videofluoroscopy (VP) versus nasopharyngoscopy (NP). METHODS: The authors retrospectively reviewed the charts of 269 consecutive patients in our cleft lip/palate clinic from 1982 to 2008 and culled those treated with a pharyngeal flap for VPI. The authors included patients who were evaluated preoperatively by both VP and NP, and had studies of suitable quality. Percentage of lateral pharyngeal wall motion was estimated with each technique and compared for each patient. RESULTS: The authors identified 25 patients who underwent both VP and NP at the same median age (4.7 years). The estimated percentage of lateral pharyngeal wall motion between the 2 techniques was significantly different ( P <0.001). Average lateral pharyngeal wall motion was estimated to be 59±25% (range: 5%-90%) by VP and only 40%±25% (range: 0%-95%) during NP. CONCLUSIONS: VP and NP are complementary, but assessment of lateral pharyngeal wall motion can vary between the 2 methods. The surgeon should be aware of the difference in estimated lateral pharyngeal wall movement when planning a procedure to correct VPI.

Kidney Tubular Ablation of Ocrl/Inpp5b Phenocopies Lowe Syndrome Tubulopathy.

Lowe syndrome and Dent disease are two conditions that result from mutations of the inositol 5-phosphatase oculocerebrorenal syndrome of Lowe (OCRL) and share the feature of impaired kidney proximal tubule function. Genetic ablation of Ocrl in mice failed to recapitulate the human phenotypes, possibly because of the redundant functions of OCRL and its paralog type 2 inositol polyphosphate-5-phosphatase (INPP5B). Germline knockout of both paralogs in mice results in early embryonic lethality. We report that kidney tubule-specific inactivation of Inpp5b on a global Ocrl-knockout mouse background resulted in low molecular weight proteinuria, phosphaturia, and acidemia. At the cellular level, we observed a striking impairment of clathrin-dependent and -independent endocytosis in proximal tubules, phenocopying what has been reported for Dent disease caused by mutations in the gene encoding endosomal proton-chloride exchange transporter 5. These results suggest that the functions of OCRL/INPP5B and proton-chloride exchange transporter 5 converge on shared mechanisms, the impairment of which has a dramatic effect on proximal tubule endocytosis.

Failed pneumoperitoneum for laparoscopic surgery following autologous Deep Inferior Epigastric Perforator (DIEP) flap breast reconstruction: a case report.

BACKGROUND: Laparoscopic abdominal surgery may prove difficult in patients who have undergone previous abdominal procedures. No reports in the medical literature have presented an aborted laparoscopic procedure for failed pneumoperitoneum following autologous flap-based breast reconstruction. CASE PRESENTATION: A 55-year-old woman presented with recurrent invasive lobular carcinoma of the right breast as well as a history of ductal carcinoma in situ of the left breast. The patient desired to proceed with bilateral skin- and nipple-sparing mastectomies with right axillary lymph node biopsy, followed by immediate bilateral autologous deep inferior epigastric perforator (DIEP) flap-based breast reconstruction. Preoperatively, a computerized tomography angiogram was obtained for reconstructive preparation, which revealed a left adrenal mass. Ensuing work-up diagnosed a pheochromocytoma. Given the concern for breast cancer progression, the patient elected to proceed first with breast cancer surgery and reconstruction prior to addressing the adrenal tumor. Subsequently, 3 months later the patient was brought to the operating room for a laparoscopic left adrenalectomy for the pheochromocytoma. With complete pharmacologic abdominal relaxation, the abdomen proved too tight to accommodate sufficient pneumoperitoneum and the laparoscopy was aborted. The patient was evaluated in the outpatient setting for assessment of abdominal wall compliance at regular intervals. Five months later, the patient was taken back to the operating room where pneumoperitoneum was established without difficulty and the laparoscopic left adrenalectomy was performed without complications. CONCLUSION: Pneumoperitoneum for laparoscopic surgery subsequent to autologous DIEP flap-based breast reconstruction may prove difficult as a result of loss of abdominal wall compliance. Prior to performing laparoscopy in such patients, surgeons should consider the details of the patient's previous reconstructive procedure and assess potential risk factors for difficulty with insufflation. Lastly, careful abdominal examination should be performed to indicate whether laparoscopy for elective procedures should be delayed until abdominal wall compliance normalizes.

