Evolutionarily related small viral fusogens hijack distinct but modular actin nucleation pathways to drive cell-cell fusion

Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP–mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

Aquareovirus and orthoreovirus are two genera of the Reoviridae family of segmented double-stranded RNA viruses that form multinucleated syncytia after infection, which can increase viral spread and pathogenicity (1–4). To drive cell-cell fusion, both aquareovirus and orthoreovirus express a nonstructural, fusion-associated small transmembrane (FAST) protein on the plasma membrane of infected cells. The FAST protein is not required for viral entry, and expression of FAST protein alone is sufficient to cause cells to fuse with naïve neighboring cells, forming large multinucleated syncytium (1, 2, 5–12), confirming they are bona fide cell-cell fusogens. Although they have similar function and topology in the membrane, FAST proteins from aquareovirus and orthoreovirus share minimal sequence identity (13). Based on phylogenetic analysis, they are hypothesized to have evolved from a common, likely nonfusogenic, ancestor 510 million years ago (4, 13, 14). Separate gain-of-function events are believed to have produced fusogenic proteins in both aquareovirus and orthoreovirus, with further divergence or acquisition events resulting in the diversity of FAST proteins found in reoviruses today (13).Aquareovirus and orthoreovirus FAST proteins are single-pass membrane proteins of fewer than 200 residues comprised of a mostly disordered cytoplasmic tail, a transmembrane domain, and a small ectodomain of fewer than 40 residues (1, 2). The membrane-disruptive ectodomains of FAST proteins typically have solvent-exposed hydrophobic residues and/or myristoylation motifs that are necessary for cell-cell fusion (5, 15–17). In contrast to other cell-cell fusogens that fuse membranes by pulling them together using conformational changes in their ∼10 nm-tall ectodomains, the ectodomains of FAST proteins have minimal predicted secondary structure, are unlikely to undergo conformational changes to drive membrane fusion (1, 2), and extend only ∼1 nm above the bilayer (5, 18). How such short fusogens can overcome the ∼2 nm repulsive hydration barrier and larger barrier presented by cell surface proteins to reach and fuse with an opposing membrane (5, 18) has been a long-standing question for FAST proteins and other short cell-cell fusogens, such as myomixer and myomaker that are involved in myoblast fusion (19–22).Recently, we found that the FAST protein from reptilian orthoreovirus, p14, hijacks the host cell actin cytoskeleton to drive cell-cell fusion by forming localized branched actin networks (23). This is accomplished through a c-src phosphorylated tyrosine motif, YVNI, in p14’s disordered cytoplasmic tail that binds to a host adaptor protein, Grb2, which then binds to N-WASP and nucleates branched actin assembly. We hypothesize that this directly couples local actin-generated forces to push p14’s short, fusogenic ectodomain into the opposing cell’s plasma membrane (23). While all FAST family proteins have similarly short ectodomains, it is unclear if this is a general strategy used by other FAST proteins to drive cell-cell fusion.Here, we report that a FAST protein from the divergent aquareovirus, p22, also hijacks the host actin cytoskeleton but does so using a molecular strategy distinct from that of the orthoreovirus FAST protein p14. Instead of binding to Grb2, we find that p22 binds to Intersectin-1 through an SH3 binding motif in its cytoplasmic tail, which binds Cdc42 to activate N-WASP–mediated branched actin assembly. We show that despite minimal sequence identity, the p22 cytoplasmic tail can be functionally swapped with that of p14, suggesting that while the cytoplasmic tails of the two FAST proteins evolved independently, they serve a similar function. By directly coupling the ectodomain to a different actin nucleator, we suggest that actin’s functional role is applying mechanical pressure to a fusogenic ectodomain at the plasma membrane. This biophysical role may be shared across other members of the FAST protein family and could be more generally employed by other cell-cell fusogens.     