Bilateral Coronoidectomy by Craniofacial Approach for Hecht Syndrome-Related Trismus.

Hecht Syndrome is an autosomal dominant distal arthrogryposis caused by mutation in the MYH8 locus characterized by trismus and pseudocamptodactyly. Hecht-associated trismus is thought to result from bilateral hyperplasia of the mandibular coronoid processes. Although several interventions to address trismus have been pursued, no consensus exists regarding optimal management. In this report, the authors present a 7-month-old male with Hecht Syndrome referred for management of trismus. By age 2, interincisal opening had progressively decreased from 12 to 5 mm despite physical therapy. Nutrition was limited to liquids, oral hygiene was compromised, and aspiration risk was present. Computed tomography examination revealed enlarged coronoid processes extending medially and superiorly to the zygomatic arches. To release bony impaction of the coronoid processes against the zygoma and to prevent reossification of the temporalis tendon insertion, resection of the enlarged coronoids and distal temporalis muscles as well as placement of Alloderm spacers were performed via an open craniofacial transzygomatic approach. Jaw motion rehabilitation was used following surgery. Two years postoperatively, the patient had no signs of recurrence and good functional stability of jaw excursion. He was able to chew and swallow solid foods, protrude his tongue, use utensils, and perform regular oral hygiene, none of which were possible before surgery. This case demonstrates that open bilateral coronoidectomy can be a successful and durable management option for trismus in patients with Hecht Syndrome. The open transzygomatic approach is safe, has low morbidity, and provides direct access and adequate exposure for coronoid resection, spacer placement, and prevention of temporalis reinsertion.

A Review of the Current State and Future Directions for Management of Scalp and Facial Vascular Malformations.

Vascular malformations are structural abnormalities that are thought to result from errors in vasculogenesis and angiogenesis during embryogenesis. Vascular malformations of the scalp present unique management challenges due to aesthetic and functional implications. This review examines the pathophysiology, clinical presentation, and management techniques for six common types of vascular malformations of the face and scalp : infantile hemangioma, capillary malformations, venous malformations, lymphatic malformations, arteriovenous malformations, and arteriovenous fistulas. These lesions range from common to rare, and have very different natural histories and management paradigms. There has been increasing understanding of the molecular pathways that are altered in association with these vascular lesions and these molecular targets may represent novel strategies of treating lesions that have historically been approached from a structural perspective only.

Melanoma in infants, caused by a gene fusion involving the anaplastic lymphoma kinase (ALK).

We describe the first cases of pediatric melanoma with ALK fusion gene arising within giant congenital melanocytic nevi. Two newborn boys presented with large pigmented nodular plaques and numerous smaller satellite nevi. Additional expansile nodules developed within both nevi and invasive melanomas were diagnosed before 10 months of age in both boys. Oncogenic driver mutations in NRAS and BRAF were absent in both cases. Instead, oncogenic ZEB2::ALK fusion genes were identified in both the nevus and melanoma developing within the nevus. In both cases, tumors were noted by ultrasound in utero, demonstrated significant nodularity at birth, and progressed to melanoma in the first year of life suggesting that congenital nevi with ALK fusion genes may behave more aggressively than those with other mutations. As ALK kinase inhibitors are effective against a range of tumors with similar ALK fusion kinases, identifying ALK fusion genes in congenital melanocytic nevi may provide an opportunity for targeted therapy.

A PH domain within OCRL bridges clathrin-mediated membrane trafficking to phosphoinositide metabolism.

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5-phosphatase domain followed by an ASH and a RhoGAP-like domain. Their divergent NH2-terminal portions remain uncharacterized. We show that the NH2-terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin-coated pits, was earlier shown to contain another binding site for clathrin in its COOH-terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2-terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin-binding site in OCRL maps to an unusual clathrin-box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin-dependent membrane trafficking.

Kaposiform Lymphangiomatosis: Pathologic Aspects in 43 Patients.

Kaposiform lymphangiomatosis is an uncommon generalized lymphatic anomaly with distinctive clinical, radiologic, histopathologic, and molecular findings. Herein, we document the pathology in 43 patients evaluated by the Boston Children's Hospital Vascular Anomalies Center from 1999 to 2020. The most frequent presentations were respiratory difficulty, hemostatic abnormalities, and a soft tissue mass. Imaging commonly revealed involvement of some combination of mediastinal, pulmonary, pleural, and pericardial compartments and most often included spleen and skeleton. Histopathology was characterized by dilated, redundant, and abnormally configured lymphatic channels typically accompanied by dispersed clusters of variably canalized, and often hemosiderotic, spindled lymphatic endothelial cells that were immunopositive for D2-40, PROX1, and CD31. An activating lesional NRAS variant was documented in 9 of 10 patients. The clinical course was typically aggressive, marked by hemorrhage, thrombocytopenia, diminished fibrinogen levels, and a mortality rate of 21%.

Airway Hemangiomas in PHACE Syndrome: A Multicenter Experience.

OBJECTIVE: To describe the prevalence and clinical characteristics of airway findings in a multi-institutional cohort of PHACE patients. STUDY DESIGN: Multicenter retrospective case series. SETTING: Multidisciplinary vascular anomalies clinics at 2 institutions. METHODS: Data were collected from the electronic medical record, including clinical presentation, airway findings, treatment, and outcomes. RESULTS: Of 55 PHACE patients, 22 (40%) had airway hemangiomas. Patients with airway involvement were more commonly female (P = .034, odds ratio [OR] 23, 95% confidence interval [CI] 1.3-410) and of Caucasian ethnicity (P = .020, OR 5.3, 95% CI 1.3-21). Anatomically, patients with bilateral S3 involvement had higher rates of airway disease (P = .0012, OR 15, 95% CI 2.9-77). Most patients with airway hemangiomas had stridor (68%). Of the patients managed in the propranolol era (2008 or later, n = 35), 14 had airway involvement. All 14 were treated with propranolol, whereas 13 (62%) of 21 nonairway patients were treated with propranolol. The average treatment duration was longer in the airway patients (22.1 vs 16.7 months). All patients who underwent tracheostomy (n = 4) did so before 2008. CONCLUSION: Risk factors for airway involvement in PHACE include female gender, Caucasian ethnicity, and stridor. Since the widespread use of propranolol, fewer patients have required surgical management of their airway disease. Given the high prevalence of airway involvement even in patients without stridor, assessment of the airway is a crucial component of a comprehensive PHACE workup.

Association of Demographic Factors and Infantile Hemangioma Characteristics With Risk of PHACE Syndrome.