Comparison of the accuracy of noninvasive hemoglobin monitoring for preoperative evaluation between adult and pediatric patients: a retrospective study

We measured noninvasive hemoglobin (SpHb) levels during the pre-anesthesia visit in patients planning elective surgery. Differences between SpHb and laboratory-measured hemoglobin (Hb_lab) were compared between adult and pediatric patients. In the pre-anesthesia visiting office, we routinely monitor noninvasive Hb levels with oxygen saturation and heart rate using Masimo Radical-7® Pulse CO-Oximetry (Masimo Corp., Irvine, CA, USA). We attached the R1 20 (body weight, 10–50 kg) or R1 25 (body weight?>?30 kg) probe on the index finger. After signal stabilization, SpHb and perfusion index (PI) were recorded. We retrospectively reviewed the recorded data and included patients who visited the anesthesiologist within 24 h after venous sampling. Bias was calculated by subtracting Hb_lab from SpHb. We compared the biases of adult and pediatric patients (<?18 years) and evaluated correlation coefficients between the bias and Hb_lab. Records of 105 patients were reviewed and 100 data points of 50 patients in each group were analyzed. The median?±?interquartile range bias was ??2.6?±?2.2 and ??1.2?±?1.5 g/dL in adult and pediatric patients, respectively (P?<?0.001); the corresponding mean?±?standard deviation PIs were 4.4?±?3.1 and 5.9?±?2.7, respectively (P?=?0.19). Bias was inversely proportional to Hb_lab irrespective of age. The correlation coefficient between the bias and Hb_lab was ??0.81 in adults and ??0.54 in pediatric patients (P?<?0.001). SpHb and Hb_lab measured during pre-anesthesia visits showed a smaller difference in pediatric than in adult patients. Lower Hb_lab corresponded to higher accuracy.     

Definitive radiotherapy for primary vaginal cancer: correlation between treatment patterns and recurrence rate

The purpose of this study was to determine the outcomes and optimal practice patterns of definitive radiotherapy for primary vaginal cancer. Between 1993 and 2012, 49 patients were treated with definitive radiotherapy for primary vaginal cancer in three hospitals. Of these, 15 patients (31%) had clinically positive regional lymph node metastasis. A total of 34 patients (70%) received external beam radiotherapy with high-dose-rate brachytherapy (interstitial or intracavitary), and 8 (16%) (with small superficial Stage I tumors) were treated with local radiotherapy. The median follow-up was 33 months (range: 1–169 months). The 3-year overall survival (OS), disease-free survival (DFS), and loco-regional control (LRC) rates were 83%, 59% and 71%, respectively. In multivariate analysis, the histological type (P = 0.044) was significant risk factors for LRC. In Federation of Gynecology and Obstetrics (FIGO) Stage I cases, 3 of 8 patients (38%) who did not undergo prophylactic lymph node irradiation had lymph node recurrence, compared with 2 of 12 patients (17%) who underwent prophylactic pelvic irradiation. For Stage III–IV tumors, the local recurrence rate was 50% and the lymph node recurrence rate was 40%. Patients with FIGO Stage I/II or clinical Stage N1 had a higher recurrence rate with treatment using a single modality compared with the recurrence rate using combined modalities. In conclusion, our treatment outcomes for vaginal cancer were acceptable, but external beam radiotherapy with brachytherapy (interstitial or intracavitary) was needed regardless of FIGO stage. Improvement of treatment outcomes in cases of FIGO Stage III or IV remains a significant challenge.     

Imaging sonographic findings of in a case of proliferating trichilemmal tumor of a finger: A case report

Proliferating trichilemmal tumor (PTT) is a rare tumor that originates from the outer root sheath of a hair follicle. About 90% of PTTs occur on the scalp. The sonographic findings of PTT in the subungual region have not been reported previously. In our case, sonography showed a heterogeneous mass containing echogenic foci with no detectable intratumoral vascularity. These echogenic foci probably represent keratin and cholesterol. © 2017 Wiley Periodicals, Inc. J Clin Ultrasound 46 :215–217, 2018     

Results of conservative management for consecutive esotropia after intermittent exotropia surgery

Purpose

To examine the clinical course of consecutive esotropia (ET) using conservative management, after intermittent exotropia (IXT) surgery.

Methods

This study included 149 out of 151 consecutive patients with ET after IXT surgery, who were managed conservatively. The clinical course of consecutive ET was examined and the patients were classified into two groups based on the duration of esodeviation: (1) >3 weeks (persistent ET group, n=56) and (2) <3 weeks (transient ET group, n=93). Patient characteristics and treatment outcomes, including the recurrence of exotropia and stereopsis, were compared between the two groups.

Results

All patients with ET were managed with full-time alternate occlusion and/or with a Fresnel prism. In 149 patients out of 151 consecutive patients, 82% of ET disappeared at 12-month follow-up and all at the last follow-up visit (31.4±23.5 months). At the final visit, a recurrence of exotropia of >10 prism dioptres was significantly less frequent in the persistent ET group than in the transient ET group (25% vs 62%, respectively; P=0.01). However, stereopsis outcome was not significantly different between the two groups, and stereopsis change was not affected by age.