Importance: A 2010 prospective study of 108 infants estimated the incidence of PHACE (posterior fossa malformations, hemangioma, arterial anomalies, cardiac defects, eye anomalies) syndrome to be 31% in children with facial infantile hemangiomas (IHs) of at least 22 cm2. There is little evidence regarding the associations among IH characteristics, demographic characteristics, and risk of PHACE syndrome. Objectives: To evaluate demographic characteristics and comorbidities in a large cohort of patients at risk for PHACE syndrome and assess the clinical features of large head and neck IH that may be associated with a greater risk of a diagnosis of PHACE syndrome. Design, Setting, and Participants: This multicenter, retrospective cohort study assessed all patients with a facial, head, and/or neck IH who were evaluated for PHACE syndrome from August 1, 2009, to December 31, 2014, at 13 pediatric dermatology referral centers across North America. Data analysis was performed from June 15, 2017, to February 29, 2020. Main Outcomes and Measures: The main outcome was presence or absence of PHACE syndrome. Data included age at diagnosis, sex, patterns of IH presentation (including size, segment location, and depth), diagnostic procedures and results, and type and number of associated anomalies. Results: A total of 238 patients (mean [SD] age, 2.96 [4.71] months; 184 [77.3%] female) were included in the analysis; 106 (44.5%) met the criteria for definite (n = 98) or possible (n = 8) PHACE syndrome. A stepwise linear regression model found that a surface area of 25 cm2 or greater (odds ratio [OR] 2.99; 95% CI, 1.49-6.02) and involvement of 3 or more locations (OR, 17.96; 95% CI, 6.10-52.85) to be statistically significant risk factors for PHACE syndrome. Involvement of the parotid gland (OR, 0.39; 95% CI, 0.18-0.85) and segment S2 (OR, 0.38; 95% CI, 0.16-0.91) was associated with a lower risk. Race and ethnicity may also be associated with PHACE syndrome risk, although more studies are needed. Conclusions and Relevance: This cohort study further described factors associated with both a higher and lower risk of PHACE syndrome. The presence of multiple anatomical sites and large surface area were associated with greater risk, whereas S2 or parotid IHs were associated with lower, but still potential, risk. These findings can help in counseling families and decision-making regarding evaluation of infants with large head and neck IHs.

Methicillin-resistant Staphylococcus aureus: a pervasive pathogen highlights the need for new antimicrobial development.

Staphylococcus aureus (S. aureus) has entered the spotlight as a globally pervasive drug-resistant pathogen. While historically associated exclusively with hospital-acquired infections in immunocompromised hosts, the methicillin-resistant form of S. aureus has been spreading throughout communities since the 1990s. Indeed, it has now become a common household term: MRSA. S. aureus has developed numerous mechanisms of virulence and strategies to evade the human immune system, including a host of surface proteins, secreted enzymes, and toxins. In hospital intensive care units, the proportion of MRSA-related S. aureus infections has increased strikingly from just 2 percent in 1974 to 64 percent in 2004. Its presence in the community has been rising similarly, posing a significant public health burden. The growing incidence of MRSA unfortunately has been met with dwindling efforts to develop new, more effective antibiotics. The continued emergence of resistant strains of bacteria such as MRSA demands an urgent revival of the search for new antibiotics.

Selective recognition of protein tetraserine motifs with a cell-permeable, pro-fluorescent bis-boronic acid.

There is considerable interest in novel cell imaging tools that avoid the use of fluorescent proteins. One widely used class of such reagents are "pro-fluorescent" biarsenical dyes such as FlAsH, ReAsH, CrAsH, and Cy3As. Despite their utility, biarsenicals are plagued by high background labeling and cytotoxicity and are challenging to apply in oxidizing cellular locale. Here we demonstrate that [(3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3',6'-diyl)bis(iminomethylene-2,1-phenylene)]bis-(9CI), a rhodamine-derived bisboronic acid (RhoBo) described initially as a monosaccharide sensor, functions as a cell-permeable, turn-on fluorescent sensor for tetraserine motifs in recombinant proteins. RhoBo binds peptides or proteins containing Ser-Ser-Pro-Gly-Ser-Ser with affinities in the nanomolar concentration range and prefers this sequence to simple monosaccharides by >10,000-fold. RhoBo fails to form fluorescent complexes with constituents of the mammalian cell surface, as judged by epifluorescent, confocal, and TIRF microscopy, but fluoresces brightly within the Ser-Ser-Pro-Gly-Ser-Ser-rich cell interior. These results suggest that current efforts to identify optimal serine-rich sequences for RhoBo will allow it to function effectively as a selective small-molecule label for appropriately tagged proteins either upon or within living cells.