Conclusions

By using conservative management only, persistent consecutive ET after IXT surgery disappeared in most cases by the 1-year follow-up visit after surgery. Recurrence of exotropia was significantly less frequent in patients with persistent ET, yet the sensory outcome was not affected by the duration of consecutive ET or age.     

Pathologic Changes in Wild Birds Infected with Highly Pathogenic Avian Influenza A(H5N8) Viruses,South Korea, 2014

In January 2014, an outbreak of infection with highly pathogenic avian influenza (HPAI) A(H5N8) virus began on a duck farm in South Korea and spread to other poultry farms nearby. During this outbreak, many sick or dead wild birds were found around habitats frequented by migratory birds. To determine the causes of death, we examined 771 wild bird carcasses and identified HPAI A(H5N8) virus in 167. Gross and histologic lesions were observed in pancreas, lung, brain, and kidney of Baikal teals, bean geese, and whooper swans but not mallard ducks. Such lesions are consistent with lethal HPAI A(H5N8) virus infection. However, some HPAI-positive birds had died of gunshot wounds, peritonitis, or agrochemical poisoning rather than virus infection. These findings suggest that susceptibility to HPAI A(H5N8) virus varies among species of migratory birds and that asymptomatic migratory birds could be carriers of this virus.     

Associations of the infancy body mass index peak with anthropometry and cardiometabolic risk in Mexican adolescents

Background: Early-life growth dynamics are associated with future health. Little is known regarding timing and magnitude of the infancy body mass index (BMI) peak with adiposity and metabolic biomarkers during adolescence.

Aim: To examine associations of the infancy BMI peak with anthropometry and cardiometabolic risk during peripuberty.

Methods: Among 163 ELEMENT participants, this study estimated age and magnitude of the infancy BMI peak from eight anthropometric measurements from birth–36?months using Newton’s Growth Models, an acceleration-based process model. Associations were examined of the infancy milestones with anthropometry and cardiometabolic risk at 8–14?years using linear regression models that accounted for maternal calcium supplementation and age; child’s birthweight, sex, and age; and the other infancy milestone.

Results: Median age at the infancy BMI peak was 9.6?months, and median peak BMI was 16.5?kg/m². Later age and larger magnitude of the peak predicted higher BMI z-score, waist circumference, and skinfold thicknesses; i.e. each 1?month of age at peak and each 1?kg/m² of peak BMI corresponded with 0.04 (0.01–0.07) and 0.33 (0.17–0.48) units of higher BMI z-score, respectively. Later age at peak was also a determinant of worse glycaemia and higher blood pressure.

Conclusion: Later age and larger magnitude of the infancy BMI peak are associated with higher adiposity at 8–14?years of age. Later age but not magnitude of the BMI peak are related to a worse cardiometabolic profile during peripuberty.     

Dipeptidyl Peptidase 10, a Novel Prognostic Marker in Colorectal Cancer

Purpose

The dipeptidyl peptidase IV (DPPIV) gene family exhibits multiple functions and is involved in the pathogenesis of various diseases. It has attracted pharmaceutical interest in the areas of metabolic disorders as well as cancer. However, clinicopathologic significance of DPPIV family in colorectal cancer is not fully understood.

Materials and Methods

The clinical relevance of DPPIV and DPP10 expression was determined by immunohistochemical staining, and by assessing its clinicopathologic correlation in 383 colorectal cancer patients with known clinical outcomes.

Results

DPPIV was not expressed in normal colon mucosa, but it showed luminal expression in 52 of the 383 colorectal cancers (13.5%). DPPIV expression in tumors was associated with right-sided location of the colon (p=0.010) and more advanced tumor stage (p=0.045). DPP10 was expressed in normal colonic mucosa, but its expression varied in primary colorectal cancer tissues. Loss of DPP10 expression was found in 11 colorectal cancers (CRCs) (2.9%), and multivariate analysis showed that loss of DPP10 expression was an independent factor for poor patient prognosis (p=0.008).

Conclusion

DPP10 may play a role in disease progression of colorectal cancer and loss of DPP10 expression in primary CRC is significantly associated with poor survival outcomes.